Immunogenic compositions of disease-associated antigens for vaccines, antibody production and immunodiagnostic assays

Abstract

According to the present invention, 21 amino acids are deleted from the C-terminal region of the S2 subunit of the SARS-CoV2-S protein, and the S1 subunit fused to the S2 is transferred to the cell membrane; in this manner, the mass presentation of the antigen S1 subunit of the SARS-CoV2-S protein in the cell membrane in its native structure and its use as a whole cell or cell membrane have been determined as a new vaccination regimen of SARS-CoV-2. The S2 subunit of the SARS-CoV2-S protein is designed as a carrier, any bacteria, viruses and tumor proteins with antigen characteristics are fused and transferred to a cell membrane, and the scheme is a comprehensive vaccination scheme covering all bacteria, viruses and even tumors.

Description

[0001] The present invention relates to a vaccination method developed in large quantities, safely and effectively in a short period of time against all viruses (especially Covid-19), bacteria and tumor-related antigens.

[0002] Preparation of cell membranes presenting the covid-19 spike protein in their structure for use in vaccination, antibody production and preparation of ELISA for antibody detection of COVID-19.

[0003] Instruction Manual Summary

[0004] The present invention is based on deleting 21 amino acids from the C-terminal region of the S2 subunit of the SARS-CoV2-S protein and transporting the S1 subunit fused to S2 to the cell membrane. In this way, the massive presentation of the antigenic S1 subunit of the SARS-CoV2-S protein in its native structure in the cell membrane and its use as a whole cell or cell membrane have been identified as a new vaccination scheme for SARS-CoV-2. Designing the S2 subunit of the SARS-CoV2-S protein as a carrier, fusing any bacterial, viral and tumor protein with antigenic properties and transporting it to the cell membrane will be a comprehensive vaccination scheme covering all bacteria, viruses and even tumors. Background Art

[0005] The Covid-19 pandemic caused by the novel coronavirus SARS-CoV-2 has caused massive global disruption. The most important hope for reducing the risk of illness and infection is believed to be the development of an effective and sizable SARS-CoV2 vaccine.

[0006] The S protein, the most critical surface protein of SARS-CoV-2, is a large trimeric transmembrane glycoprotein with many glycosylation modifications that form a unique corona structure on the surface of the virus. The S protein consists of S1 subunit and S2 subunit. The total length of SARS-CoV2-S is 1273 amino acids and consists of a signal peptide at the N-terminus (amino acids 1-13), S1 subunit (residues 14-685) and S2 subunit (residues 686-1273); the last two regions are responsible for receptor binding and membrane fusion, respectively. In the S1 subunit, there is an N-terminal domain (residues 14-305) and a receptor binding domain (residues 319-541);

[0007] The S2 subunit consists of the fusion peptide (FP) (residues 788-806), heptad repeat sequence 1 (HR1) (residues 912-984), HR2 (residues 1163-1213), TM domain (residues 1213-1237) and cytoplasmic domain (residues 1237-1273) (Huang et al., 2020). The S1 subunit mainly contains the receptor binding domain (RBD), which is responsible for receptor recognition. Mutations involving RBD-related amino acids lead to changes in virus species preference and infection characteristics. The S2 subunit contains elements necessary for membrane fusion. The S protein plays a key role in the binding of the virus to the host cell membrane receptor and the fusion of the virus with the host cell membrane receptor. Given the key role of the S protein in SARS-CoV2 infection and adaptive immunity, the S protein is an important target site for neutralizing antibodies and a key target for vaccine design.

[0008] Roughly speaking, many vaccines received emergency use authorization (EUA) from the U.S. FDA three years after the first case of COVID-19 was identified. The new technologies used to develop these vaccines also helped to speed up the development of vaccine candidates.

[0009] Of the 64 COVID-19 vaccines in clinical trials, 44 are based on S protein, of which 29 are based on full-length S protein (65.91%, 29 / 44), 14 are based on S protein RBD (31.82%, 14 / 44), and 1 is based on S protein S-2P (2.27%, 1 / 44). In addition, Moderna mRNA-1273 vaccine and Pfizer-BioNtech BNT162b2 vaccine were designed based on the nucleotide sequence of S protein (Golob et al., 2021).

[0010] Three major whole inactivated virus (WIV) vaccines are beginning to be widely distributed. Several protein-based and protein particle-based vaccines are showing promising results (Hotez and Bottazzi, 2021)

[0011] Traditionally, vaccines take more than 10 years to progress from target identification and platform selection to completion of Phase I, II, and III clinical trials and ultimately regulatory approval. The massive SARS-CoV2 infection and death toll worldwide demand a different approach. In May 2020, the U.S. government established Operation WarpSpeed, a public-private partnership to accelerate the development, production, and distribution of COVID-19 vaccines, therapeutics, and diagnostics (Golob et al., 2021).

[0012] Despite the urgency of the situation, the development of SARS-CoV2 vaccines proceeds through the same steps as other vaccines: target identification, platform selection, design of vaccine candidates, and phased human clinical trials. In Phase I and Phase II clinical trials, immune responses to SARS-CoV2 vaccines are primarily determined based on the detection of antibodies against the S protein and RBD. Protection against SARS-CoV2 infection is assumed based on the detection of antibody titers similar to those found in convalescent plasma and ELISA antibody test results comparable to the effective levels in the virus neutralization assay.

[0013] Phase I / II trials focus on dose finding, safety, and measurement of immunogenicity (e.g., development of neutralizing antibodies against the SARS-CoV2-S protein). Phase III trials need to be double-blind, randomized, and placebo-controlled, and a minimum of 2 months of safety data is required for consideration of emergency use authorization (EUA) (Golob et al., 2021).

[0014] Virus-specific antibodies (IgA and IgM, followed by IgG) against viral surface glycoproteins (primarily the spike (S) glycoprotein) are tested within 7 to 10 days after onset of illness. Studies have shown that neutralizing antibodies are short-lived, declining to undetectable levels within a few months.

[0015] Currently reported efficacy estimates are based on data from the first 3-4 months after the first dose of the candidate vaccine. Therefore, in order to stop the spread of the virus, at least 60% of the entire population should be immunized within 2-3 months. However, the above-mentioned vaccine production methods cannot produce enough vaccine to vaccinate such a large population in a short period of time. Therefore, ongoing challenges to achieving vaccination goals include producing enough vaccine to vaccinate a large population and supply chain issues, timely and equitable distribution, and the continued threat of emerging virus variants.

[0016] Therefore, there is a need for effective vaccine methods that can be produced in large quantities in a short period of time.

[0017] Some studies have shown that the SARS-CoV2 spike protein alone crosses the blood-brain barrier (Rhea et al., 2021).

[0018] Furthermore, researchers found that the S1 subunit of the SARS-CoV2 S protein, without a complete virion or genome, triggers growth signals and mediates cell signaling in cultured human blood vessels from the lung, promoting proliferation and / or hypertrophy of vascular smooth muscle and endothelial cells, leading to complex cardiovascular outcomes (Suzuki et al., 2020).

[0019] The present invention relates to a method for mass production of safe and effective SARS-CoV2 vaccines in a short period of time.

[0020] The present invention is based on the intracellular target signaling region of the coronavirus spike protein described by Lontok et al. (Lontok et al., 2004).

[0021] According to their findings, coronavirus budding at the ER-Golgi intermediate compartment (ERGIC) requires the accumulation of viral envelope proteins at this point in the secretory pathway. They demonstrated that the spike (S) protein from the group 3 coronavirus infectious bronchitis virus (IBV) contains a typical double lysine ER retrieval signal (-KKXX-COOH) in its cytoplasmic tail. This signal allows the retention of chimeric reporter proteins in the ERGIC and allows the transport of full-length S proteins as well as chimeras to the plasma membrane when this protein is mutated. Recently, Boson et al. suggested that envelope proteins (E) and membrane proteins (M) regulate the intracellular trafficking of S and its intracellular processing. Indeed, data showed that upon co-expression of E or M, S relocalized to the ERGIC or Golgi compartments, as observed in SARS-CoV-2 infected cells, which prevented the formation of syncytia. They also showed that the C-terminal retrieval motif in the cytoplasmic tail of S is required for M-mediated retention of S in the ERGIC and that E induces S retention by regulating the cellular secretory pathway. They also showed that the S protein prepared with a 21-amino acid deletion in the C-terminal region was transported to the plasma membrane and that the S protein was substantially localized in the cell membrane, which resulted in the formation of syncytia.

[0022] They concluded that the C-terminal portion of the SARS-CoV2-S cytoplasmic tail is essential for retention of the S protein in the ERGIC compartment (Boson et al., 2020).

[0023] Therefore, I hypothesized that when the SARS-CoV2-SΔ21 protein is ectopically expressed in cells without M expression, it will be densely localized in the cytoplasmic membrane and not retained in the ERGIC. In addition, the absence of the E protein will increase the secretion of SARS-CoV2-S to the cell membrane and prevent the retention of SARS-CoV2-S in the ERGIC. Importantly, the SARS-CoV2-SΔ21 protein will be located in the cell membrane in its most native form compared to conditions in which the S protein was obtained by other different methods (Hotez and Bottazzi, 2021).

[0024] Therefore, I hypothesize that the antigenic S1 subunit of the SARS-CoV2-S protein is presented in large quantities in the cell membrane of eukaryotic organisms in its native structure and its use as whole cells or cell membranes as a new vaccination strategy for SARS-CoV-2. The S1 of SARS-CoV2 presented in large quantities in the cell membrane in its native structure will provide a much stronger and more specific immune response.

[0025] Most importantly, in this way, it is possible to prepare almost unlimited amounts of the native form of the SARS-CoV2-S antigen using cell lines in a short period of time.

[0026] In addition, because the S protein will be attached to the cell membrane and cannot circulate freely, it will not be able to pass the blood-brain barrier and may not exhibit the above-mentioned cell signal stimulation effect.

[0027] Vaccination with whole dead tumor cells (autologous or allogeneic) in cancer patients has been tested for a long time and its clinical application has been approved and used. Neller et al. examined the clinical results of 173 published peer-reviewed immunotherapy trials using melanoma, renal cell carcinoma and hepatocellular carcinoma, lung cancer, prostate cancer, breast cancer, colorectal cancer, cervical cancer, pancreatic cancer or ovarian cancer. They showed that neither allogeneic nor autologous whole tumor cells caused any significant side effects (Neller et al., 2008)

[0028] Therefore, after SARS-CoV2-SΔ21 is expressed in allogeneic cancer cells, it will be possible to use it as whole cell (dead) or cell membrane extracts for vaccination. It is also one of the purposes of the present invention to use immortalized autologous and / or allogeneic primary tissue cells or stem cells to express SARS-CoV2-SΔ21.

[0029] One of the advantages of using live cells expressing SARS-CoV2-S is that the presence of heat shock proteins, uric acid, HMGB1 (high mobility group protein 1), ATP, and double-stranded genomic DNA from dying cells has an adjuvant effect, as shown in previous studies (Radford et al., 2014).

[0030] Recently, the application of exosomes in cancer vaccines and treatments has become increasingly important. Exosomes are nanometer-sized membrane vesicles derived from late endosomal compartments that are able to transfer proteins, lipids, and RNA between cells. Most studies investigating exosomes as cancer vaccines or treatments have focused on fully antigen-loaded exosomes that are able to induce immune responses (Nicolini et al., 2021). In addition, recent findings have shown that B cells and dendritic cells (DCs) release exosomes that express major histocompatibility complex (MHC) class I and class II as well as co-stimulatory molecules (CD80 / 86) and can induce peptide-specific T cell proliferation in an MHC-dependent manner (Liu et al., 2019).

[0031] Therefore, the S protein is expressed in large quantities in the cell membrane in its native form and its use for vaccination purposes is similar to the exosome vaccine approach. In addition, it is known that in contrast to exosomes, all tissues contain MHCI expression in their cell membranes. Summary of the invention

[0032] Exosomes are nanometer-sized membrane vesicles that can transfer proteins, lipids, and RNA between cells. Fully antigen-loaded exosomes can induce immune responses.

[0033] The present invention is developed based on the logic of immune response, which is the result of protein (antigen) transport loaded on exosomes. In the present invention, instead of transporting antigens through exosomes, it is based on transporting proteins (antigens) by loading them into cell membranes, providing antigen presentation and inducing immune responses.

[0034] The C-terminal part of the SARS-CoV2-S cytoplasmic tail is essential for the retention of the S protein in the ERGIC compartment. Therefore, in the present invention, it is assumed that the amino acid deletion of the C region of the SARS-CoV2-S protein will allow the SARS-CoV2-S protein to occur in the cell membrane without being retained in the ERGIC. Importantly, they will be present in the cell membrane in their most natural form. Therefore, the present invention is based on the S2 subfragment protein of SARS-CoV2-S with the last 21 amino acids removed, which transports the antigen protein (S1 subfragment) to the cell membrane and presents it densely there ( Figure 1b , Figure 1c ).

[0035] According to the first aspect of the present invention, SARS-CoV2 spike Δ21 overexpression places the SARS-CoV2-S protein in the cell membrane ( Figure 1f to Figure 1j ).

[0036] Therefore, the present invention provides a complete SARS-CoV2-S protein (Spike Δ21) fragment with the last 21 amino acids removed, which is overexpressed in eukaryotic cells and presented in / with the cell membrane as a new coronavirus SARS-CoV2 vaccine.

[0037] Another embodiment of the present invention is the expression of the SARS-CoV2-SΔ21 gene using a vector system designed for a tetracycline-inducible retroviral mammalian expression system, which is controlled by adding doxycycline (Dox) to the culture medium.

[0038] The present invention confirms that, based on the expression level of SARS-CoV2-S protein, cell-cell fusion was observed in Hep3B cells 24 hours after dox addition, and further unification of cells was observed after 48 hours, destroying the cell structure ( Figure 2a Although the expression of SARS-CoV2-SΔ21 was not as significant as in Hep3B cells, fusion was also produced in Renca and MAD109 cells ( Figure 1i and Figure 1j )

[0039] The present invention also demonstrates that cells expressing SARS-CoV2 Spike Δ21 provide substantial Spike protein expression in the cell membrane, as determined by ELISA assays in addition to dot blot and IHC assays ( Figures 3a to 3e ).

[0040] Therefore, the present invention provides an ELISA assay to measure the levels of anti-SARS-CoV2-S antibodies (IgM, IgG and IgA) in the serum of humans infected or vaccinated with SARS-CoV2.

[0041] The present invention also proposes for the first time that due to overexpression of SARS-CoV2-SΔ21 in cells, the S1 protein is transported to the cell membrane and replaces the main membrane proteins SPTA1 and CDH2. It can be determined that cell membrane proteins including SPTA1 or CDH2 are reduced at a rate similar to the increase in the amount of S protein in the cell membrane. These results were confirmed by dot blot and ELISA ( Figure 4a to Figure 4h )

[0042] The present invention also proposes to use allogeneic cancer cells expressing SARS-CoV2-SΔ21 in their membranes as vaccines to induce a large amount of SARS-CoV2-S protein-specific antibodies ( Figure 5a , Figure 5b ).

