KR20230041699A - Immunogenic composition against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - Google Patents

Abstract

An immunogenic composition against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is provided, particularly an immunogenic composition comprising a recombinant SARS-CoV-2 S protein and an adjuvant.

Description

Immunogenic composition against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

CROSS REFERENCES OF RELATED APPLICATIONS

This application claims priority to and benefit from U.S. Provisional Application No. 63/040,696, filed on June 18, 2020, and PCT Application No. PCT/US21/20277, filed on March 1, 2021, the disclosures of which are incorporated herein by reference in their entirety. is used as

field of invention

The present invention relates to an immunogenic composition against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), particularly an immunogenic composition comprising a recombinant SARS-CoV-2 S protein and an adjuvant.

Description of Prior Art

In late 2019, a novel coronavirus emerged and was identified as the cause of a cluster of respiratory infections. This viral pathogen did not match all other known viruses and was later officially named "Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)". The official name of the disease caused by SARS-CoV-2 is Coronavirus Disease 2019 (COVID-19). Common symptoms of COVID-19 include fever, dry cough, fatigue, malaise, muscle or body aches, sore throat, diarrhea, conjunctivitis, headache, loss of taste or smell, skin rash, and shortness of breath. Most cases present with mild symptoms, but some progress to acute respiratory distress syndrome (ARDS), triggered by cytokine storms, multi-organ failure, septic shock, and blood clots. The first confirmed death from coronavirus infection occurred on 9 January, and as of 13 June 2021, 175,306,598 confirmed cases of COVID-19 have been reported to WHO, including 3,792,777 deaths. The numbers are still growing rapidly.

A COVID-19 vaccine is needed to reduce the risk of contracting SARS-CoV-2 without limiting daily activities. In particular, a COVID-19 vaccine that can rapidly induce an immune response against SARS-CoV-2 is urgently needed.

Summary of Invention

In one aspect, the present invention provides a severe acute respiratory syndrome coronavirus comprising an antigenic recombinant protein and an adjuvant selected from the group consisting of aluminum containing adjuvants, unmethylated cytosine-phosphate-guanosine (CpG) motifs, and combinations thereof. Provided is an immunogenic composition against virus 2 (SARS-CoV-2), wherein the antigenic recombinant protein substantially has a proline substitution at residues 986 and 987 and a "GSAS" substitution at residues 682 to 685 SARS-CoV-2. It consists of residues 14 to 1208 of the S protein and the C-terminal T4 fibritin trimerization domain.

In another aspect, the invention provides an immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a subject in need thereof, comprising administering to the subject an effective amount of an immunogenic composition of the invention. provides a method for inducing

In another aspect, the invention provides a method for protecting a subject in need thereof from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, comprising administering to the subject an effective amount of an immunogenic composition of the invention. provides a way

In another aspect, the invention provides a method for preventing a subject in need thereof from developing COVID-19 disease, comprising administering to a subject an effective amount of an immunogenic composition of the invention.

In another aspect, the invention provides use of the immunogenic composition of the invention for inducing an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a subject in need thereof.

In another aspect, the invention provides use of the immunogenic composition of the invention for protecting a subject from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

In another aspect, the present invention provides use of the immunogenic composition of the present invention for preventing a subject from suffering from COVID-19 disease.

These and other aspects will become apparent from the following description of the preferred embodiments taken in conjunction with the following drawings.