[0043] The present invention also confirmed that the use of living cells, dead (ultraviolet treatment, freeze-thaw) cells or cell membranes alone of allogeneic cells expressing SARS-CoV2-S protein induced a large amount of SARS-CoV2-S protein-specific antibodies (data not shown).

[0044] The present invention also confirms that the use of the above cells or cell membranes also induced a large amount of SARS-CoV2-S protein-specific antibodies (data not shown) because the cell membrane was obtained by different methods such as optical, mechanical, electrical and chemical lysis techniques.

[0045] Results: The mean endpoint titers of S protein-specific IgG in mice immunized with a single vaccination of RencaWhC expressing SARS-CoV2-S (WhC-S) containing CFA were significantly higher than those observed in mice receiving PBS containing CFA ( Figure 5a In addition, in mice immunized with CM expressing SARS-CoV2-S (CM-S), S protein-specific IgG, although not significant, was observed to be slightly increased compared with the group of mice immunized with WhC-S ( Figure 5c ). In addition, the addition of CFA to WhC-S and CM-S immunizations slightly increased S protein-specific IgG, although not significantly ( Figure 5d ).

[0046] It was determined that vaccination with CM-S produced a statistically much stronger immune response than vaccination with BNT162b2. It was also determined that the difference between the two vaccination systems was higher in single-dose vaccination ( Figure 5e ). Furthermore, vaccination with CM-S produced a statistically significantly stronger immune response compared to the human convalescent serum (hcPS) pool, even with a single dose of CM-S vaccination ( Figure 5e ).

[0047] In both neutralization tests performed with cell membranes presenting the SARS-CoV2-S protein (CM-S) and with NM coated with RBD units of the SARS-CoV2-S protein, statistically significant differences were observed in the dual vaccination with CM-S compared with the BNT162b2 vaccine and hcPS. All neutralization tests were completed with sera obtained after the booster dose ( Figure 6a and Figure 6b ).

[0048] Similar to the above neutralization test, in the live SARSCoV-2 neutralization assay, statistically significant differences were observed in the dual vaccination with CM-S compared to the BNT162b2 vaccine and hcPS. The average NT50 (50% neutralization titer) values ​​in CM-S were 3.8 and 4.2 times the values ​​observed in the BNT162b2 vaccine and hcPS, respectively. Figure 6c ).

[0049] statement

[0050] In one embodiment of the present invention, the number of amino acids removed from the S2 subfragment protein of SARS-CoV2-S may be less than 21 amino acids, such as 20, 19, 18, 17, 16, 15, 14, 13 or fewer amino acids.

[0051] In another embodiment of the present invention, the number of amino acids removed from the S2 subfragment protein of SARS-CoV2-S may be more than 21 amino acids, such as 22, 23, 24, 25, 26, 27 or more amino acids.

[0052] Another embodiment of the present invention is the S2 subunit of SARS-CoV2-S, which transports proteins that fuse with the cell membrane and may not require all segments of the subunit including the FP, HR1, HR2, TM and CT domains to perform the transport function to the cell membrane.

[0053] Another embodiment of the present invention is to preferably use a vector system designed for a tetracycline-inducible mammalian expression system for the expression of the SARS-CoV2-SΔ21 gene, which is controlled by adding Dox to the culture medium. Other inducible vector systems can also be used for this purpose.

[0054] Another embodiment of the present invention is that the inducible vector system may be recombinant virus related, such as retrovirus, lentivirus or adenovirus related vectors, bacteriophage or any other viral system.

[0055] Another embodiment of the present invention is that recombinant virus-related vector systems that provide sustained expression of SARS-CoV2-S protein, such as retrovirus, lentivirus or adenovirus-related vector systems, can also be used, as long as their expression does not damage the cells used.

[0056] Another embodiment of the present invention is that the expression of the SARS-CoV2-SΔ21 gene will preferably be carried out using a vector system containing the EF1 promoter. CMV and other vector systems containing promoters that function in mammalian cells can also be used.

[0057] In one embodiment of the present invention, a cell membrane (CM) prepared as described in the present invention is used to present SARS-CoV2-SΔ21 obtained from an allogeneic cancer cell line in its structure as a SARS-CoV2 vaccine.

[0058] In one embodiment of the present invention, dead cells (WhC) prepared as described in the present invention are used to present SARS-CoV2-SΔ21 obtained from an allogeneic cancer cell line in their structure as a SARS-CoV2 vaccine.

[0059] In one embodiment of the present invention, WhC (live) prepared as described in the present invention is used to present SARS-CoV2-SΔ21 obtained from an allogeneic primary tissue cell line in its membrane (WhC-S) as a SARS-CoV2 vaccine.

[0060] In one embodiment of the present invention, CM prepared as described in the present invention is used to present SARS-CoV2-SΔ21 obtained from an allogeneic primary tissue cell line in its structure (CM-S) as a SARS-CoV2 vaccine.

[0061] In one embodiment of the present invention, CM-S prepared as described in the present invention is used to present SARS-CoV2-SΔ21 obtained from a primary immortalized allogeneic cell line in its structure as a SARS-CoV2 vaccine.

[0062] In one embodiment of the present invention, living cells with MHC antigens that generate an allogeneic immune response in a recipient are used to present SARS-CoV2-SΔ21 obtained from a primary immortalized allogeneic cell line in their structure as a SARS-CoV2 vaccine.

[0063] In one embodiment of the present invention, WhC (live) prepared as described in the present invention is used to present SARS-CoV2-SΔ21 obtained from an allogeneic stem cell line in its membrane as a SARS-CoV2 vaccine, and the allogeneic stem cell line includes an embryonic stem cell line, a mesenchymal stem cell line, a fetal stem cell line, an adult stem cell line, and an amniotic membrane stem cell line.

[0064] In one embodiment of the present invention, a WhC (live) having an MHC antigen that produces an allogeneic immune response in a recipient prepared as described in the present invention is used to present SARS-CoV2-SΔ21 obtained from an allogeneic stem cell line in its membrane as a SARS-CoV2 vaccine, wherein the allogeneic stem cell line includes an embryonic stem cell line, a mesenchymal stem cell line, a fetal stem cell line, an adult stem cell line, and an amniotic membrane stem cell line.

[0065] In one embodiment of the present invention, CM-S prepared as described in the present invention is used to present SARS-CoV2-SΔ21 obtained from allogeneic stem cells in its structure as a SARS-CoV2 vaccine, and the allogeneic stem cells include embryonic stem cell lines, mesenchymal stem cell lines, fetal stem cell lines, adult stem cell lines, and amniotic stem cell lines.

[0066] In one embodiment of the present invention, living cells (allogeneic, primary, immortalized) of antigen presenting cells (APCs) are used to present SARS-CoV2-SΔ21 in their structures as SARS-CoV2 vaccines, including macrophages, B cells, and especially dendritic cells prepared as described in the present invention. APC cells will be selected to elicit an immune response in the recipient due to the MHC antigens they carry.

[0067] In one embodiment of the present invention, CM-S (allogeneic, secondary, cancer, immortal) prepared as described in the present invention is used to present SARS-CoV2-SΔ21 obtained from APC in its structure as a SARS-CoV2 vaccine.

[0068] In one embodiment of the present invention, WhC (live) prepared as described in the present invention is used to present SARS-CoV2-SΔ21 obtained from autologous stem cells, primary tissue cells (immortalized) lines in its membrane as a SARS-CoV2 vaccine.

[0069] In one embodiment of the present invention, CM-S prepared as described in the present invention is used to present SARS-CoV2-SΔ21 obtained from autologous stem cells or primary tissue cell lines in its structure as a SARS-CoV2 vaccine.

[0070] In one embodiment, the present invention provides that expression of SARS-CoV2-S protein on CM leads to displacement of other CM proteins and reduces them in CM, which ultimately prevents the formation of unwanted background antibodies.

[0071] In one embodiment of the present invention, using CM-S expressing SARS-CoV2-S protein as a vaccine will prevent the formation of unwanted background antibodies.

[0072] The use of autologous or allogeneic cells having SARS-CoV2-S in the membrane for immunization according to the present invention will prevent the formation of unwanted background antibodies and will ensure the acquisition of pure antibodies specific for the target SARS-CoV2-S.

[0073] In another embodiment, the present invention provides a method for producing antibodies against the SARS-CoV-2-S protein of SARS-CoV-2.

[0074] In another embodiment, the present invention provides a method for an immunodiagnostic test kit for identifying antibodies and / or antigens as disease markers, for example, by searching for Covid-19 S-specific antibodies by ELISA, wherein the wells are coated with cells or cell membranes presenting the SARS-CoV2-S protein obtained by the method of the present invention.

[0075] In the methods of the invention where a protein (antigen) is obtained and then a specific antibody is obtained, a variety of assays can be used. For example, various immunoassays can be used to detect the antibodies or proteins (antigens) of the invention. Such immunoassays generally involve measuring the antigen / antibody complex formation between a protein or peptide (i.e., antigen) and its specific antibody.

[0076] According to another aspect of the invention, the opportunity is provided to perform an ELISA test using cells grown in cell culture plates that express the SARS-CoV2-S protein in their membrane. In this test, permeabilization will not be necessary, since the SARS-CoV2-S protein is strongly and naturally present in the cell membrane ( Figure 3a to Figure 3d ).

[0077] According to another aspect of the invention, an opportunity is provided to use cell membranes containing SARS-CoV2-SΔ21 in the development of ELISA systems or other antibody screening assays. In this assay, cell membranes containing a large amount of SARS-CoV2-S protein in its native structure are used to coat ELISA plates.

[0078] According to another aspect of the present invention, there is provided the opportunity to use cell membranes containing SARS-CoV2-S protein to produce antibodies against S protein, wherein the cells presenting SARS-CoV2-S protein in their membranes may be allogeneic.

[0079] In another aspect of the invention, the opportunity is provided to use intact allogeneic cancer cells, primary cells, stem cells prepared as described in the present invention, presenting large amounts of SARS-CoV2-S protein in its native structure in its membrane to be used for the production of antibodies against the S protein.

[0080] In one embodiment of the invention, living cells of allogeneic cells (cancer cells, primary cells, stem cells) prepared as described in the invention, containing SARS-CoV2-SΔ21 in their structure, are used to produce antibodies against the S protein. The cells will be selected to elicit an immune response in the recipient due to the MHC antigens they carry.

[0081] In one embodiment of the present invention, living cells of APCs prepared as described herein, including macrophages, B cells, and in particular dendritic cells, containing SARS-CoV2-SΔ21 in their membranes, are used to produce antibodies against the S protein, wherein the APCs will be selected to elicit an immune response in the recipient due to the MHC antigens they carry.

[0082] The scope of the present invention also includes all nucleotide changes that may occur in S1 or S2 due to codon optimization algorithms. The nucleic acid sequence derivatives of the present invention have at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the native nucleic acid sequence.

[0083] The scope of the present invention also includes the following:

[0084] Different epitopes of SARS-CoV2-S1, including RBD or part of RBD or capsid protein, can also be presented by fusing the coding sequences of different epitopes of S1 to S2 for presentation on the cell membrane.

[0085] The scope of the present invention is not intended to be limited to SARS-CoV2 related vaccination programs, antibody production, development of ELISA systems or other antibody screening tests. The present invention provides vaccination programs, antibody production, development of ELISA systems or other antibody screening test programs because this enables the antigenic epitopes of all viruses to be presented on the cell membrane by the SARS-CoV2-S2 protein.

[0086] Therefore, the present invention covers all viruses including HIV, since any viral proteins with antigenic properties are fused to the SARS-CoV2-S2 protein, ensuring their expression in the cell and, with the help of SARS-CoV2-S2, transporting them to the cell membrane and presenting them there in a concentrated manner.

[0087] In another embodiment, the present invention provides vaccination programs, antibody production, development of ELISA systems or other antibody screening test programs, since any bacterial proteins with antigenic properties are fused to the S2 protein, ensuring their expression in the cell, and with the help of S2 they are transported to the cell membrane and presented there in a concentrated manner.

[0088] Therefore, the present invention covers vaccination schemes for all bacteria, since any bacterial proteins with antigenic properties are fused to the SARS-CoV2-S2 protein, ensuring their expression in the cell and, with the help of SARS-CoV2-S2, transporting them to the cell membrane and presenting them there in a centralized manner.

[0089] On the other hand, the present invention also provides a method for treating or preventing viral infection, by fusing multiple viral proteins (more than one in the same cell membrane) showing antigenic structures different from the SARS-CoV2-S2 protein, ensuring their expression in the cell, and with the help of the SARS-CoV2-S2 protein, transporting them to the cell membrane and presenting them to the immune system in a concentrated manner, resulting in a vaccination scheme.

[0090] On the other hand, the present invention also provides a method for treating or preventing bacterial infections, by fusing a plurality of bacterial proteins showing different antigenic structures with the S2 protein, ensuring their expression in the cell, and with the help of S2, transporting them to the cell membrane where they present themselves centrally to the immune system, resulting in a vaccination scheme.

[0091] While the bacterial or viral-associated protein does not need to be intact, it may be possible to present smaller epitopes that display antigenic properties by binding to the S2 subunit of SARS-CoV2-S.

[0092] Therefore, in another aspect, the present invention also provides a method in which it is possible to present more than one antigenic structure of a virus or bacterium by using the S2 subunit of SARS-CoV2-S. This will naturally result in a much stronger vaccination regimen.

[0093] In another aspect, the present invention also provides a method for treating or preventing cancer, which results in a vaccination scheme due to the fusion of tumor-associated proteins with S2 proteins, ensuring their expression in cells, and transporting them to the cell membrane with the help of S2, where they present themselves centrally to the immune system. Here, the tumor antigen-expressing cells can be living autologous primary stem cells, allogeneic primary stem cells and cancer cell lines from donors with different MHC antigens. In addition, all cell types can be used to ectopically express tumor-associated proteins and transport them with the help of S2 proteins and incorporate them into CM, which is then used as a natural carrier.

[0094] On the other hand, the present invention also provides a method for treating or preventing cancer, by fusing a variety of tumor-associated proteins (or epitopes) showing different antigenic structures with the SARS-CoV2-S2 protein, ensuring their expression in the cell, and transporting them to the cell membrane with the help of S2, where they present themselves to the immune system in a concentrated manner, resulting in a much stronger vaccination scheme.

[0095] Although the present invention describes the S2 subunit of the SARS-CoV2-S protein as an antigenic protein carrier, different DNA sequences of plasma membrane proteins such as vesicular stomatitis virus G (VSV-G) can also perform the same function, which is a well-studied plasma membrane protein (Miyanohara, 2012). Therefore, the scope of the present invention is not limited to the use of the S2 subunit of the SARS-CoV2-S protein as an antigenic epitope carrier, and one or more other protein molecules that carry proteins to the cell membrane in the cell can also be used for the same purpose. DETAILED DESCRIPTION

[0096] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, as the scope of the present invention is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art.