The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to indicate the same or similar elements of an embodiment.
1 shows the results of a neutralization assay using serum from mice immunized with SARS-CoV-2 S-2P recombinant protein in the presence or absence of aluminum phosphate adjuvant.
Figure 2 shows the results of a neutralization assay using sera from mice immunized with different formulations of SARS-CoV-2 S-2P recombinant protein.
Figure 3 shows the induction of neutralizing antibodies by CpG 1018 and aluminum hydroxide-enhanced SARS-CoV-2 S-2P at 2 weeks after the second injection. BALB/c mice (N=6 per group) were administered 2 dose levels of Chinese hamster ovary (CHO) cell-expressing SARS-CoV-2 S-2P supplemented with CpG 1018, aluminum hydroxide, or a combination of the two at 3-week intervals. Immunization and antisera were harvested 2 weeks after the second injection. A neutralization assay was performed with a pseudovirus expressing the SARS-CoV-2 spike protein to determine the ID ₅₀ (left) and ID ₉₀ (right) titers of neutralizing antibodies against antiserum. ^** p < 0.01, ^*** p < 0.001.
Figure 4 shows total anti-S IgG titers in mice immunized with S-2P with adjuvant. Serum from BALB/c mice (N=6 per group) of FIG. 3 immunized with 0, 1 or 5 μg of S-2P together with CpG 1018, aluminum hydroxide or a combination of the two were analyzed by ELISA (enzyme-linked immunosorbent assay). quantified for the total amount of anti-S IgG. ^*** p < 0.001.
5 shows neutralization of wild-type SARS-CoV-2 virus by antibodies induced by SARS-CoV-2 S-2P supplemented with CpG 1018 and aluminum hydroxide. Antisera were collected as described in FIG. 4 (N=6 per group) and a neutralization assay was performed with wild-type SARS-CoV-2 to determine neutralizing antibody titers. ^** p < 0.01, ^*** p < 0.001.
Figure 6 shows inhibition of pseudoviruses carrying the D614D (wild-type) or D614G (mutant) versions of the Spike protein by mice immunized with S-2P together with CpG 1018 and aluminum hydroxide. Antisera from BALB/c mice immunized with 1 or 5 μg of S-2P along with 10 μg CpG 1018 and 50 μg aluminum hydroxide were collected as in FIG. 5 (N=5 per group due to assay volume). Neutralization assays were performed with pseudoviruses with D616D or D614G spike proteins.
7 shows IFN-γ/IL-4, IFN-γ/IL-5, and IFN-γ/IL-6 ratios. IFN-γ, IL-4, IL-5 and IL-6 values from cytokine assays (N=6 per group) were used to calculate ratios. Ratio values greater than 1 indicate a Th1 bias and ratios less than 1 indicate a Th2 biased response. ^* p < 0.05, ^** p < 0.01.
8A-8B show neutralizing antibody titers by pseudovirus assay in hamsters 2 weeks after secondary immunization. Hamsters (N=10 per group) received either vehicle control (PBS), 1 μg (LD) or 5 μg (HD) of S-2P supplemented with 150 μg CpG 1018 and 75 μg aluminum hydroxide, or adjuvant alone at 3-week intervals. were immunized twice. Antisera were harvested 2 weeks after the second injection and a neutralization assay was performed with a pseudovirus expressing the SARS-CoV-2 spike protein to determine ID ₉₀ titers of neutralizing antibodies (Figure 8A) and total anti-S IgG antibody titers by ELISA. determined (FIG. 8B). Results are expressed as the geometric mean with error bars representing statistical significance and 95% confidence intervals calculated with the Kruskal-Wallis test with corrected Dunn's multiple comparisons. Dotted lines indicate the lower and upper limits of detection (ID ₉₀ to 40 and 5120, IgG ELISA to 100 and 1,638,400). ^*** p < 0.001, ^**** p < 0.0001.
9A-9B show the viral load of hamsters on day 3 or day 6 (dpi) after infection with SARS-CoV-2. Hamsters were euthanized at 3 or 6 dpi and lung tissue samples were collected for viral load determination by quantitative PCR of viral genomic RNA (FIG. 9A) and TCID ₅₀ assay for viral titers (FIG. 9B). Results are expressed as the geometric mean with error bars representing statistical significance and 95% confidence intervals calculated with the Kruskal-Wallis test with Dunn's multiple comparisons corrected. The dotted line represents the lower detection limit and limit (100). ^* p < 0.05, ^** p < 0.01.
Figure 10 shows the lung pathology scores of hamsters on day 3 or day 6 (dpi) after infection with SARS-CoV-2. Hamsters were euthanized at 3 or 6 dpi and lung tissue samples were collected for sectioning and staining. Histopathology sections were scored as described in Methods and the results tabulated. Results are expressed as the mean of lung pathology scores, with error bars representing statistical significance and standard error calculated by one-way ANOVA test with Tukey's multiple comparisons. ^**** p < 0.0001.
11 shows a summary of solicited adverse events in the Phase 1 clinical trial. Participants were asked to record requested local and systemic side effects on the participant's diary card for up to 7 days after each vaccination. Adverse events (AEs) requested were tabulated and graded as mild, moderate or severe.
12A-12C shows a summary of the humoral immune response in the Phase I clinical trial. Anti-spike IgG was measured by ELISA in the sera of participants vaccinated with 5, 15 or 25 μg of MVC-COV1901 (FIG. 12A), in a pseudovirus neutralization assay (FIG. 12B) or in a live virus neutralization assay (FIG. 12C). Neutralization titers were measured. Human convalescent serum (HCS) from 35 recovered COVID-19 patients was analyzed in the same assay for comparison and the NIBSC 20/130 standard was used as standard in the live virus neutralization assay (asterisk in Figure 12C). Bars represent geometric mean titers and error bars represent 95% confidence intervals.
Figure 13 shows a summary of cellular immune responses in phase 1 clinical trials. Cells were stimulated with the S1 peptide pool of peptides and incubated at 37° C. for 24-48 hours. Cells stimulated with CD3-2 mAb served as a positive control. ELISpot assay was used to detect IFN-γ (left panel) or IL-4 (right panel). The average of the spot-forming units (SFU) counted in triplicate peptide pool stimulation was calculated and normalized by subtracting the average of the negative control replicates (control media). Results were expressed as SFU per million PBMCs. Bars represent mean values and error bars represent standard deviations.
14 shows neutralization of SARS-CoV-2 pseudoviruses carrying wild-type or B.1.351 (beta) mutant spike proteins by antisera from rats vaccinated with enhanced S-2P. Rats were immunized three times at two-week intervals with the indicated amounts of supplemented S-2P. Antisera from 5 males were pooled into one sample and antisera from 5 females were pooled into another sample. This resulted in two pooled samples (N=2) for each dose group. Antisera were harvested 2 weeks after the second immunization (day 29) or 2 weeks after the third immunization (day 43), pooled as described above, Neutralization assays were performed with pseudoviruses expressing SARS-CoV-2 Wuhan wild-type or B.1.351 mutant spike proteins to determine ID ₅₀ and ID ₉₀ titers of neutralizing antibodies. Results are presented as bars representing geometric mean titers with symbols representing values for each sample.
15A-15F show neutralization of SARS-CoV-2 pseudoviruses carrying wild-type or mutant spike proteins by antisera from clinical trial subjects vaccinated with different doses of the MVC-COV1901 vaccine. Serum samples from the Phase 1 clinical trial of MVC-COV1901 subjects were collected 4 weeks after the second immunization (56 days after the first immunization). ID ₅₀ neutralizing titers for low dose (LD; FIG. 15A), medium dose (MD; FIG. 15B), high dose (HD; FIG. 15C), low dose (LD; FIG. 15D), medium dose (MD; FIG. 15E), high dose ( ID ₉₀ neutralization titers for HD; Figure 15F) and all dose groups were determined in a pseudovirus neutralization assay. Results were expressed as dots representing individual serum sample neutralization titers. A Kruskal-Wallis test with Dunn multiple comparisons corrected was performed and the statistical significance of the variants relative to the wild type is indicated above each column. ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to immunogenic compositions against SARS-CoV-2. The immunogenic composition comprises an antigenic recombinant protein and an aluminum-containing adjuvant and/or an adjuvant containing an unmethylated cytosine-phosphate-guanosine (CpG) motif. The antigenic recombinant protein comprises residues 14 to 1208 and the C-terminal T4 fibritin trimerization domain of the SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and "GSAS" substitutions at residues 682 to 685 .