[0097] Hereinafter, elements of the present invention will be described. These elements are listed together with specific embodiments, however, it should be understood that they can be combined in any manner and in any quantity to create additional embodiments. The various described embodiments and preferred embodiments should not be interpreted as limiting the present invention to only the embodiments clearly described. This specification should be understood to support and encompass embodiments that combine the embodiments clearly described with any number of disclosed and / or preferred elements. In addition, any arrangement and combination of all described elements in this application should be considered to be disclosed by the description of the application, unless otherwise indicated in context.

[0098] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry and recombinant DNA techniques as explained in the art (e.g., Molecular Cloning: A Laboratory Manual, 3rd edition, ed. Sambrook and Russel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2001).

[0099] Throughout the specification and in the claims that follow, unless the context otherwise requires, the word "include" and variations such as "comprise" and "contain" will be understood to imply the inclusion of the members. The terms "one / kind" and "the / said" and similar indicators used in the context of describing the present invention (especially in the context of the claims) are interpreted as covering both the singular and the plural, unless otherwise specified herein or clearly contradictory to the context. The enumeration of the numerical range here is only for the purpose of being used as a shorthand method for citing each individual value falling within the range individually. Unless otherwise specified herein, each individual value is incorporated into the specification as if it were listed separately in this article. Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better describe the present invention and is not intended to limit the scope of the present invention. Any language in the specification should not be understood to indicate that any element not protected by the claims is necessary for the practice of the present invention.

[0100] This specification refers to several documents in full. Each document cited herein, whether above or below, is incorporated herein by reference in its entirety. Nothing herein should be construed as admitting that the present invention is not entitled to be earlier than such disclosure due to prior invention.

[0101] The present invention provides a method based on deleting 21 amino acids from the C-terminal region of the S2 subunit of the SARS-CoV2 spike protein (SARS-CoV2-SΔ21) and transporting the S1 subunit naturally fused with S2 to the cell membrane (CM).

[0102] The present invention provides nucleic acid molecules encoding chimeric S1 subunits fused to the S2Δ21 subunit of the SARS-CoV2-S protein. Chimeric S1 can be prepared to include all active mutations identified to date and that may appear in the future.

[0103] The chimeric SARS-CoV2-S protein of the present invention can be produced as a recombinant protein in a eukaryotic cell system and presented in CM.

[0104] The present invention also provides an immunogenic composition comprising cells having chimeric SARS-CoV2-SΔ21 or cells having CM of SARS-CoV2-SΔ21.

[0105] The present invention also provides a method for generating an immune response against SARS-CoV2-SΔ21 in a subject, comprising administering to the subject an effective amount of cells or CM having a chimeric SARS-CoV2-SΔ21 protein, including any combination thereof, thereby generating an immune response against SARS-CoV2 in the subject.

[0106] The present invention also provides a method for treating and / or preventing coronavirus infection in a subject, comprising administering to the subject an effective amount of a composition of the present invention comprising cells having SARS-CoV2-SΔ21 and / or CM having SARS-CoV2-SΔ21.

[0107] Also provided herein is a method for preventing a disease or condition caused by SARS-CoV2 infection in a subject, comprising administering to the subject an effective amount of cells or CM having SARS-CoV2-SΔ21 protein, including any combination thereof, thereby preventing the subject from having a disease or condition caused by coronavirus infection.

[0108] In addition, the present invention provides a method for protecting a subject from SARS-CoV2 infection, comprising administering to the subject an effective amount of cells or CM having SARS-CoV2-S protein, including any combination thereof, thereby protecting the subject from coronavirus infection.

[0109] Thus, the present invention provides a method of inducing or eliciting an immune response in a subject, comprising administering to the subject an effective amount of a composition of the present invention comprising cells having SARS-CoV2 S and / or CM having SARS-CoV2-S. Thus, the present invention provides a vaccination program that can be used to immunize a subject against infection by SARS-CoV-2, as well as to vaccinate a subject against newly formed (mutant) SARS-CoV-2.

[0110] The subject of the present invention is any mammal capable of producing an immune response to coronavirus. The subject of the present invention may be a mammal, and in a specific embodiment is a human, which may be an infant, child, adult or elderly. The terms "subject" and "individual" are used interchangeably and relate to mammals. For example, in the context of the present invention, mammals are humans, non-human primates, domestic animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., experimental animals such as mice, rats, rabbits, and any other animals. The term "animal" as used herein also includes humans. The term "subject" may also include patients with disease, i.e., animals, preferably humans."Subjects at risk of being infected by coronavirus" or "subjects at risk of being infected by coronavirus" are any subjects that may or have been exposed to coronavirus.

[0111] As is well known to those of ordinary skill in the art, the cells of the present invention or their CM can be administered, delivered and / or introduced into a subject according to any method currently known or later identified for the administration, introduction and / or delivery of cells or CM.

[0112] The term "cell membrane" (also called plasma membrane or cytoplasmic membrane, and historically known as protoplasmic membrane) is a biological membrane that separates the interior of all cells from the external environment, protecting the cells from their environmental influences. The cell membrane is composed of a lipid bilayer located between phospholipids to maintain their fluidity at various temperatures. There are a variety of protein receptors and recognition proteins, such as antigens, on the surface of the membrane. Most membrane proteins must be inserted into the membrane in some way. To achieve this, the N-terminal "signal sequence" of amino acids guides the protein to the endoplasmic reticulum, which inserts the protein into the lipid bilayer. Once inserted, the proteins are then transported to their final destination in the vesicle, where the vesicle fuses with the target membrane.

[0113] The term "exosome" refers to nanometer-sized membrane vesicles derived from late endosomal compartments. Cells release exosomes to the extracellular environment, and exosomes represent an important mode of cell-to-cell communication by acting as vehicles capable of transferring proteins, lipids, and RNA. Therefore, antigen-loaded exosomes are used as a cancer vaccine approach in new studies.

[0114] Therefore, similar to the use of exosomes in vaccination, the present invention provides a method of using CM as a carrier molecule to carry antigens, such as in the embodiment of the vaccination regimen established in the present invention by presenting SARS-CoV2-S protein on CM.

[0115] The exact amount of cells with SARS-CoV2-S or CM with SARS-CoV2-S required varies from subject to subject, depending on the species, age, weight and general condition of the subject, the specific primary autologous or allogeneic cells used, the mode of administration thereof, etc. Therefore, it is not possible to specify an exact amount for each cell. However, given the teachings herein, a person of ordinary skill in the art can determine an appropriate amount using only routine experimentation. As used herein, "effective amount" refers to an amount of a compound or composition sufficient to produce a desired effect, which can be a therapeutic, preventive and / or beneficial effect. A non-limiting example of an effective amount of cells of the invention or CM per cell that can be administered to a subject is about 10 3 to about 10 10 cells or CM per cell, preferably about 10 3 to about 10 9 cells or CM per cell, particularly about 10 4 to about 10 6 The amount of CM per cell or per cell may vary, depending on the age, species and / or condition of the subject being treated. As is well known to those of ordinary skill in the art, for any given antigen or vaccine, an optimal dose needs to be determined, for example, based on the production method and the immune response generated.

[0116] Ideally, the subject will receive a single injection. If additional injections are needed, the injections may be repeated weekly / monthly for an indefinite period of time and / or until the efficacy of the treatment has been determined. As described herein, the efficacy of the treatment may be determined by assessing symptoms and clinical parameters and / or by detecting a desired immune response.

[0117] Embodiments of the invention include antibodies produced by a subject of the invention who has generated an immune response to a SARS-CoV2-S protein of the invention, wherein the antibody is specific for an epitope present on the SARS-CoV2-S protein. Such antibodies may be specific for an epitope in any portion of the SARS-CoV2-S protein of the invention as described herein.

[0118] Cells or CM can be delivered subcutaneously, intradermally or intramuscularly. As is well known in the art, the most appropriate route in any given case will depend on factors such as the species, age, sex and general condition of the subject, the nature and severity of the condition being treated and / or the nature of the specific composition being administered (i.e., cells, CM, dose-cell, CM / per cell).

[0119] The scope of the present invention is not intended to be limited to vaccination programs related to SARS-CoV2-S, because the chimeric structure formed by the SARS-CoV2 S2 subunit (which serves as a carrier of the CM) and any viral protein with antigenic properties can be prepared to cover all viruses. Therefore, the present invention covers all viruses, including HIV.

[0120] Therefore, the scope of the present invention covers vaccination programs for all infectious diseases due to the formation of a chimeric structure formed by the SARS-CoV2 S2 subunit (which serves as a carrier of the CM) and a protein with antigenic properties of any infectious agent (disease-associated antigen).

[0121] The term "disease" or "disorder" refers to an abnormal condition affecting an individual's body. A disease is generally understood to be a medical condition associated with specific symptoms and signs. A disease may be caused by factors that originate from an external source, such as an infectious disease, or it may be caused by an internal dysfunction, such as an autoimmune disease. As used herein, the term "disease" or "disorder" particularly includes conditions that would benefit from expression of a peptide or protein (as described above), for example, as evidenced by alleviation and / or improvement of symptoms.

[0122] The term "infectious disease" refers to any disease that can be transmitted from individual to individual or from organism to organism and is caused by a microbial agent (e.g., the common cold). Examples of infectious diseases include: viral infectious diseases such as AIDS (HIV), coronavirus, hepatitis A, B, or C, herpes, herpes zoster (varicella), rubella (rubella virus), yellow fever, dengue fever and other flaviviruses, influenza virus; hemorrhagic infectious diseases (Marburg virus or Ebola virus) and severe acute respiratory syndrome (SARS); bacterial infectious diseases such as Legionnaires' disease (Legionella); sexually transmitted diseases (e.g., chlamydia or gonorrhea); gastric

[0123] ulcers (Helicobacter); cholera (Vibrio); tuberculosis; diphtheria; infections caused by E. coli, Staphylococci, Salmonella or Streptococci (tetanus); infections caused by protozoan pathogens, such as malaria, sleeping sickness, leishmaniasis; toxoplasmosis, i.e. infections caused by Plasmodium, Trypanosoma, Leishmania and Toxoplasma; or fungal infections, such as those caused by Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.

[0124] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that will stimulate the host's immune system to produce a cellular antigen-specific immune response and / or a humoral antibody response to the disease. Therefore, disease-associated antigens can be used for therapeutic purposes. Disease-associated antigens are preferably associated with microbial infection, typically a microbial antigen, or associated with cancer, typically a tumor. The antigen can be a disease-associated antigen, such as a tumor-associated antigen, a viral antigen, or a bacterial antigen.

[0125] According to the present invention, the term "disease" also refers to a cancer disease. The term "cancer disease" or "cancer" (medical term: malignant tumor) refers to a class of diseases in which a group of cells show uncontrolled growth (division beyond normal limits), invasion (invasion and destruction of adjacent tissues), and sometimes metastasis (spread to other parts of the body through lymph or blood). These three malignant characteristics of cancer distinguish them from benign tumors, which are self-limited and do not invade or metastasize. Most cancers form tumors, i.e., swellings or lesions formed by abnormal growth of cells (called neoplastic cells or tumor cells), but some cancers such as leukemia do not form. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, gliomas, and leukemias. More specifically, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the sexual and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal cancer, spinal tumors, gliomas, meningiomas and pituitary adenomas. According to the present invention, the term "cancer" also includes cancer metastasis.

[0126] The term "antigen-related disease" refers to any disease involving an antigen, e.g., a disease characterized by the presence and / or expression of an antigen. The antigen-related disease may be an infectious disease, an autoimmune disease, or a cancer disease or simply cancer. As described above, the antigen may be a disease-associated antigen, such as a tumor-associated antigen, a viral antigen, or a bacterial antigen.

[0127] Therefore, the scope of the present invention also covers vaccination programs for all tumors, thanks to the formation of a chimeric structure formed by the SARS-CoV2 S2 subunit (which serves as a carrier of the CM) and any protein with antigenic properties of that tumor (tumor-associated antigen).

[0128] The term "viral antigen" refers to any viral component that has antigenic properties, ie, is capable of eliciting an immune response in an individual. A viral antigen may be a viral ribonucleoprotein or an envelope protein.

[0129] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, ie, is capable of stimulating an immune response in an individual. Bacterial antigens may be derived from the bacterial cell wall or cytoplasmic membrane.

[0130] On the other hand, the present invention also provides a method for treating or preventing viral or bacterial infections, by fusing a plurality of viral or bacterial proteins showing different antigenic structures with the S2 protein to ensure their expression in the cells, and transporting them to the CM with the help of S2 and presenting them to the immune system in a concentrated manner, thereby obtaining a vaccination program.

[0131] While the bacterial or viral-associated protein does not need to be intact, it may be possible to present smaller epitopes that display antigenic properties by binding to the S2 subunit of SARS-CoV2-S.

[0132] Therefore, in another aspect, the present invention also provides a method in which it is possible to present more than one antigenic structure of a virus or bacteria by using the S2 subunit of SARS-CoV2 S. This will naturally result in a much more powerful vaccination regimen.

[0133] On the other hand, the present invention also provides a method for treating or preventing cancer, by fusing a variety of tumor-associated proteins (or epitopes) showing different antigenic structures with the S2 subunit of the SARS-CoV2-S protein, ensuring their expression in cells, and transporting them to the CM with the help of S2 and presenting them to the immune system there in a concentrated manner, resulting in a much stronger vaccination program. This method can be applied by fusing different antigenic proteins or peptides to the SARS-CoV2-S2 protein in a frameshift manner in a single vector system. This method can also be achieved using more than one vector system in the same cell, which vector systems have different selection markers to express each antigenic protein fused to the SARS-CoV2-S2 protein.

[0134] As used herein, the term "immune response" relates to the immune system, such as a response to an immunogenic organism, such as a bacterium or virus, a cell or a substance. The term "immune response" includes an innate immune response and an adaptive immune response. Preferably, the immune response is associated with the activation of immune cells, the induction of cytokine biosynthesis and / or antibody production. Preferably, the immune response includes the steps of activating antigen presenting cells, such as dendritic cells and / or macrophages, presenting antigens or fragments thereof by the antigen presenting cells, and activating cytotoxic T cells as a result of such presentation.

[0135] In another embodiment, the present invention provides a method for producing an antibody. The antibody can be used as a therapeutic agent, diagnostic agent or reagent for various diseases. As a general method for preparing antibodies, a method of administering an antigen to a mammal such as a mouse and obtaining the antibody from the animal serum is used. In the production of antibodies, the target antibody may not be effectively obtained. The two main difficulties are that the amount of the antigen is not enough to immunize the mammal, and it is not fully purified. Therefore, it is desirable to prepare a large amount of fully purified antigens for immunization. However, in reality, many antigens are difficult to purify, and it is difficult to prepare a sufficient amount of antigens. In other words, the antigen preparation method is often an obstacle in antibody production. As a method for expressing a large amount of protein, the method using baculovirus has attracted people's attention. By using these methods, the desired membrane protein can be expressed in large quantities on the baculovirus membrane.