In some embodiments, residues 14 to 1208 of the SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and "GSAS" substitutions at residues 682 to 685 are amino acid sequences of SEQ ID NO: 1 or at least relative to SEQ ID NO: 1 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of an amino acid sequence.

In some embodiments, the C-terminal T4 fibritin trimerization motif is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence.

In some embodiments, the antigenic recombinant protein comprises the amino acid sequence of SEQ ID NO: 5 or 6 or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% relative to SEQ ID NO: 5 or 6 do.

In some embodiments, the aluminum-containing adjuvant is aluminum hydroxide, aluminum oxyhydroxide, aluminum hydroxide gel, aluminum phosphate, aluminum phosphate gel, aluminum hydroxyphosphate, aluminum hydroxyphosphate sulfate, amorphous aluminum hydroxyphosphate sulfate, potassium aluminum sulfate , aluminum monostearate, or combinations thereof.

In some embodiments, a 0.5 ml dose of the immunogenic composition comprises between about 250 and about 500 μg Al ³⁺ or about 375 μg Al ³⁺ .

In some embodiments, the unmethylated CpG motif comprises a synthetic oligodeoxynucleotide (ODN) of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or a combination thereof.

In some embodiments, a 0.5 ml dose of the immunogenic composition comprises about 750 to about 3000 μg of oligonucleotides, or the immunogenic composition comprises about 750 μg, about 1500 μg or about 3000 μg of oligonucleotides.

The invention also relates to a method of inducing an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a subject in need thereof comprising administering to the subject an effective amount of an immunogenic composition of the invention. It is about.

The present invention also relates to a method for protecting a subject in need thereof from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection comprising administering to the subject an effective amount of an immunogenic composition of the present invention. .

The present invention also relates to a method for preventing a subject in need thereof from developing COVID-19 disease, comprising administering to a subject an effective amount of an immunogenic composition of the present invention.