[0136] However, in addition to exogenous membrane proteins (background antigens), membrane proteins derived from baculovirus are expressed on the baculovirus membrane thus obtained. Therefore, when using budding baculovirus as an immunogen to produce antibodies, antibodies against membrane proteins derived from baculovirus are also produced. As a result, it is difficult to effectively produce antibodies against target membrane proteins by known immunization methods. Therefore, in order to use the target membrane protein expressed on the baculovirus membrane as an antigen, it is necessary to fully purify the membrane protein. However, it is generally difficult to purify exogenous membrane proteins from budding baculovirus. Therefore, it can be said that it is impossible to obtain highly purified membrane proteins sufficient for immunization in practice. For antigens that are difficult to purify, it is difficult to obtain the desired antibodies by conventional methods.

[0137] In one embodiment of the present invention, using CM expressing SARS-CoV2-S protein as a vaccine will prevent the formation of unwanted background antibodies.

[0138] Importantly, the present invention provides that expression of SARS-CoV2-S protein on CM leads to displacement of other CM proteins and their reduction in CM, which ultimately prevents presentation of other CM proteins and formation of unwanted background antibodies.

[0139] The use of autologous or allogeneic cells with antigen in the membrane for immunization of animals according to the present invention will prevent the formation of unwanted background antibodies and will ensure that pure antibodies specific for the target antigen are obtained. As mentioned above, the most important benefit of the present invention is the availability of large amounts of proteins in their native structural form. Therefore, the present invention will solve the above-mentioned problem (production of antibodies using baculovirus) by obtaining and using large amounts of pure and native proteins present in CM for immunization.

[0140] Therefore, the present invention provides a method for producing antibodies against proteins of all disease-related factors, including viruses, bacteria, tumors and other diseases.

[0141] In another embodiment, the present invention provides a method for an immunodiagnostic test kit for identifying antibodies and / or antigens as disease markers. For example, searching for SARS-CoV2-S specific antibodies by enzyme-linked immunosorbent assay (ELISA) is an important and frequently used immunodiagnostic method, wherein the wells are coated with cells or CM presenting SARS-CoV2-S protein obtained by the method of the present invention.

[0142] In the methods of the invention where a protein (antigen) is obtained and then a specific antibody is obtained, a variety of assays can be used. For example, various immunoassays can be used to detect the antibodies or proteins (antigens) of the invention. Such immunoassays generally involve measuring the antigen / antibody complex formation between a protein or peptide (i.e., antigen) and its specific antibody.

[0143] The immunoassays of the present invention can be competitive or non-competitive, both types of assays being well known and well developed in the art. In competitive binding assays, an antigen or antibody competes with a detectably labeled antigen or antibody for specific binding to a capture site bound to a solid surface. The concentration of labeled antigen or antibody bound to the capture agent is inversely proportional to the amount of free antigen or antibody present in the sample.

[0144] In some embodiments, the antibodies and / or proteins may be conjugated or otherwise linked or attached (e.g., covalently or non-covalently) to a solid support (e.g., a bead, plate, slide, dish, membrane, or well) according to known techniques. The antibodies may also be conjugated or otherwise linked or attached to a detectable group such as a radiolabel (e.g., 35 S. 125 I. 32 P. 13 H. 14 C. 131 I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), gold beads, chemiluminescent labels, ligands (e.g., biotin), and / or fluorescent labels (e.g., fluorescein).

[0145] Various organic and inorganic polymers, whether natural or synthetic, can be used as the material of solid surface. The non-limiting example of polymer includes polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly (ethylene terephthalate), rayon, nylon, poly (vinyl butyrate), polyvinylidene fluoride, silicone, polyoxymethylene, cellulose, cellulose acetate, nitrocellulose etc. Other materials that can be used include but are not limited to paper, glass, ceramics, metal, metalloid, semiconductor material, cement etc. In addition, the material forming gel can be used, such as protein (for example, gelatin), lipopolysaccharide, silicate, agarose and polyacrylamide. It is also suitable to form the polymer of several aqueous phases, such as dextran, polyalkylene glycol or surfactant such as phospholipid, long chain (12-24 carbon atoms) alkyl ammonium salt etc. When the solid surface is porous, various pore sizes can be adopted according to the property of the system.

[0146] A variety of immunoassay systems can be used, including but not limited to radioimmunoassay (RIA), ELISA assay, enzyme immunoassay (EIA), "sandwich" assay, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, immunofluorescence assay, fluorescence activated cell sorting (FACS) assay, immunohistochemistry assay, protein A immunoassay, protein G immunoassay, protein L immunoassay, biotin / avidin assay, biotin / streptavidin assay, immunoelectrophoresis assay, precipitation / flocculation reaction, immunoblot (protein blot; dot / slot blot); immunodiffusion assay; liposome immunoassay, chemiluminescent assay, library screening, expression arrays, immunoprecipitation, competitive binding assay and immunohistochemical staining. These and other assays are described in Hampton et al. (Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn. (1990)) and Brown TA (Genomes. 2nd ed., Oxford, Wiley-Liss: 2002), Auld et al. (The assay Guidance Manual; https: / / www.ncbi.nlm.nih.gov / books / ), and other literature.

[0147] As described above, the present invention provides a method for producing antibodies against proteins of all disease-related factors including viruses, bacteria, tumors and other diseases.

[0148] Thus, in another embodiment, the present invention provides passive antibody therapy methods against viruses, bacteria and other agents.

[0149] In passive antibody therapy, specific antibodies needed to kill a specific factor are delivered directly into the body.

[0150] As used herein, the term "antibody" refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD and IgE. Antibodies can be monoclonal or polyclonal and can be of any species of origin, including, for example, mouse, rat, rabbit, horse, goat, sheep or human, or can be chimeric or humanized antibodies.

[0151] According to the present invention, nucleic acid or nucleic acid molecule refers to a nucleic acid preferably a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). According to the present invention, nucleic acid includes genomic DNA, cDNA, mRNA, recombinantly prepared and chemically synthesized molecules. According to the present invention, nucleic acid can be in the form of a single-stranded or double-stranded and linear or covalently closed circular molecule. According to the present invention, the term "nucleic acid" also includes chemical derivatization of nucleic acid on nucleotide bases, sugars or phosphates.

[0152] In a preferred embodiment, the nucleic acid molecule according to the present invention is a vector. The term "vector" is used in its most general sense here, and comprises any intermediate carrier of nucleic acid, for example, so that the nucleic acid can be introduced into a prokaryotic and / or eukaryotic host cell, and is integrated into the genome under appropriate circumstances. Such vectors are preferably replicated and / or expressed in cells. Vectors include plasmids, phagemids or viral genomes. As used herein, the term "plasmid" generally relates to a construct of extrachromosomal genetic material, typically a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0153] In a preferred embodiment of the invention, recombinant viral techniques are used. They provide useful alternatives to non-viral methods, which have low transfer efficiencies in certain cell types (e.g., primary cultured cells and epithelial cells). The most commonly used vectors are retroviruses, lentiviruses, or adenoviruses; however, other viruses include adeno-associated viruses (AAV), herpes viruses, vaccinia viruses, and several RNA viruses.

[0154] Viral techniques for gene transfer exploit the methods used by wild-type organisms to enter and integrate into the host genome.

[0155] While adenoviral vectors can be used for transient assays, retroviral vectors stably integrate into the genome of dividing target cells, allowing the introduced gene to be transmitted and expressed in all daughter cells.

[0156] Another embodiment of the present invention is that the expression of the SARS-CoV2-SΔ21 gene or a gene product with antigenic properties to be used for any disease as described herein will preferably be carried out using a vector system containing an EF1 promoter. The term "promoter" or "promoter region" refers to a DNA sequence upstream (5') of the coding sequence of a gene, which controls the expression of the coding sequence by providing recognition and binding sites for RNA polymerase. The promoter region may include other recognition or binding sites for other factors involved in regulating the transcription of the gene. Examples of preferred promoters according to the present invention are EF1, CMV, pGK and other promoters that function in mammalian cells. Promoters can control the transcription of prokaryotic or eukaryotic genes.

[0157] A promoter can be "inducible" and initiate transcription in response to an inducer, or it can be "constitutive" if transcription is not controlled by the inducer. If the inducer is not present, an inducible promoter is expressed only to a very small extent or not at all. In the presence of an inducer, the gene is "turned on" or the level of transcription increases. This is usually mediated by the binding of specific transcription factors. A preferred embodiment of the present invention is the inducible expression of the target gene (here SARS-CoV2 S). This inducible system can be used in a plasmid or viral vector system. Although a tetracycline inducible vector system is used, other induction systems are also included in the present invention.

[0158] Another embodiment of the present invention is that recombinant virus-related vector systems that provide sustained expression of SARS-CoV2-SΔ21 protein, such as retrovirus, lentivirus or adenovirus-related vector systems, can also be used, as long as their expression does not damage the cells used.

[0159] The term "isolated nucleic acid" means, according to the invention, that the nucleic acid has been (i) amplified in vitro, e.g., by polymerase chain reaction (PCR), (ii) produced by cloning recombination, (iii) purified, e.g., by cleavage and gel electrophoresis fractionation, or (iv) synthesized, e.g., by chemical synthesis. An isolated nucleic acid is a nucleic acid that can be used for manipulation by recombinant DNA techniques. (e.g., as described in Sambrook et al., eds., "Molecular Cloning: A Laboratory Manual," 3rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0160] In the context of the present invention, the term "recombinant" means "prepared by genetic engineering". Preferably, in the context of the present invention, a "recombinant object" such as a recombinant nucleic acid does not occur naturally.

[0161] As nucleic acid, in particular DNA, for expressing more than one peptide or protein, it is possible to use nucleic acid types in which different peptides or proteins are encoded in different nucleic acid molecules or nucleic acid types in which the peptides or proteins are encoded in the same nucleic acid molecule.

[0162] The term "expression" is used according to the invention in its most general sense and includes the production of RNA and / or peptides or proteins, for example by transcription and / or translation. With respect to RNA, the term "expression" or "translation" particularly relates to the production of peptides or proteins. It also includes partial expression of nucleic acids. Furthermore, expression can be transient, stable and / or stably induced.

[0163] According to the present invention, the term "translation" relates to the process in the cellular ribosomes by which a strand of messenger RNA directs the assembly of an amino acid sequence to produce a peptide or protein.

[0164] Terms such as "enhancement of expression", "enhanced expression" or "overexpression" in the context of the present invention refer to the amount of a peptide or protein expressed by a given number of vector molecules being higher than the amount of the peptide or protein expressed by the same number of vector molecules. The amount of a peptide or protein can be given in moles or weight (e.g., in grams), or in mass or polypeptide activity, for example, as catalytic activity if the peptide or protein is an enzyme, or as binding affinity if the peptide or protein is an antibody or antigen or receptor. In one embodiment, terms such as "enhancement of expression", "enhanced expression" or "increased expression" in the context of the present invention refer to the amount of a peptide or protein expressed by a given number of vector molecules in a given period of time being higher than the amount of the peptide or protein expressed by the same number of vector molecules in the same period of time.

[0165] The term "codon optimization" is a method in genetic engineering to improve gene expression by changing synonymous codons based on the organism's codon preference. As used herein, "improvement" refers to the codon-optimized nucleotide sequence used in non-natural cells producing more protein than the non-codon-optimized native nucleotide sequence. As used herein, "nucleotide sequence" describes the expression of a complete protein or any sequence within the same framework in a vector as an amino acid epitope or as a complete protein.

[0166] The expression level and / or duration of expression of an RNA can be determined by measuring the amount (such as the total amount expressed and / or the amount expressed over a given period of time) and / or the time of expression of a peptide or protein encoded by the RNA, for example by using an ELISA procedure, an immunohistochemical procedure, a quantitative image analysis procedure, Western blot, mass spectrometry, a quantitative immunohistochemical procedure or an enzyme assay.

[0167] The term "transfection" refers to the introduction of nucleic acid (particularly DNA) into a cell. For the purposes of the present invention, the term "transfection" also includes the introduction of nucleic acid into a cell or the uptake of nucleic acid by such cells. The term "transduction" refers to retroviruses that have been used as vectors for transferring genes into eukaryotic cells, which is a process called transduction. Therefore, according to the present invention, cells for transfecting nucleic acid may be present in vitro or in vivo, for example, the cells may form part of an organ. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. Since the nucleic acid introduced during the transfection process is usually not integrated into the nuclear genome, the exogenous nucleic acid will be diluted or degraded by mitosis. Cells that allow free amplification of nucleic acid greatly reduce the dilution rate. If it is desired that the transfected nucleic acid is actually retained in the genome of the cell and its daughter cells, stable transfection must be performed as in the present invention.

[0168] According to the present invention, any technique for introducing nucleic acid into (i.e., transferring or transfecting into) cells in vitro can be used. Preferably, nucleic acid is transfected into cells by standard techniques. Such techniques include calcium phosphate-DNA precipitation, electroporation, lipofection, and microinjection.

[0169] According to the present invention, preferably, the introduction of a nucleic acid encoding a protein or peptide into a cell or the uptake of a nucleic acid encoding a protein or peptide by a cell results in the expression of the protein or peptide. The cell may express the encoded peptide or protein intracellularly (e.g., in the cytoplasm and / or nucleus), or, as in the present invention, the encoded peptide or protein may be transported to the CM and presented.

[0170] According to the present invention, the term "host cell" refers to any cell that can be transformed or transfected with exogenous nucleic acid. Particularly preferred are mammalian cells, such as cells from humans, mice, hamsters, pigs, goats, primates. Cells can be derived from a variety of tissue types, and include primary cells and cell lines. Specific examples include fibroblasts, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. Stem cell lines are a group of stem cells that are cultured in vitro and can proliferate indefinitely. Stem cell lines are derived from animal or human tissues and come from one of three sources: embryonic stem cells, adult stem cells, or induced stem cells.

[0171] In other embodiments, the host cell is an antigen presenting cell, particularly a dendritic cell, a monocyte or a macrophage.

[0172] According to the present invention, host cells can be derived from immortalized primary cells, which are derivatives of primary cells, which ultimately avoid normal cell aging and acquire the ability of continuous cell division due to mutation events. Immortalized cell lines can be generated from cells isolated from tumors, or mutations can be introduced to immortalize primary cells. Immortality of cell lines can be achieved by different methods, including ectopic expression of telomerase or telomerase reverse transcriptase (TERT), by mutating the p53 gene and the pRb gene, or introducing oncogenes. Viral vectors can be used for all mentioned methods, i.e., introducing TERT and oncogenes or mutating p53 / pRb.