The invention also relates to the use of an immunogenic composition of the invention for inducing an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a subject, said method comprising the immunogenic composition of the invention and administering an effective amount to a subject in need thereof.

In some embodiments, the immune response comprises the production of neutralizing antibodies to SARS-CoV-2 and a Th1 biased immune response.

The present invention also relates to the use of an immunogenic composition of the invention for protecting a subject from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the method comprising administering an effective amount of the immunogenic composition of the invention to and administering to a subject in need thereof.

The present invention also relates to the use of the immunogenic composition of the present invention for preventing a subject from suffering from COVID-19 disease, the method comprising administering to a subject in need thereof an effective amount of the immunogenic composition of the present invention. do.

In some embodiments, the immunogenic composition is administered by intramuscular injection.

The meaning of technical and scientific terms described herein can be clearly understood by those skilled in the art.

As used herein, the singular forms "a", "an" and "the" include plural references unless otherwise indicated. For example, “an” excipient includes one or more excipients.

As used herein, the phrase “comprising” is not limiting, i.e., embodiments may include additional elements. In contrast, the phrase “consisting of” is restrictive, ie, such embodiments do not include additional elements (except trace impurities). The phrase “consisting essentially of” is limiting in part, ie, such embodiments may include further elements that do not materially alter the basic characteristics of such embodiments.

As used interchangeably herein, the terms “polynucleotide” and “oligonucleotide” refer to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), modified oligonucleotides nucleotides and oligonucleosides or combinations thereof. Oligonucleotides can be composed of linear or circular shapes, or oligonucleotides can include both linear and circular segments. Oligonucleotides are generally polymers of nucleosides linked through phosphodiester linkages, although alternative linkages such as phosphorothioate esters can also be used in oligonucleotides. A nucleoside consists of a purine (adenine (A) or guanine (G) or a derivative thereof) or pyrimidine (thymine (T), cytosine (C) or uracil (U) or a derivative thereof) base bound to a sugar. The four nucleoside units (or bases) in DNA are called deoxyadenosine, deoxyguanosine, thymidine and deoxycytidine. Nucleotides are phosphate esters of nucleosides.

As used herein, the term "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)" refers to the coronavirus strain that causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a positive sense single-stranded RNA virus with a genome size of 29,903 bases. Each SARS-CoV-2 virion is between 50 and 200 nanometers in diameter and has four structural proteins known as the S (spike), E (envelope), M (membrane) and N (nucleocapsid) proteins. The N protein holds the RNA genome and the S, E and M proteins together make up the viral envelope. Spike protein is a protein involved in enabling the virus to attach to and fuse with the host cell membrane; Specifically, the S1 subunit catalyzes attachment and the S2 subunit catalyzes fusion.

As used herein, the term "immunogenic composition against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)" refers to a composition that stimulates or induces an immune response against SARS-CoV-2. Immune responses include, but are not limited to, production of neutralizing antibodies to SARS-CoV-2 and Th1 biased immune responses.

As used herein, the term "aluminum containing adjuvant" refers to an adjuvant comprising aluminum. In some embodiments, the aluminum containing adjuvant is aluminum hydroxide, aluminum oxyhydroxide, aluminum hydroxide gel, aluminum phosphate, aluminum phosphate gel, aluminum hydroxyphosphate, aluminum hydroxyphosphate sulfate, amorphous aluminum hydroxyphosphate sulfate, potassium aluminum sulfate, aluminum monostearate or combinations thereof. In some embodiments, the aluminum containing adjuvant is an aluminum containing adjuvant approved by the FDA for administration to humans. In some embodiments, the aluminum-containing adjuvant is an aluminum hydroxide adjuvant approved by the FDA for administration to humans. In some embodiments, the aluminum-containing adjuvant is an aluminum phosphate adjuvant approved by the FDA for administration to humans.

As used herein, the term "unmethylated cytosine-phosphate-guanosine (CpG) motif" refers to a CpG containing oligonucleotide in which C is unmethylated and which contributes to a measurable immune response as determined in vitro, in vivo and/or ex vivo. refers to In some embodiments, the CpG containing oligonucleotide contains a palindromic hexamer following the general formula 5'-purine-purine-CG-pyrimidine-pyrimidine-3'. In some preferred embodiments, the unmethylated cytosine-phosphate-guanosine (CpG) motif has an oligonucleotide of SEQ ID NO: 8 (5'-TGACTGTGAACGTTCGAGATGA-3') in which the C of CG is unmethylated. In some embodiments, the CpG containing oligonucleotide contains a TCG in which the C is unmethylated and is 8 to 100 nucleotides, preferably 8 to 50 nucleotides, or preferably 8 to 25 nucleotides in length. In some preferred embodiments, the unmethylated cytosine-phosphate-guanosine (CpG) motif has an oligonucleotide of SEQ ID NO: 9 (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′) in which the C of TCG is unmethylated. Examples of unmethylated cytosine-phosphate-guanosine (CpG) motifs further include 5'-GGTGCATCGATGCAGGGG GG-3' (SEQ ID NO: 10), 5'-TCCATGGACGTTCCTGAGCGTT-3' (SEQ ID NO: 11), 5'-TCGTCGTTCGAACGACGTTGAT-3 '(SEQ ID NO: 12), and 5'-TCGTCGACGATCGGC GCGCGCCG-3' (SEQ ID NO: 13). The CpG-containing oligonucleotides described herein are pharmaceutically acceptable salt forms thereof unless otherwise indicated. In one preferred embodiment, the CpG containing oligonucleotide is in sodium salt form.

An “effective amount” or “sufficient amount” of a substance is an amount sufficient to achieve beneficial or desired results, including clinical results, and thus “effective amount” depends on the context in which it is applied. Regarding administration of the immunogenic composition, an effective amount contains an adjuvant and SARS-CoV-2 S-2P recombinant protein sufficient to induce an immune response. An effective amount can be administered in one or more doses.

The terms "individual" and "subject" refer to a mammal. "Mammal" includes, but is not limited to, humans, non-human primates (eg monkeys), farm animals, sport animals, rodents (eg mice and rats) and pets (eg dogs and cats).

The term "dose" as used herein with reference to an immunogenic composition refers to a measured portion of an immunogenic composition taken (administered or received) by a subject at any one time.

The terms "isolated" and "purified" as used herein refer to material that has been removed from at least one naturally associated component (eg, removed from its original environment). The term "isolated" when used in reference to a recombinant protein refers to a protein that has been removed from the culture medium of the host cell that produced the protein.

“Stimulation” of a response or parameter refers to eliciting and/or enhancing (e.g., TLR compared to the absence of a TLR agonist) that response or parameter when compared to other identical conditions except for the parameter of interest, or alternatively when compared to other conditions. increase in TLR signal in the presence of an agonist). For example, "stimulation" of an immune response means an increase in the response. Depending on the parameter measured, the increase can be from a factor of 5 to more than 500 times or from 5, 10, 50 or 100 times to 500, 1,000, 5,000 or 10,000 times.

As used herein, the term “immunization” refers to the process of increasing a mammalian subject's response to an antigen to enhance its ability to resist or overcome infection.

As used herein, the term “vaccination” refers to the introduction of a vaccine into the body of a mammalian subject.

“Adjuvant” refers to a substance that, when added to a composition comprising an antigen, non-specifically enhances or enhances the immune response to an antigen in a recipient upon exposure.

The invention is further illustrated by the following examples, which are provided for purposes of demonstration and not limitation. Those skilled in the art will recognize in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example

Example 1 Preparation of an immunogenic composition against SARS-CoV-2

Proline substitution at residues 986 and 987, "GSAS" substitution at the furin cleavage site (residues 682 to 685) (SEQ ID NO: 14) and C-terminal T4 fibritin trimerization domain (SEQ ID NO: 2), HRV3C protease cleavage site (SEQ ID NO: 14) No. 3), a plasmid having a polynucleotide encoding residues 1 to 1208 of the SARS-CoV-2 S protein with an 8x His Tag and a Twin-Strep Tag (SEQ ID NO: 4) (Wuhan-Hu-1 strain; GenBank: MN908947 ) were transfected into ExpiCHO-S cells (Thermo Fisher Scientific, Waltham, MA, USA).

ttingen, Germany)를 사용하여 상청액으로부터 단백질을 정제하였다. HRV3C 프로테아제(1% wt/wt)를 단백질에 첨가하고 반응물을 4℃에서 밤새 인큐베이션하였다. 소화된 단백질을 Superose 6 16/70 컬럼(GE Healthcare Biosciences, Chicago, IL, USA)을 사용하여 추가로 정제하였다. 이후 정제된 SARS-CoV-2 S-2P 재조합 단백질(서열 번호 5 또는 6)을 비메틸화 CpG 모티프(CpG 1018, 서열 번호 8) 및/또는 수산화알루미늄(Al(OH)₃) 또는 인산알루미늄(AlPO₄)과 같은 알루미늄 함유 아주반트와 함께 SARS-CoV-2에 대한 면역원성 조성물로서 제형화하였다.After 6 days, cell cultures were harvested and cultured on Strep-Tactin resin (IBA Lifesciences, G.

ttingen, Germany) was used to purify the protein from the supernatant. HRV3C protease (1% wt/wt) was added to the protein and the reaction was incubated overnight at 4°C. The digested protein was further purified using a Superose 6 16/70 column (GE Healthcare Biosciences, Chicago, IL, USA). Thereafter, the purified SARS-CoV-2 S-2P recombinant protein (SEQ ID NO: 5 or 6) contains an unmethylated CpG motif (CpG 1018, SEQ ID NO: 8) and/or aluminum hydroxide (Al(OH) ₃ ) or aluminum phosphate (AlPO ₄ ) as an immunogenic composition against SARS-CoV-2 with an aluminum containing adjuvant.