[0173] The term "pharmaceutically active peptide or protein" includes peptides or proteins that can be used to treat a subject, wherein the expression of the peptide or protein will be beneficial, for example, in improving the symptoms of a disease or condition. The term "immunologically active compound" relates to any compound that preferably changes humoral immunity by inducing and / or inhibiting immune cell maturation, inducing and / or inhibiting cytokine biosynthesis and / or by stimulating B cells to produce antibodies, thereby changing the immune response. Immunologically active compounds have effective immunostimulatory activity, including but not limited to antiviral and antitumor activity, and can also downregulate other aspects of the immune response, such as moving the immune response away from the TH2 immune response, which can be used to treat a variety of TH2-mediated diseases. Immunologically active compounds can be used as vaccine adjuvants.

[0174] In some embodiments of the invention, the composition may be administered with an adjuvant. As used herein, "adjuvant" describes a substance that can be any immunomodulatory substance that can be combined with a polypeptide or nucleic acid vaccine to enhance, improve or otherwise modulate the immune response of a subject without adverse effects on the subject.

[0175] Non-limiting examples of adjuvants that can be used in the vaccines of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as Freund's complete and incomplete adjuvants, block copolymers (CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.), AMPHIGEN TM Adjuvant, saponin, Quil A or other saponin fractions, monophosphoryl lipid A and avridine lipid-amine adjuvant. Non-limiting examples of oil-in-water emulsions that can be used for the vaccine of the present invention include modified SEAM62 and SEAM 1 / 2 formulations. Modified SEAM62 is a product containing 5% (v / v) squalene (Sigma), 1% (v / v) SPAN TM85 detergent (ICI surfactant), 0.7% (v / v) TWEEN TM 80 detergent (ICI surfactant), 2.5% (v / v) ethanol, 200 pg / ml Quil A, 100 μg / ml cholesterol in water oil emulsion, and 0.5% (v / v) lecithin modified SEAM 1 / 2 is a 5% (v / v) squalene, 1% (v / v) SPAN TM 85 detergent, 0.7% (v / v) Tween 80 detergent, 2.5% (v / v) ethanol, 100 μg / ml Quil A and 50 μg / ml cholesterol in an oil-in-water emulsion. Other immunomodulators that may be included in the vaccine include, for example, one or more interleukins, interferons or other known cytokines.

[0176] The present invention also includes "variants" of the peptides, proteins or amino acid sequences described herein.

[0177] For the purposes of the present invention, "variants" of an amino acid sequence include amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants.

[0178] Amino acid insertion variants include insertion of single or two or more amino acids in a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion and appropriate screening of the resulting product is also possible.

[0179] Amino acid addition variants include amino- and / or carboxyl-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more stretches of a protein.

[0180] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion may be at any position of the protein. Amino acid deletion variants comprising a deletion at the N-terminus and / or C-terminus of the protein are also referred to as N-terminal and / or C-terminal truncated variants.

[0181] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a protein into peptides), and the association (e.g., by binding) of one or more of these fragments with MHC molecules for presentation by cells (preferably antigen presenting cells) to specific T cells.

[0182] As used herein, "coronavirus" refers to the genus in the Coronaviridae family, which is in turn classified into the order Nidovirales. Coronaviruses are large, enveloped positive-strand RNA viruses. They have the largest genome of all RNA viruses and replicate by a unique mechanism that results in high-frequency recombination. Coronaviruses include antigenic groups I, II, and III. Non-limiting examples of coronaviruses include SARS coronaviruses, including Covid-19, MERS coronaviruses, transmissible gastroenteritis virus (TGEV), human respiratory coronaviruses, porcine respiratory coronaviruses, canine coronaviruses, feline enteric coronaviruses, feline infectious peritonitis virus, rabbit coronaviruses, mouse hepatitis virus, sialolacrimal gland inflammation virus, porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, avian infectious bronchitis virus, and turkey coronaviruses, as well as chimeras of any of the above viruses (see Lai and Holmes, "Coronaviridae: The Viruses and Their Replication", in Fields Virology (4th edition, 2001)).

[0183] As used herein, a "coronavirus permissive cell" can be any cell in which a coronavirus can at least replicate, including naturally occurring cells and recombinant cells. In some embodiments, a permissive cell is also a cell that a nested virus or coronavirus can infect. A permissive cell can be a cell modified by recombinant means to produce the cell surface receptor ACE-2 for SARS-CoV2 herein.

[0184] The term "antigen" refers to a factor comprising an epitope, against which an immune response is produced. The term "antigen" particularly includes peptides and proteins. The term "antigen" also includes factors that become antigenic and sensitizing only by conversion (e.g., in the middle of a molecule or by completing with a body protein). Antigens are preferably presentable by cells of the immune system, such as antigen presenting cells, such as dendritic cells or macrophages. In addition, antigens or their processed products are preferably recognized by T or B cell receptors, or by immunoglobulin molecules such as antibodies. In a preferred embodiment, the antigen is a disease-associated antigen, such as a tumor-associated antigen, a viral antigen or a bacterial antigen.

[0185] In one embodiment, the disease-associated antigen is a tumor-associated antigen. In this embodiment, the present invention can be used to treat cancer or cancer metastasis. On the other hand, the present invention provides a method for treating or preventing cancer, by fusing one or more tumor-associated proteins showing different antigenic structures with the S2Δ21 segment of the SARS-CoV2-S protein, ensuring their expression in cells, and transporting them to the cell membrane with the help of S2Δ21 and presenting them to the immune system there, a vaccination scheme is obtained. Preferably, the diseased organ or tissue is characterized by diseased cells such as cancer cells expressing disease-associated antigens, and / or characterized by the association of disease-associated antigens with their surfaces. Antigen immunization with complete or substantially complete tumor-associated antigens or fragments thereof (such as MHC class I and class II peptides or nucleic acids, in particular CM) makes it possible to induce MHC class I and / or class II responses, and thus stimulate T cells, such as CD830 cytotoxic T lymphocytes capable of lysing cancer cells and / or CD4+T cells. This immunization can also induce a humoral immune response (B cell response), leading to the production of antibodies against tumor-associated antigens. In one embodiment, the term "tumor-associated antigen" refers to a cancer cell component that can be derived from the cytoplasm, CM and nucleus. In particular, it refers to those antigens which are produced intracellularly or on tumor cells as CM antigens, preferably in large amounts.

[0186] The term "treatment" refers to any treatment that improves the health status of an individual and / or prolongs (increases) the life span of an individual. The treatment may eliminate the disease in an individual, prevent or slow the progression of the disease in an individual, inhibit or slow the progression of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual currently suffering from or previously suffering from the disease.

[0187] In particular, the term "treatment of a disease" includes curing, shortening the duration, ameliorating, slowing or inhibiting the progression or worsening of the disease or its symptoms.

[0188] The term "immunotherapy" relates to a treatment which preferably involves a specific immune response and / or immune effector functions.

[0189] The terms "immunization" or "vaccination" describe the process of treating a subject for therapeutic or prophylactic reasons.

[0190] A vaccine is a biological preparation that provides automatic acquired immunity against a specific infectious disease. Vaccines usually contain factors similar to pathogenic microorganisms and are usually made of weakened or killed forms of microorganisms, their toxins or one of their surface proteins. Vaccines that can be produced using recombinant DNA technology are called recombinant vaccines. Recombinant vaccines can be produced in many different ways. These types of vaccines are made with the help of expression systems such as bacteria, insects, yeast, plants, mammals and acellular. Although various types of vaccines can be prepared by recombinant DNA technology, recombinant vaccines can be divided into two major categories. DNA vaccines usually consist of synthetic DNA containing genes encoding disease factor proteins. Typically, plasmid DNA used as vaccines is propagated in bacteria such as Escherichia coli, and they are isolated and purified for injection. This "naked" DNA is usually injected intramuscularly or intradermally. The principle behind DNA vaccines is that antigens can be directly expressed by host cells in a way that simulates viral infection and causes an immune response in the host. Recombinant (protein subunit) vaccines are subunit vaccines that contain only a portion of pathogenic organisms. Typically, these are synthetic peptides representing protein components that induce immune responses. But they can also be composed of protein subunits (antigens) expressed in heterologous expression systems (E. coli, yeast, insects, etc.) using recombinant protein expression technology. Prokaryotic expression systems for vaccine antigen production include bacteria such as E. coli, and eukaryotic systems include mammals, yeast or insect cells. Before selecting the correct system for vaccine antigen expression, several factors should be considered. Among them, the presence or absence of expression level, selection markers and post-translational modifications are important factors that interfere with the production effectiveness of recombinant antigens as vaccines.

[0191] Therefore, the present invention provides a new antigen expression and presentation system that provides proteins (antigens) in their native structure in large quantities in eukaryotic cell membranes, and its use as whole cells or CM will be a new vaccination strategy.

[0192] The present invention demonstrates that presenting S1 of SARS-CoV2 in large amounts in CM in its native structure will provide a much stronger and more specific immune response.

[0193] Most importantly, in this way, it is possible to prepare almost unlimited amounts of the native form of the SARS-CoV2-S antigen using cell lines in a short period of time.

[0194] In addition, because the S protein will be attached to the cell membrane and cannot circulate freely, it will not be able to pass the blood-brain barrier and may not exhibit the above-mentioned cell signal stimulation effect.

[0195] On the other hand, the present invention also provides a method for treating or preventing viral infections, by fusing multiple viral proteins (more than one in the same cell membrane) showing different antigenic structures with the SARS-CoV2-S2 protein, ensuring their expression in the cell, and with the help of the SARS-CoV2-S2 protein, transporting them to the cell membrane and presenting them centrally to the immune system, resulting in a vaccination scheme.

[0196] This method can be applied by fusing different antigenic proteins or peptides to the SARS-CoV2-S2 protein in a frameshift manner in a single vector system. This method can also be implemented in the same cell using vector systems that have different selection markers to express each antigenic protein fused to the SARS-CoV2-S2 protein.

[0197] The agent stimulates the body's immune system to recognize the agent as a threat, destroy it, and further recognize and destroy any microorganisms associated with the agent that it may encounter in the future. Vaccines can be prophylactic (to prevent or ameliorate the effects of future infection with natural or "wild" pathogens), or therapeutic (to combat an already established disease, such as cancer).

[0198] The administration of vaccines is known as vaccination. Vaccination is the most effective way to prevent infectious diseases; widespread immunity due to vaccination is largely responsible for the eradication of smallpox worldwide and has limited the spread of diseases such as polio, measles, and tetanus in much of the world.

[0199] The term "carrier" refers to the natural organic cell or CM with which the active ingredient is combined to promote, enhance or enable an application.

[0200] As described in detail, CM and / or whole cells are used as vectors in the present invention.

[0201] The present invention is described in detail by the following drawings and examples, which should be interpreted only in an illustrative manner rather than in a limiting manner. On the basis of the description and examples, further embodiments are readily understood by those skilled in the art and are also within the scope of the present invention.

[0202] Example

[0203] Confirmation of cell membrane separation

[0204] Cell membranes and cytoplasm prepared according to the specific protocols in Materials and Methods were placed in equal amounts on nitrocellulose membranes and then studied by a dot blot system using antibodies specific for proteins in the cell membrane or cytoplasm. Cog2 (conserved oligomeric Golgi complex subunit 2) is required for correct Golgi morphology and function, is a 730 amino acid component of the COG complex, and is located on the cytoplasmic side of the Golgi. Calreticulin and calnexin are nascent proteins associated with the endoplasmic reticulum. They are also integral proteins and are present in different amounts in the cell membrane depending on the cell type.

[0205] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme associated with glycolysis and is mainly present in the cytoplasm. However, GAPDH has been shown to be present in the cell membrane, although in small amounts. Figure 1-A shows that, although GAPDH and Cog2 are almost absent from the cell membrane but present in the cytoplasm, calreticulin and calnexin are present in both the cell membrane and the cytoplasm as expected.

[0206] Overexpression of the SARS-CoV2 spike Δ21 protein places the spike protein in the cell membrane

[0207] As previously described, when the ER retrieval signal in the cytoplasmic tail of the S protein is mutated, the full-length S protein is allowed to translocate to the plasma membrane. Therefore, we used a dot blot assay to investigate the expression of the S protein on the cell membrane of cells expressing SARS-CoV2 Spike wt or SARS-CoV2 Spike Δ21. 1 × 10 6 TA-Hep3B cells containing pRetroX-Tight-Pur-hSARS-CoV2-S wt or pRetroX-Tight-Pur-hSARS-CoV2-SΔ21 and TA-Renca cells and TA-MAD109 cells containing pRetroX-Tight-Pur-mSARS-CoV2-S wt or pRetroX-Tight-Pur-mSARS-CoV2-SΔ21 were inoculated with doxacillin (dox) 24 hours after the cells were inoculated. Then, after 12, 24, and 48 hours, the cells were prepared and used for different studies. Figure 1b to Figure 1eThe dot blot results are shown, in which almost all of the spike protein was present in the cell membrane in cells expressing spike Δ21, while in cells expressing spike wt, no spike protein was detected in the cell membrane of Hep3B cells and Renca cells. In addition, as a result of testing with IHC (without permeabilization process), SARS-CoV2 S protein was intensively detected in cells expressing SARS-CoV2 SΔ21, while SARS-CoV2 S protein was not detected in cells expressing SARS-CoV2 S wt ( Figure 1f to Figure 1g ). Figure 1h to Figure 1j Shown is the amount of SARS-CoV2S protein over time after induction with dox in TA-Hep3B cells containing pRetroX-Tight-Pur-hSARS-CoV2-SΔ21 and TA-Renca cells and TA-MAD109 cells containing pRetroX-Tight-Pur-mSARS-CoV2-SΔ21.

[0208] Cell fusion occurs in the presence of SΔ21 expression

[0209] Hep3B cells containing pRetroX-Tight-Pur-S wt or pRetroX-Tight-Pur-SΔ21 were plated into 24-well plates at 3 × 10 cells per well. 5 cells. After 24 hours, the medium in the wells was removed and replaced with medium containing dox. Twenty-four hours after dox addition, cells were stained with Hoechst (1 μg / ml) or IHC stained with anti-SARS-CoV2-S antibody and observed by fluorescence microscopy. Cell-cell fusion was observed in Hep3B cells containing pRetroX-Tight-Pur-SΔ21 24 hours after dox addition ( Figure 2a and Figure 2b ), and

[0210] Further unification of the cells was observed after 48 hours, with disruption of the cell structure ( Figure 1h Although not as pronounced as in Hep3B cells, SΔ21 expression also produced fusion in Renca cells and MAD109 cells ( Figure 1i and Figure 1J ).

[0211] Cells expressing SARS-CoV2 SpikeΔ21 provided abundant Spike protein expression as determined in the cell membrane by ELISA assay.