Example 2 Immunogenicity of Immunogenic Compositions Against SARS-CoV-2 in Mice

This example provides a description of a preclinical study to evaluate the immunogenicity of the immunogenic composition against SARS-CoV-2 from Example 1 in mice.

A. Preliminary test - 1-SARS-CoV-2 S-2P recombinant protein formulated with aluminum phosphate

Materials and Methods

mouse immunization. 6-8 week old BALB/c mice (The National Laboratory Animal Center, Taiwan) (N=5/group) were vaccinated with SARS-CoV-2 S-2P recombinant protein at weeks 0 and 3. SARS-CoV-2 S-2P recombinant protein diluted in PBS (final concentration 1 μg or 10 μg/mL) was mixed with aluminum phosphate (final concentration 0.5 mg aluminum/mL). Mice were inoculated intramuscularly with 100 μL (50 μL to each hind limb). Two weeks after the final immunization, serum was collected for antibody response determination.

pseudovirus production. A cDNA encoding the spike protein (SEQ ID NO: 7) of the Wuhan-Hu-1 strain was synthesized using the QuikChange XL kit (Stratagene, San Diego, CA, USA) and then inserted into the CMV/R plasmid. Sequencing was used to confirm the CMV/R-SARS-CoV-2 spike plasmid. HEK293T cells were obtained from ATCC and cultured in DMEM supplemented with 10% FBS, 2 mM glutamine and 1% penicillin/streptomycin at 37° C. and 5% CO ₂ . To produce the SARS-CoV-2 pseudovirus, the CMV/R-SARS-CoV-2 spike plasmid was mixed with the packaging plasmid pCMVDR8.2 and the transduction plasmid using Fugene 6 Transfection Reagent (Promega, Madison, WI, USA). HEK293T cells were cotransfected with pHR CMV-Luc. 72 hours after transfection, the supernatant was collected, filtered and frozen at -80°C.

Pseudovirus Infectivity and Neutralization Assay. Huh7.5 cells (RRID: CVCL_7927) were cultured at 37° C. and 5% CO ₂ in DMEM supplemented with 10% FBS, 2 mM glutamine and 1% penicillin/streptomycin. Pseudovirus infectivity was assessed in Huh7.5 cells plated overnight in 96-well black/white isoplates (PerkinElmer, Waltham, MA, USA). Two-fold serial dilutions of pseudovirus were added in triplicate to resting Huh7.5 cells. After 2 hours incubation, fresh medium was added. Cells were lysed at 72 hours and luciferase substrate (Promega) was added. Luciferase activity was measured as relative luciferase units (RLU) at 570 nm on a SpectramaxL (Molecular Devices, San Jose, CA, USA). For neutralization experiments, serial dilutions (1:40, 4-fold, 8-fold dilutions) of mouse serum were mixed with various pseudovirus strains previously titrated to a target of 50,000 RLU. From the RLU readings, triplicate averages were taken at each dilution to generate a sigmoidal curve; A 50% neutralization titer (IC ₅₀ ) was calculated as uninfected cells were considered 100% neutral and cells transduced only with virus were considered 0% neutral.

result

The neutralization assay results are shown in FIG. 1 . SARS-CoV-2 S-2P recombinant proteins formulated with aluminum phosphate (0.1 μg/mouse and 1 μg/mouse) induced greater neutralization than recombinant proteins (0.1 μg/mouse and 1 μg/mouse) alone. These data demonstrate that aluminum phosphate significantly increases the immunogenicity of the SARS-CoV-2 S-2P recombinant protein as an antigen in a vaccine against coronavirus disease (COVID-19).