[0212] In the next experiment, 1 × 10 1 Up to 1×10 4TA-Hep3B cells, TA-Renca cells, and TA-MAD109 cells containing pRetroX-Tight-Pur-hSARS-CoV2-S wt, pRetroX-Tight-Pur-hSARS-CoV2-SΔ21, and pRetroX-Tight-Pur-mSARS-CoV2-S wt and pRetroX-Tight-Pur-mSARS-CoV2-SΔ21 were added with dox after 24 hours and fixed with 4% paraformaldehyde after 12, 24, and 48 hours. The ELISA protocol was used to measure the levels of IgG, total anti-SARS-CoV2-S antibodies in the serum of people with large amounts of anti-SARS-CoV2-S antibodies detected using a commercial ELISA assay. Here, the ELISA assay protocol was completed without permeabilization. The ELISA assay showed that the signal increased with the amount of cells expressing SARS-CoV2-SΔ21 and the duration of dox administration ( Figure 3a and Figure 3b ). However, in the ELISA test cells expressing SARS-CoV2-S wt, in the Renca cell line ( Figure 3c ) and Hep3B cell lines ( Figure 3d ) Almost no signal was detected in the ELISA assay. When anti-S1 antibody (Biolegend) was used, the signal increased with the amount of cells expressing SARS-CoV2-SΔ21 ( Figure 3d ). In addition, it was determined that the permeabilization process did not increase the amount of signal in cells expressing SARS-CoV2-SΔ21 (data not shown).

[0213] SARS-CoV2-SΔ21 replaces other cell membrane proteins

[0214] Next, we asked the question of how high levels of S protein in the cell membrane affect other membrane proteins. In other words, how does the cell membrane handle such a large amount of S protein, and how is this possible?

[0215] To this end, we studied the amounts of two basic proteins that make up the cell membrane structure, including spectrin alpha erythroblastic (SPTA1), a member of a family of molecular scaffold proteins that connect the plasma membrane to the actin cytoskeleton and play a role in the determination of cell shape, and N-cadherin (CDH2), a transmembrane protein expressed in a variety of tissues, in the cell membrane and cytoplasm in cells with or without SARS-CoV2-S SΔ21 expression.

[0216] The cell membrane and cytoplasm of TA-Hep3B expressing rtT (control cells), TA-Hep3B containing pRetroX-Tight-Pur-hSARS-CoV2-SΔ21 and TA-Renca (control cells), and TA-Renca containing pRetroX-Tight-Pur-mSARS-CoV2-SΔ21 were isolated after 36 hours of dox treatment, and the amounts of SPTA1 and CDH2 in the cell membrane and cytoplasm were studied by dot blotting using SPTA1 or CDH2-specific antibodies.

[0217] Figures 4a to 4d It was shown that the levels of SPTA1 and CDH2 proteins in the cell membrane of Hep3B cells and Renca cells expressing SΔ21 were as low as 1 / 2-1 / 3 compared with those in Hep3B cells and Renca cells that did not express SΔ21. Therefore, due to the overexpression of SARS-CoV2-SΔ21 in cells, the S1 protein was transported to the cell membrane and replaced the major membrane proteins SPTA1 and CDH2. In addition to confirming the dot blot results, SARS-CoV2-S protein expression was detected by ELISA at 0, 12, 24, and 48 hours after the addition of dox ( Figure 4e ) and cell membrane proteins (SPTA1 and CDH2) ( Figure 4f and Figure 4g ).like Figure 4f and Figure 4g As shown, it was determined that the cell membrane proteins including SPTA1 or CDH2 were increased in the cell membrane with the increase of S protein in the cell membrane ( Figure 4e ) decreased at a similar rate. In addition, the same results were obtained in ELISA tests using SARS-CoV2-S negative patient serum as a source of autoantibodies against the cell membrane. It was determined that the cell membrane proteins bound to human serum antibodies decreased in the cell membrane at a similar rate to the increase in the amount of S protein ( Figure 4h ).

[0218] Immunization of mice

[0219] Balb / c mice group and C57BU6 mice group were treated with 5×10 4 TA-Renca-mSARS-CoV2-SΔ21 whole cells (WhC) (3 animals / group) or from 5×10 4 Cell membranes (CM) obtained from TA-Renca-mSARS-CoV2-SΔ21 cells with and without complete Freund's adjuvant (CFA) were immunized subcutaneously (SC) at the Laboratory Animal Care Unit of Ankara University Medical College (4 animals were used per group). Cells and cell membranes were obtained 36 h after addition of dox on growing cell cultures. In addition, 5 × 10 4TA-Renca-mSARS-CoV2-SΔ21 cells or from 5×10 4 The cell membrane obtained by the TA-Renca-mSARS-CoV2-SΔ21 cell line immunized 4 animals / group, and PBS+CFA was used as a placebo to immunize 4 animals / group. In the same time period, 4 mice were immunized with the determined Covid-19mRNAS vaccine (BNT162b2) administered for the ml / kg dose described in adults, and the other 4 groups of mice were immunized with the whole inactivated virus (WIV) (Sinovac-Coronavac Covid-19) administered for the ml / kg dose described in adults. Blood samples were collected from the tail vein on the 15th and 28th days and used to measure antibody titers and virus neutralization assays.

[0220] Antibody measurement by ELISA in immunized mice

[0221] The total levels of IgG, anti-SARS-CoV2-S antibodies in individual immunized mice were measured by ELISA. Blood samples were collected from the tail vein on days 15 and 28 and used to measure antibody titers and virus neutralization assays.

[0222] Results: The mean endpoint titers of S protein-specific IgG in mice immunized with a single vaccination of RencaWhC expressing SARS-CoV2-S (WhC-S) containing CFA were significantly higher than those in Balb / C mice receiving PBS containing CFA ( Figure 5a ) and C57BL / 6 mice ( Figure 5b ). In addition, in mice immunized with CM expressing SARS-CoV2-S (CM-S), S protein-specific IgG was observed to be slightly increased, although not significant, compared with the group of mice immunized with WhC-S ( Figure 5c ). In addition, the addition of CFA to WhC-S and CM-S immunizations slightly increased S protein-specific IgG, although not significantly ( Figure 5d ).

[0223] It was determined that vaccination with CM-S produced a statistically stronger immune response than vaccination with BNT162b2 ( Figure 5e It was also determined that the differences between the two groups of vaccination systems were higher in the single-dose vaccination ( Figure 5e ). Furthermore, vaccination with CM-S produced a statistically significantly stronger immune response compared to the human convalescent serum (hcPS) pool, even with a single dose of CM-S vaccination ( Figure 5e ).

[0224] In both neutralization tests performed with cells presenting SARS-CoV2-S protein (WhC-S) and NM coated with RBD units of SARS-CoV2-S protein, statistically significant differences were observed in the sera of humans doubly vaccinated with BNT162b2 (mRNA-S-PS), sera of the mouse group doubly vaccinated with BNT162b2 (mRNA-S), and hcPS ( Figure 6a and Figure 6b ). All neutralization tests were performed with sera obtained after the booster dose.

[0225] Live SARSCoV-2 Neutralization Assay

[0226] Similar to the above neutralization test, in the live SARSCoV-2 neutralization assay, statistically significant differences were observed between mice doubly vaccinated with CM-S and the mouse group doubly vaccinated with BNT162b2 (mRNA-S), humans doubly vaccinated with BNT162b2 (mRNA-S-PS), and the hcPS group ( Figure 6c ).

[0227] The average NT50 (50% neutralization titer) values ​​in CM-S were 8.2, 3.7, and 4.5 times the values ​​observed in the sera of the mouse group doubly vaccinated with BNT162b2 (mRNA-S), the sera of humans doubly vaccinated with BNT162b2 (mRNA-S-PS), and hcPS, respectively. Figure 6c ).

[0228] Immune response type assessment

[0229] As mentioned above, a variety of SARS-CoV-2 vaccine types have been developed, such as DNA-based formulations, RNA-based formulations, recombinant subunits containing viral epitopes, adenovirus-based vectors, and purified inactivated viruses. In order to fully evaluate the immunogenicity and safety of various SARS-CoV-2 vaccine antigens, preclinical animal model experiments are necessary. In humans, induced virus neutralization ability is the gold standard for determining vaccine efficacy. However, in mouse vaccine evaluation models, the detection of IgG, IgG1, and IgG2a antibody titers and IgG1-IgG2a ratios better correlate with vaccine efficacy than neutralization alone. A deeper understanding of the immune response in animal models will help evaluate the protective ability, toxicity, and safety of SARS-CoV-2 vaccines.

[0230] An increased IgG1 / IgG2a ratio indicates a Th2 type humoral immune response, while an increased IgG2a / IgG1 ratio tends to favor a Th1 type cellular immune response. A balance of the Th1 / Th2 immune response will help improve the protective effect of the vaccine and avoid pathological enhancement after immunization. Increased induction of both antibody isotypes as measured by ELISA correlates better with vaccine efficacy than neutralization alone.

[0231] Figure 6d It was shown that CM-S elicited 4.2-fold and 4.9-fold more IgG1 than mice vaccinated with mRNA-S and WIV, respectively, and CM-S also elicited 4.3-fold and 3.2-fold more IgG2a than mice vaccinated with mRNA-S and WIV, respectively. Interestingly, although the neutralizing antibody titers of WIV were lower than those of mRNA-S vaccination (data not shown), it secreted more IgG2a compared to mRNA-S vaccination. In addition, while the IgG2a / IgG1 ratio was altered in favor of a Th2-type humoral immune response in animals vaccinated with CM-S and mRNA-S, it was altered in favor of a Th1-type cellular immune response in mice vaccinated with WIV ( Figure 6d ).

[0232] Materials and methods

[0233] cell

[0234] Hep3B human hepatocellular carcinoma cell line, Renca cell line derived from renal cortical adenocarcinoma in Balb / c mice, MAD109 mouse lung cancer cell line were used in these experiments. Cells were grown in DMEM or RPMI medium supplemented with 10% FBS, 1% Pen / Strep, 1% L-glutamine. All cells were incubated at 37°C, 5% CO2.

[0235] Mouse codon-optimized SARS-CoV2-S-wt and SARS-CoV2-S-Δ21 cDNA preparation

[0236] Mouse codon-optimized SARS-CoV2-S wt and SARS-CoV2-SΔ21 cDNA preparation

[0237] The SARS-CoV2 strain Whuan-Hu-1 (Genebank NC 045512) spike genome was codon-optimized for mice (mARS-CoV-2S wt) and cloned using GeneScript ( https: / / www.genscript.com ) were prepared as mARS-CoV-2S wt and mARS-CoV-2SΔ21.

[0238] Codons optimized for H. sapiens SARS-CoV2-S-wt were a gift from Gerald Pao (Addgene plasmid #141347; http: / / n2t.net / addgene:141347 ; RRID: Addgene_141347). Codons optimized for Homo sapiens SARS-CoV2-S-Δ21 were a gift from Jesse Bloom (Addgene plasmid #155130; http: / / n2t.net / addgene:155130; RRID:Addgene_155130).

[0239] Overexpression of SARS-CoV2-S wt and SARS-CoV2-SΔ21 in cells using the Retro-X Tet-On Advanced Inducible Expression System

[0240] The Retro-X Tet-OnAdvanced inducible expression system requires the presence of two retroviral constructs in the cells, namely the tetracycline-dependent rtTA transactivator (pRetroX-Tet-On-Advanced) and the target construct driven by the transactivator (pRetroX-Tight-Pur + target gene). Therefore, we first established the Hep3B human origin cell line and Renca and MAD109 mouse cell lines expressing the rtTA transgene. For this purpose, Hek293T cells were transfected with pRetroX-Tet-On-Advanced and the auxiliary vectors pCMV-VSV-G and pUMVC using the CalPhos mammalian transfection kit (631312-Takarabio). After 48 hours, the supernatant containing the rtTA virus was collected and infected with the target cells in the presence of 10 mg / ml polybrene (TR-1001, Sigma-Aldrich). After 24 hours, the medium of the cells was replaced with medium containing 0.2 mg / ml-1 mg / ml neomycin (G418-RO, Sigma-Aldrich), followed by selection for 10-15 days.

[0241] The living cells were then combined in banks and named TA-Hep3B, TA-Renca, and TA-MAD109.

[0242] Codons optimized for human SARS-CoV2-S wt and SARS-CoV2-SΔ21 (hSARS-CoV2-S and SARS-CoV2-SΔ21) genes and codons optimized for mouse SARS-CoV2-S wt and SARS-CoV2-SΔ21 (mSARS-CoV2-S and mSARS-CoV2-SΔ21) genes were cloned into a modified pBSK (pBSK-clo2D) subcloning vector plasmid.

[0243] The hSARS-CoV2-S wt, hSARS-CoV2-SΔ21, mSARS-CoV2-S wt, mSARS-CoV2-SΔ21 genes were inserted into the pBSK-Clo2D vector construct, which was pre-prepared with cloning sites of the pBSK(-) vector such as Kpnl, Apal, Xhol, Sall, Pmel, BamHl EcoRV, EcoRl, Xbal, Notl, Hindlll, Mfel, EcoRl, Pstl, Smal, BamHl, Xbal, BstXl and Sac1 for routine DNA cloning in my laboratory. The blunt-ended SARS-CoV2-S gene was inserted into the pBSK-clo2D plasmid cut with EcoRV and dephosphorylated with a dephosphorylase (FastAp thermosensitive alkaline phosphatase (Thermoscientific-ThermoFisher USA)). pBSK-hSARS-CoV2-S wt, pBSK-hSARS-CoV2-SΔ21, pBSK-mSARS-CoV2-S wt, and pBSK-mSARS-CoV2-SΔ21 vectors were obtained. Sequence analysis was performed to search for mutations in each vector.

[0244] Codons optimized for human hSARS-CoV2-S wt and hSARS-CoV2-SΔ21 genes, and codons optimized for mouse mSARS-CoV2-Swt and mSARS-CoV2-SΔ21 genes were cloned into the pRetroX-Tight-Pur vector.

[0245] hSARS-CoV2-S wt and hSARS-CoV2-SΔ21, as well as mSARS-CoV2-S wt and mSARS-CoV2-SΔ21 genes were obtained from pBSK-hSARS-CoV2-S wt, pBSK-hSARS-CoV2-SΔ21, pBSK-mSARS-CoV2-S wt, and pBSK-mSARS-CoV2-SΔ21 vectors, respectively, by cutting with BamHI enzyme. The pRetroX-Tight-Pur retroviral vector was cut with BamHI restriction enzyme, dephosphorylated, and ligated with hSARS-CoV2-S wt and hSARS-CoV2-SΔ21, mSARS-CoV2-S wt, and mSARS-CoV2-SΔ21 cDNAs with compatible sticky ends. After the directional constructs were determined, E. coli DH5a was used to replicate pRetroX-Tight-Pur-hSARS-CoV2-S wt, pRetroX-Tight-Pur-hSARS-CoV2-SΔ21, pRetroX-Tight-Pur-mSARS-CoV2-S wt, and pRetroX-Tight-Pur-mSARS-CoV2-SΔ21 vectors in large quantities.