B. Preliminary test - 2-SARS-CoV-2 S-2P recombinant protein formulated with a combination of CpG and aluminum hydroxide

Materials and Methods

mouse immunization. 6-8 week old BALB/c mice (The National Laboratory Animal Center, Taiwan) (N=6/group) were vaccinated with SARS-CoV-2 S-2P recombinant protein at weeks 0 and 3. SARS-CoV-2 S-2P recombinant protein diluted in PBS (final concentration 10 μg or 50 μg/mL) was mixed with CpG 1018 (SEQ ID NO: 8) (to a final concentration of 0.1 mg/mL), aluminum hydroxide (0.5 mg aluminum /mL), or a combination of CpG 1018 (to a final concentration of 0.1 mg/mL) and aluminum hydroxide (to a final concentration of 0.5 mg aluminum/mL), respectively. Mice were inoculated intramuscularly with 100 μL (50 μL to each hind limb). Two weeks after the final immunization, serum was collected for antibody response measurement.

Pseudovirus production, pseudovirus infectivity and neutralization assays. The method is described in Section A.

result

The results of the neutralization experiment are shown in FIG. 2 . SARS-CoV-2 S-2P recombinant protein formulated with a combination of CpG and aluminum hydroxide induced the highest neutralizing activity at both low (1 μg/mouse) and high dose (5 μg/mouse) doses. In addition, the SARS-CoV-2 S-2P recombinant protein formulated with aluminum hydroxide alone (1 μg/mouse) produced greater neutralization than the recombinant proteins formulated with CpG alone (1 μg/mouse and 5 μg/mouse). induced. Recombinant proteins formulated with CpG alone showed neutralizing activity in a dose-dependent manner. These data demonstrate that CpG and/or aluminum hydroxide significantly increase the immunogenicity of the SARS-CoV-2 S-2P recombinant protein as an antigen in a vaccine against coronavirus disease (COVID-19).

C. Development of a Stable Pre-fusion SARS-CoV-2 Spike Protein Antigen Adjuvanted

Materials and Methods

Pseudovirus production and titration. To produce the SARS-CoV-2 pseudovirus, a plasmid expressing the full-length wild-type Wuhan-Hu-1 strain SARS-CoV-2 spike protein (SEQ ID NO: 7) was transfected using TransIT-LT1 transfection reagent (Mirus Bio). HEK293T cells were co-transfected with the packaging and reporter plasmids pCMVΔ8.91 and pLAS2w.FLuc.Ppuro (RNAi Core, Academia Sinica). The D614G variant was generated by changing the nucleotide at position 23403 (Wuhan-Hu-1 reference strain) from A to G using site-directed mutagenesis. Mock virus was produced by omitting the p2019-nCoV spike (WT). 72 hours after transfection, the supernatant was collected, filtered and frozen at -80°C. A cell viability assay was used to estimate the transduction unit (TU) of the SARS-CoV-2 pseudotyped lentivirus in response to limiting dilution of the lentivirus. Briefly, HEK-293 T cells stably expressing the human ACE2 gene were plated in 96-well plates 1 day prior to lentiviral transduction. For pseudovirus titration, different amounts of pseudovirus were added to the culture medium containing polybrene. Spin infections were performed at 1100xg in 96-well plates for 30 min at 37°C. After incubating the cells at 37° C. for 16 hours, the culture medium containing virus and polybrene was removed and replaced with fresh complete DMEM containing 2.5 μg/ml puromycin. After treatment with puromycin for 48 hours, the culture medium was removed and cell viability was detected using 10% AlarmaBlue reagent according to the manufacturer's instructions. The viability of uninfected cells (no puromycin treatment) was set at 100%. Viable cells versus diluted viral dose were plotted to determine viral titer (transduction units).

Pseudovirus-based neutralization assay. HEK293-hAce2 cells (2×10 ⁴ cells/well) were seeded in 96-well white isoplates and incubated overnight. Serum was heated at 56°C for 30 min to inactivate complement and diluted in MEM supplemented with 2% FBS at an initial dilution factor of 20 followed by 2-fold serial dilutions (a total of 8 steps up to a final dilution of 1:5120). for the dilution step). The diluted serum was mixed with an equal volume of pseudovirus (1000 TU) and incubated for 1 hour at 37° C. before adding to the plate with cells. After 1 hour incubation, the culture medium was replaced with 50 μL of fresh medium. The next day, the culture medium was replaced with 100 μL of fresh medium. Cells were lysed 72 hours after infection and relative luciferase units (RLU) were measured. Luciferase activity was detected with Tecan i-control (Infinite 500). 50% and 90% inhibition dilution titers (ID ₅₀ and ID ₉₀ ) were calculated with uninfected cells considered 100% neutral and cells transduced only with virus as 0% neutral. Both the reciprocal ID ₅₀ and ID ₉₀ geometric mean titer (GMT) were determined because ID ₉₀ titers are useful when ID ₅₀ titer levels consistently saturate at the upper limit of detection.