[0246] In the next step, Hek293T cells were transfected with pRetroX-Tight-Pur-hSARS-CoV2-S wt, pRetroX-Tight-Pur-hSARS-CoV2-SΔ21, pRetroX-Tight-Pur-mSARS-CoV2-S wt and pRetroX-Tight-Pur-mSARS-CoV2-SΔ21 vectors and auxiliary vectors pCMV-VSV-G and pUMVC using the CalPhos Mammalian Transfection Kit (631312 - Takara bio), respectively.

[0247] After 48 hours, supernatants and culture media containing viruses of pRetroX-Tight-Pur-hSARS-CoV2-S wt, pRetroX-Tight-Pur-hSARS-CoV2-SΔ21, pRetroX-Tight-Pur-mSARS-CoV2-S wt and pRetroX-Tight-Pur-mSARS-CoV2-SΔ21 vectors were collected from individual cells and infected with TA-Hep3B, TA-Renca and TA-MAD109 target cells expressing rtTA. Infectious clones were selected for 6 days with 0.5mg / ml-3mg / ml puromycin (P9620-Sigma-Aldrich). Infected clones were maintained in the presence of 250ng / ml puromycin. The expression of SARS-CoV2-S wt and SARS-CoV2-SΔ21 in individual cell lines was tracked by dot blot and immunohistochemistry (IHC).

[0248] Overexpression of SARS-CoV2-RBD using the Retro-X Tet-OnAdvanced inducible expression system

[0249] For this purpose, mouse RBD and human RBD were PCR amplified from mSARS-CoV2-SΔ21 or hSARS-CoV2-SΔ21 cDNA using a sense primer containing a Kozak sequence and an antisense primer containing a stop codon as follows:

[0250] mRBD-F 5'-GCCGCCGCCATGCAGCCAAACCGAGTCC-3', mRBD-R 5'-TTAGAAGTTCACGCACTTGTTC-3', hRBD-F 5'-GCCGCCGCCATGCAACCCACCGAGTCCATTGTG-3', hRBD-R 5'-TTAATTTACGCACTTGTTCTTC3'. They were then cloned into the pRetroX-Tight-Pur vector as described above and inserted into cells for use in the dox inducible system.

[0251] Isolation of cell membranes.

[0252] Membranes were isolated from cells using previously described methods (Zhai et al., 2017, Cao et al., 2016) with some modifications. Briefly, cells were harvested and cultured at 2.5 × 10 7The concentration of 1 cell / mL is resuspended in ice-cold Tris-magnesium buffer (TM buffer, pH 7.4, 0.01M Tris and 0.001M MgCl), and homogenized with a Dounce homogenizer to disrupt the cells. The cell homogenate is mixed with 1M sucrose to a final concentration of 0.25M sucrose, and then centrifuged at 2000g and 4°C for 10 minutes. The supernatant is collected and further centrifuged at 3000g and 4°C for 30 minutes to collect the cell membrane. The cell membrane is washed with an ice-cold TM buffer containing 0.25M sucrose, and collected by centrifugation at 3000g and 4°C for 30 minutes.

[0253] Dot blot

[0254] The cell membrane solution (also the cytoplasmic extract) was diluted to 5 μL of 1×10 3 Cells were added and applied to nitrocellulose membrane (NM). These points were then dried. NM was completely immersed in blocking solution (PBS solution containing 5% skim milk) and then incubated for 60 minutes. NM was washed three times by being completely immersed in PBS-Tween (PBS solution containing 0.05% Tween 20), 1 minute each time. NM was immersed in PBS-milk (PBS solution containing 5% skim milk) diluted 1 / 100 with antibodies or serum containing anti-SARS-Cov2S antibodies, and incubated for one hour. NM was washed three times with PBS-Tween as described above. According to the first antibody used, NM was completely immersed in PBS-milk diluted 1 / 1500 with anti-human, anti-mouse or anti-rabbit IgG antibodies (Ab) conjugated with horseradish peroxidase (Thermo Fisher Scientific, USA), and incubated for 45 minutes. Washed three more times with PBS-Tween. Finally, the blot reaction was revealed by completely immersing the membrane in HRP chemiluminescence for 5 minutes and visualized by the Bio-Rad chemiDoc system. Each NM was analyzed in duplicate. https: / / image.nih.gov . / ij) The program studies the strength of the points.

[0255] Whole cell ELISA preparation

[0256] 1 × 10 cells were seeded in a 96-well cell culture plate. 3TA-Hep3B cells, TA-Renca cells, and TA-MAD109 cells containing pRetroX-Tight-Pur-hSARS-CoV2-S wt, pRetroX-Tight-Pur-hSARS-CoV2-SΔ21, pRetroX-Tight-Pur-mSARS-CoV2-S wt, and pRetroX-Tight-Pur-mSARS-CoV2-SΔ21 were added with doxacillin (dox) after 24 hours and fixed with 4% paraformaldehyde after 36 hours.

[0257] Cell membrane ELISA preparation

[0258] According to the above protocol, cell membranes containing S protein were prepared 36 hours after dox was added to the cells. The TM buffer containing cell membranes was diluted with coating buffer (0.5 M carbonate-bicarbonate buffer, pH 9.6) and used to coat all 96 wells of the ELISA plate to 1×10 3 cells.

[0259] ELISA preparation of S1 subunit and RBD subunit of SARS-CoV2

[0260] ELISA plates were coated overnight with 20 ng / ml of SARS-CoV2 S1 (Biolegend, USA), or RBD obtained from the above cells (cells expressing SARS-CoV2-S RBD) was diluted with coating buffer and used to coat all 96 wells of the ELISA plate.

[0261] Antibody measurements were performed using whole cell or cell membrane coated ELISA.

[0262] The total level of IgG, anti-SARS-CoV2-S antibodies in infected or vaccinated individuals was measured by the above-mentioned preparative ELISA.

[0263] Ninety-six-well cell culture plates coated with cells containing mSARS-CoV2-S wt and mSARS-CoV2-SΔ21 proteins or cell membranes containing covid-19S protein were blocked with 5% milk in PBS for 1.5 h at room temperature (RT).

[0264] Serum samples from each immunization group were diluted in PBS-Tween-1% milk, added to the plate, and incubated at RT for 1 hour. The plate was then washed, a second HRP-conjugated anti-human IgG antibody (Solarbio-China; all diluted 1:1,000 in PBS-Tween-1% milk) was added, and incubated at room temperature for 45 minutes. The plate was washed, o-phenylenediamine dihydrochloride (OPD) substrate (Sigma-Aldrich-0.5mg / ml-1.0mg / ml OPD dissolved in buffer-0.05M citric acid, 0.05M sodium phosphate; pH 5) was added, and then 1μl 30% hydrogen peroxide was added per 1ml substrate), and 1M H2SO4 was added to terminate the reaction. The absorbance was read at 450nm.

[0265] Expression analysis of SARS-CoV2-S by immunofluorescence microscopy.

[0266] Immunofluorescence experiments were performed in Hep3B cells, Renca cells, and MAD109 cells expressing SARS-CoV2-SΔ21. Twenty-four hours after cell division, dox was added to cells except control cells. After fixation and blocking at 12, 24, and 48 hours after dox addition, permeabilized cells or non-permeabilized cells were incubated with SARS-CoV2-S pozitif serum from patients infected with SARS-CoV2-S or anti-S antibody (Biolegend). Anti-SARS-CoV2-S was detected with rabbit or human secondary antibodies conjugated to FITC fluorescent dye (Solarbio; dilution 1:1000). We used Hoecst (Sigma) to stain the nuclei. Images of cell sections were taken using a fluorescence microscope.

[0267] Ethical statement.

[0268] Mouse studies were performed at the Laboratory Animal Care Unit of Ankara University Faculty of Medicine under the supervision of veterinarians with the permission of the Local Ethics Committee for Animal Experimentation of Ankara University (File No. 2021-54; Permit No. 2021-1074). The animal protocol was approved by the Local Ethics Committee for Animal Experimentation of Ankara University (AU). All animal experiments and methods were performed in accordance with the guidelines and regulations for animal experimentation approved by the Local Ethics Committee for Animal Experimentation of AU. The study was performed in accordance with the ARRIVE guidelines. Live virus studies were performed in the Microbiology Reference Laboratory (Animal Biosafety Level 3 or above - ABSL3p) of the General Directorate of Public Health in Ankara, Turkey.

[0269] Animal Immunization

[0270] 5 × 10 4100 μl volume of whole cells expressing TA-Renca-mSARS-CoV2-S or from 1 × 10 4 Groups of Balb / c mice and C57BL / 6 mice (4 animals per group) were subcutaneously immunized with 100 μl of cell membranes obtained from cells expressing TA-Renca-mSARS-CoV2-SΔ21. Cells and cell membranes were obtained 36 hours after the addition of dox on the growing cell culture. On days 1 and 15, cell membranes isolated according to the above protocol and cells collected by scraping and washing 3 times with PBS were administered to mice together with complete Freund's adjuvant (CFA, Sigma-Aldrich). Incomplete Freund's adjuvant was used in the second vaccination. IgG titers in sera collected from mice immunized with cells or cell membranes containing mSARS-CoV2-SΔ21 on day 15 or day 28 after vaccination were determined by ELISA. Control groups received PBS containing CFA or cell membranes of the same amount of Renca wt cells. In addition, 1×10 4 TA-Renca-mSARS-CoV2-SΔ21 cells or from 1×10 4 The cell membrane obtained by the TA-Renca-mSARS-CoV2-SΔ21 cell line immunized 4 animals / group, and PBS+CFA was used as a placebo to immunize 4 animals / group. In the same time period, 4 mice were immunized with the determined Covid-19mRNA S vaccine (BNT162b2) administered for the ml / kg dose described in adults, and the other 4 groups of mice were immunized with the whole inactivated virus vaccine (Sinovac-CoronavacCovid-19) administered for the ml / kg dose described in adults. Blood samples were collected from the tail vein on the 15th and 28th days and used to measure antibody titers and virus neutralization assays.

[0271] Blood was collected from each individual mouse and processed to obtain serum samples for analysis of IgG antibodies, IgG isotypes, and titers of neutralizing antibodies against SARS-CoV-2.

[0272] Immunogenicity evaluation of vaccination

[0273] To detect antibody levels in sera from mice vaccinated with cellular or cell membrane vaccines containing mSARS-CoV2-SΔ21, geometric dilutions of sera used for ELISA were used for immunogenicity analysis. The endpoint titer was the reciprocal of the serum dilution whose mean OD value was three times that of the pre-immunization control sample.

[0274] Enzyme-linked immunosorbent assay.

[0275] The total levels of IgG, anti-SARS-CoV2-S antibodies in individual immunized mice were measured by ELISA. 96-well cell culture plates with cells containing mSARS-CoV2-S were blocked with 5% milk in PBS for 2 hours at room temperature (RT).

[0276] To detect antibody levels in the serum of mice vaccinated with cells or cell membrane vaccines containing mSARS-CoV2-SΔ21, two-fold serial dilutions of the serum used for ELISA were used for immunogenicity analysis. Serum samples from each immunization group were diluted in PBS-Tween-1% milk, added to the plate, and incubated for 1 hour at RT. The plate was then washed, a second HRP-conjugated anti-mouse IgG antibody (Biolegend; all diluted 1:1,000 in PBS-Tween-1% milk) was added, and incubated at room temperature for 45 minutes. The plate was washed, OPD substrate (Sigma-Aldrich) was added, and the reaction was terminated by adding 1M H2SO4. The absorbance was read at 450nm. The total IgG titer was measured as the last dilution that produced an absorbance at least three times the initial serum absorbance.

[0277] Neutralization assay.

[0278] The ability of sera obtained from mice immunized with the vaccine candidate TA-Renca-mSARS-CoV2-SΔ21 cell membranes (CM) to neutralize the SARS-CoV2 virus was initially determined by two different protocols. In the first protocol, sera from 1×10 6 Whole cells (1 ml PBS in total) obtained from TA-Renca-mSARS-CoV2-SΔ21 cells were used for neutralization assays after washing 3 times with PBS. 100 μL of cell membranes were incubated with two-fold serial dilutions of mouse serum at 37 ° C for 1 hour. The mixture was then centrifuged and the supernatant was used for ELISA in 96-well plates coated with RBD, and the relative neutralization titer was determined. ID50 was determined as the highest dilution of serum that resulted in a 50% decrease in the OD450 signal in the ELISA compared to the negative serum control.

[0279] In the second protocol, cytoplasmic proteins of RBD units containing SARS-CoV2-S overexpressed in TA-Renca under dox control were adsorbed to NM (nitrocellulose membrane). For neutralization assays, NM with RBD were incubated with two-fold serial dilutions of mouse serum for 1 h at 37°C. The mixture was then centrifuged (to remove neutralizing antibodies bound to RBD), and the supernatant was used for ELISA containing SARS-CoV2-S antigen on 96-well plates.

[0280] Live SARS-CoV2 neutralization assay.

[0281] The presence of virus-specific neutralizing antibodies in mice vaccinated with CM-S, mRNA-S and WIV vaccinations was evaluated by virus neutralization assay. Serial dilutions (twice in DMEM) of serum samples were mixed with equal-volume 100TCID50 virus (1:10000) titers in duplicate and incubated at 37°C for 1 hour. Subsequently, serum-virus mixtures were inoculated on 90% confluence Vero E6 cells of one-day age and grown in 96-well plates. Infected cells were further incubated for four days under the same conditions. When 100% CPE was observed in the virus control wells, the test was evaluated by an inverted microscope.

[0282] IHC

[0283] The cells were fixed in a buffered formalin (4%) solution. Purified SARS-CoV2 IgG or anti-SARS-CoV2 S1 antibody (Biolegend) from convalescent human plasma was used as the primary antibody, and then incubated with FITC-labeled anti-human IgG secondary antibody (SigmaAldrich, USA) for one hour. Finally, the cells on the slide were observed using a fluorescence microscope.

[0284] Statistical analysis.

[0285] All data are expressed as mean ± standard error of the mean. For all analyses, P values ​​were obtained from Student's t test (unpaired, two-tailed) or Spearman rank correlation test. All charts were generated using Microsoft Excel. Data are based on the results of at least three independent experiments. Error bars show standard deviation. BRIEF DESCRIPTION OF THE DRAWINGS

[0286] Figure 1 SARS-CoV2 SΔ21 expression ensures that the spike protein is located in the cell membrane, a. Confirming cell membrane (CM) separation. b., c. SARS-CoV2 SΔ21 expression localizes the entire spike protein to Hep3B CM and Renca CM, respectively, as shown by dot blots. d., e. SARS-CoV2 S WT expression localizes the entire spike protein to the cytoplasm of Hep3B cells and Renca cells, respectively, as shown by dot blots. f., g. Although SARS-CoV2 SΔ21 expression localizes the entire spike protein to Hep3B CM and Renca CM, this does not occur in cells with SARS-CoV2 S wt expression, as shown by IHC. h., i., and j. SARS-CoV2 SΔ21 expression localizes the entire spike protein to the membrane of Hep3B CM, Renca CM, and MAD109 cells, as shown by IHC. After cells containing pRetroX-Tight-Pur-SARS-CoV2-SΔ21 were induced with dox, spike protein expression at different time intervals was shown by IHC.

[0287] Figure 2 Cell fusion occurs in the presence of SARS-CoV2-SΔ21 expression. Twenty-four hours after the addition of dox, Hep3B cells containing pRetroX-Tight-Pur-hSARS-Cov2 S wt or pRetroX-Tight-Pur-hSARS-Cov2 SΔ21 cells were stained with Hoechst (1 μg / ml) or IHC stained with anti-SARS-CoV2-S antibody and observed by fluorescence microscopy.

[0288] Figure 3 Evaluation of immunogenicity of cells expressing SARS-CoV2-SΔ21. a., b. ELISA assays showed that the signal increased with the amount of cells expressing SARS-CoV2-SΔ21 (a) and the duration of dox administration (b) (hcPS (convalescent patient serum) was used as the primary antibody). c. and d. ELISA assay cells expressing SARS-CoV2-S wt, almost no signal was detected in Hep3B cells and Renca cells, respectively. e. In an ELISA assay using a commercial anti-S1 antibody (Biolegend), the signal increased with the amount of cells expressing SARS-CoV2-SΔ21.

[0289] Figure 4 SARS-CoV2-SΔ21 replaces major cell membrane proteins, including SPTA1 and CDH2

[0290] After treatment with dox for 36 hours, the cell membrane and cytoplasm of TA-Hep3B expressing rtT (control cells), TA-Hep3B containing pRetroX-Tight-Pur-hSARS-CoV2-SΔ21 (control cells), and TA-Renca containing pRetroX-Tight-Pur-mSARS-CoV2-SΔ21 were isolated, and the amount of SPTA1 and CDH2 proteins in the cell membrane and cytoplasm of the cells was investigated by dot blot using SPTA1 and CDH2 specific antibodies. a. and b. show the dot blot and graph of the abundance of SPTA1 and CDH2 proteins in the cytoplasm and cell membrane of TA-Hep3B (control) and TA-Hep3B-SΔ21 cells, respectively, and c. and d. show the dot blot and graph of the abundance of SPTA1 and CDH2 proteins in the cytoplasm and cell membrane of TA-Renca (control) and TA-Renca-SΔ21 cells, respectively. e.-h. After the addition of dox, SARS-CoV2-S protein expression (e) and the amount of cell membrane proteins (f, g) and autoantibodies (h) (using mSARS-CoV2-S negative human serum) were detected by ELISA in Renca cells containing pRetroX-Tight-Pur-mSARS-CoV2-SΔ21 at 0, 12, 24 and 48 hours.

[0291] Figure 5 Evaluation of the immunogenicity of vaccination with whole cells (WhC) or cell membranes (CM) obtained from TA-Renca-mSARS-CoV2-SΔ21 in Balb / C mice (a) and C57BL / 6 mice (b) (sera were collected 14 days after the first vaccination with WhC-S and 14 days after the second vaccination) c. The results of vaccination with WhC-S were compared with the results of vaccination with CM-S. d. The effect of adding adjuvants to vaccination with CM-S. e. The results of the immunogenicity evaluation of vaccination with CM-S were compared with the results of vaccination with mRNA-S, WIV (whole inactivated virus) and hcPS vaccines in Balb / C mice. h. The titer of neutralizing antibodies against live virus in the same group.

[0292] Figure 6. Protective efficacy of CM-S against SARS-CoV-2 in Balb / c mice. a. Neutralizing antibody titers of CM-S, mRNA-S-PS, mRNA-S and hcPS sera (neutralization was completed using Renca cells expressing mSARS-CoV2-SΔ21). b. Neutralizing antibody titers of serum CM-S, mRNA-S-PS (serum of patients doubly immunized with mRNA-S), mRNA-S and hcPS (neutralization was completed using NM-RBD). c. Neutralizing antibody titers against live virus in the same group. d. The titer ratio of IgG2a to IgG1 was calculated in Balb / c mice vaccinated with CM-S, mRNA-S and WIV. Data are mean ± SEM (standard error of the mean). Comparison was performed by Student's t-test (paired, two-tailed). Data are a representative result of three independent experiments.

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Claims

1. The method is based on a model in which the S2 subfragment protein of SARS-CoV2-S (SARS-CoV2-SΔ21) with the last 21 amino acids removed transports the antigen protein (S1 subfragment) to the cell membrane (CM) and presents itself densely on the CM.

2. The method according to claim 1, wherein the S2 subfragment protein of SARS-CoV2-SΔ21 acts as a transporter for the S1 subfragment protein of SARS-CoV2-S with antigenic properties to the CM.

3. The method according to claim 1, wherein the S2 subfragment protein of SARS-CoV2-SΔ21 is capable of showing the same transporter activity against the CM when the number of amino acids removed from the C-terminus is less than 21 amino acids or more than 21 amino acids.

4. The method according to claim 1, wherein the S2 subfragment protein of SARS-CoV2-SΔ21 is capable of showing the same transporter activity against the CM when the S2 protein fragments including FP, HR1 and HR2 fragments are removed, or if the S2 protein fragments are arranged differently.

5. The method according to claims 1-4, wherein the S2 subfragment protein is transported by the transporter function of SARS-CoV2-SΔ21 and presented in CM, which will enable active immunization, in other words, vaccination against the SARS-CoV2-S protein.

6. The method according to claim 1, comprising expressing the S1 segment of the SARS-CoV2-S protein having antigenic properties naturally fused to the S2 segment in a cell using a plasmid expression system.

7. The method according to claim 6, wherein the plasmid expression system comprises using a vector system designed as a tetracycline-inducible mammalian expression system, wherein the tetracycline-inducible mammalian expression system is controlled by adding Dox to the culture medium.

8. The method according to claim 7, wherein the tetracycline-inducible mammalian expression plasmid expression system further comprises retrovirus, lentivirus, adenovirus-associated vector, phage or any other viral system.

9. The method according to claim 7, wherein the inducible mammalian expression plasmid expression system can be an inducible system other than the tetracycline inducible system.

10. The method of claim 6, wherein the plasmid expression system, such as a retrovirus, lentivirus or adenovirus-associated vector system provides sustained expression.

11. The method according to claim 6, wherein the plasmid expression system will preferably be performed using a vector system containing EF1 promoter or CMV and other vector systems containing promoters that function in mammalian cells.

12. The method according to claims 1-11, comprising using living cells, comprising allogeneic primary cancer cell lines, passaged cancer cell lines, immortalized primary cancer cell lines or allogeneic cancer cell lines with a vector system expressing SARS-CoV2-SΔ21 as a SARS-CoV2 vaccine.

13. The method according to claims 1-11, comprising using living cells having MHC antigens for producing an allogeneic immune response in a recipient prepared as described in the present invention, containing SARS-CoV2-SΔ21 obtained from the allogeneic primary cell line, the continuous cell line, the immortalized primary cell line or the continuous cell line in its structure as a SARS-CoV2 vaccine.

14. The method according to claims 1-11, comprising using the cell membrane (CM) (CM-S) containing SARS-CoV2-SΔ21 obtained from the allogeneic primary cell line, the passaged cell line, the immortalized cell line or the cancer cell line having a vector system expressing SARS-CoV2-SΔ21 as a SARS-CoV2 vaccine.

15. The method according to claims 1-11, comprising using whole cells (live) prepared as described in the present invention, containing SARS-CoV2-SΔ21 obtained from an allogeneic stem cell line in its membrane as a SARS-CoV2 vaccine, wherein the allogeneic stem cell line includes an embryonic stem cell line, a mesenchymal stem cell line, a fetal stem cell line, an adult stem cell line, and an amniotic membrane stem cell line.

16. The method according to claims 1-11, comprising using whole cells prepared as described in the present invention having MHC antigens for producing the allogeneic immune response in the recipient, containing SARS-CoV2-SΔ21 obtained from the allogeneic stem cell line in its structure as a SARS-CoV2 vaccine, wherein the allogeneic stem cell line includes an embryonic stem cell line, a mesenchymal stem cell line, a fetal stem cell line, an adult stem cell line, and an amniotic stem cell line.

17. The method according to claims 1-11, comprising using CM-S obtained from allogeneic stem cells as a SARS-CoV2 vaccine, wherein the allogeneic stem cells include embryonic stem cell lines, mesenchymal stem cell lines, fetal stem cell lines, adult stem cell lines, and amniotic stem cell lines that express SARS-CoV2-SΔ21 in their membranes.

18. The method according to claims 1-11, comprising using living cells, including autologous primary tissue cells and stem cells, as a SARS-CoV2 vaccine, and the autologous primary tissue cells and the stem cells include embryonic stem cell lines, mesenchymal stem cell lines, fetal stem cell lines, adult stem cell lines, and amniotic membrane stem cell lines that express SARS-CoV2-SΔ21 in their membranes.

19. The method according to claims 1-11, comprising using CM-S obtained from the autologous primary tissue cells and the stem cells as a SARS-CoV2 vaccine, wherein the autologous primary tissue cells and the stem cells include embryonic stem cell lines, mesenchymal stem cell lines, fetal stem cell lines, adult stem cell lines, and amniotic stem cell lines expressing SARS-CoV2-SΔ21 in their membranes.

20. The method according to claims 1-19, which relates to a vaccination method involving not only humans but also animals.

21. The method according to claims 1-19, which provides the opportunity to use cells that express large amounts of SARS-CoV2-S protein in its native structure in their membranes.

22. The method of claim 21, which provides for stronger active immunity and vaccination against the SARS-CoV2-S protein.

23. The method according to claims 1-19, which provides the opportunity to use whole cells expressing SARS-CoV2-S, which have been grown in 96-well plates, which will be used for immunological tests, including SARS-CoV2-S protein ELISA.

24. The method according to claims 1-19, which provides the opportunity to use CM in immunological tests including ELISA, wherein the cell membrane containing SARS-CoV2-S will be used to coat the ELISA plate.

25. The method according to claims 1-9, which provides the opportunity to use whole cells or CM containing a large amount of SARS-CoV2-S protein in its native structure for antibody production against the S protein.

26. The method of claim 25, comprising using living cells to produce antibodies, wherein the cells can be any eukaryotic cell line that expresses and presents the SARS-CoV2-S protein in the cell membrane.

27. The method of claim 25, comprising using CM to produce antibodies, wherein the cell can be any eukaryotic cell line that expresses and presents the SARS-CoV2-S protein in the cell membrane.

28. The method according to claims 1-9, comprising fusing different epitopes of S1 with antigenic properties to the S2Δ21 fragment of SARS-CoV2-S protein and expressing intracellularly using a plasmid expression system, wherein the different epitopes include RBD, mutant RBD or any specific part of the RBD of the S protein to transport to the cell membrane and provide the presentation of the structure.

29. In addition to SARS-CoV2-S vaccination, according to the method of claims 1 to 19 of the present invention, due to the fusion of any viral protein with antigenic properties with the S2Δ21 fragment of the SARS-CoV2-S protein, their expression in the cells is ensured, and with the help of the transport function of the S2Δ21 they are transported to the cell membrane and present themselves there in a concentrated manner, a vaccination scheme for all viruses including HIV is obtained.

30. According to the method of claims 1 to 19 of the present invention, a vaccination scheme for all bacteria is obtained due to the fusion of any bacterial protein with antigenic properties with the S2Δ21 fragment of the SARS-CoV2-S protein, ensuring their expression in the cells and their transport to the cell membrane with the help of the transport function of the S2Δ21 and their central presentation there.

31. The method according to claims 1-19, which also includes the treatment or prevention of cancer by establishing a vaccination program, which is obtained due to the fusion of tumor-associated proteins with antigenic properties to the S2Δ21 fragment of the SARS-CoV2-S protein, ensuring their expression in the cells and their transport to the cell membrane with the help of the transport function of the S2Δ21 and presenting themselves there.

32. The method according to claims 1 to 19 of the present invention provides an immunogenic composition of disease-associated antigens for use in vaccines, antibody production and immunodiagnostic testing, wherein the expression of SARS-CoV2-S protein on the CM results in the displacement of other CM proteins and their removal from the CM, which ultimately prevents the formation of unwanted background antibodies.

33. In addition to claim 32, by using the present invention, immunizing animals with antigens in their membranes in autologous or allogeneic cells will prevent the formation of unwanted background antibodies and will ensure the formation of antibodies specific for the target antigen.

34. The method according to claims 1-22, which in another aspect also comprises a method for treating or preventing viral and bacterial infections by providing a vaccination regimen, which is obtained due to the fusion of multiple viral or bacterial proteins showing different antigenic structures to the S2Δ21 fragment of the SARS-CoV2-S protein, ensuring their expression in the cells and their transport to the cell membrane with the help of the transport function of the S2Δ21 and there presenting themselves centrally to the immune system.

35. The method according to claims 1-22, which in another aspect also comprises a method for treating or preventing cancer by providing a vaccination scheme, which is obtained by fusing multiple tumor-associated proteins showing different antigenic structures to the S2Δ21 fragment of the SARS-CoV2-S protein, ensuring their expression in the cells, and transporting them to the cell membrane with the help of the transport function of the S2Δ21 and presenting them centrally to the immune system.

36. The method according to claim 35, wherein the cancer disease is selected from, but not limited to, carcinoma, lymphoma, blastoma, sarcoma, glioma and leukemia. More specifically, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, sexual and reproductive organ cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal cancer, spinal tumors, gliomas, meningiomas and pituitary adenomas; according to the present invention, the term "cancer" also includes cancer metastasis.

37. The method according to claims 23-30, further comprising the development of antibody production, immunodiagnostic test kits including the ELISA system or other antibody screening test procedures, as this enables all viral and bacterial antigenic epitopes to be presented in the cell membrane via the S2Δ21 fragment of the SARS-CoV2-S protein.

38. The present invention also claims that other different DNA sequences, proteins, or one or more molecules can play a similar role as the S2Δ21 fragment of the SARS-CoV2-S protein, such as VSV-G involved in transporting the protein to the CM and for this purpose.

39. The present invention claims that the above-mentioned vaccine regimen also includes the use of an adjuvant, wherein the adjuvant includes one or more of an alumina gel adjuvant, a saponin, an oil-in-water emulsion, an oil-in-water emulsion, an oil-in-water emulsion, an acrylic acid or methacrylic acid polymer, a copolymer of maleic anhydride and an alkenyl (alkenyl) derivative, a RIBI adjuvant system, a block copolymer, SAF-M, a monophosphoryl lipid A, an avridine lipid-amine adjuvant, an Escherichia coli heat-labile enterotoxin, a cholera toxin, 1314, a muramyl dipeptide or a gel adjuvant.

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