Neutralizing wild-type SARS-CoV-2. A neutralization assay using SARS-CoV-2 virus was performed as previously reported (Huang et al., J. Clin. Microbiol. 58(8): e01068-e1120, 2020). Vero E6 cells (2.5×10 ⁴ cells/well) were seeded in 96-well plates and incubated overnight. Serum was heated at 56°C for 30 minutes to inactivate complement and diluted with an initial dilution of 20 in serum-free MEM followed by further 2-fold serial dilutions for a total of 11 dilution steps to a final dilution of 1:40,960. made. Diluted serum was mixed with equal volumes of SARS-CoV-2 virus and 100 TCID _50/50 Mixed in μL (hCoV-19/Taiwan/CGMH-CGU-01/2020, GenBank accession MT192759) and incubated at 37° C. for 2 hours. The serum-virus mixture was then added to a 96-well plate with Vero E6 cells and incubated in MEM with 2% FBS for 5 days at 37°C. After incubation, cells were fixed by adding 4% formalin to each well for 10 minutes and stained with 0.1% crystal violet for visualization. Result to ID ₅₀ The log 50% endpoint for the titer and the log 90% endpoint for the ID ₉₀ titer were calculated by the Reed-Muench method.

Immunization of mice. Female BALB/c and C57BL/6 mice were maintained at National Laboratory Animal Center, Academia Sinica, Taiwan and BioLASCO Taiwan Co., Ltd. Ltd. For antigen formulation, SARS-CoV-2 S-2P protein was mixed with equal volumes of CpG 1018 (SEQ ID NO: 8), aluminum hydroxide, PBS or CpG 1018 + aluminum hydroxide. Mice aged 6 to 9 weeks were injected twice (per mouse) at 3-week intervals as previously described (Pallesen et al., Proc. Natl. Acad. Sci. USA, 114(35): E7348-E7357, 2017). Intramuscular injection of 50 μL into each of the left and right quadriceps muscles) was immunized. Total serum anti-S IgG and anti-RBD IgG titers were measured by E. coli-expressed fragments of the S protein containing the S-2P antigen and RBD region, respectively. Detection was done by direct ELISA using custom coated 96-well plates.

Cytokine analysis. Two weeks after the second injection, mice were euthanized and splenocytes were isolated and stimulated with S-2P protein (2 μg/well) as previously described (Lu et al. Immunology, 130(2): 254-261, 2010). did For the detection of IFN-γ, IL-2, IL-4 and IL-5, culture supernatants were harvested from 96-well microplates and mouse IFN-γ Quantikine ELISA kit, mouse IL-2 Quantikine ELISA kit, mouse IL-4 Cytokine levels were analyzed by ELISA using the Quantikine ELISA kit and the Mouse IL-5 Quantikine ELISA kit (R&D System). OD450 values were read with Multiskan GO (Thermo Fisher Scientific).

Dose range finding study for single and repeated dose intramuscular injection (IM) in Sprague Dawley (SD) rats. Crl:CD Sprague-Dawley (SD) rats were prepared by BioLASCO Taiwan Co., Ltd. Ltd. Animal studies were conducted at the Testing Facility for Biological Safety of TFBS Bioscience Inc., Taiwan. SD rats aged 6 to 8 weeks were immunized with 5 μg, 25 μg or 50 μg of S-2P supplemented with 1500 μg CpG 1018 alone or 750 μg CpG 1018 combined with 375 μg aluminum hydroxide. Test article or vehicle control was administered intramuscularly (0.25 mL/site, two quadriceps) to each rat on day 1 (single dose study) and day 15 (repeat dose study). The observation period was 14 days (single dose study) and 28 days (repeat dose study). Parameters assessed included clinical signs, local irritation test, moribund state/mortality, body temperature, body weight, and food intake over the lifespan. Blood samples were taken for blood analysis including coagulation tests and serum chemistry. All animals were euthanized and necropsied for gross lesion examination, organ weights and histopathological evaluation of injection sites and lungs.

statistical analysis. For neutralization assays, geometric mean titers are shown as bar heights and 95% confidence intervals are shown as error bars. For cytokine and rat data, bar or symbol height represents SD+mean indicated by error bars. Dotted lines indicate the upper and lower limits of detection. The analysis package of Prism 6.01 (GraphPad) was used for statistical analysis. Data were compared to different adjuvants at the same S-2P dose level or to the same adjuvant system with different antigen doses. The Kruskal-Wallis test with Dunn's multiple comparisons corrected was used for nonparametric tests between more than two experimental groups. The Mann-Whitney U-test was used to compare the two experimental groups. Spearman's rank correlation coefficient was used for the correlation between antibody titer and neutralizing titer. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

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Induction of potent neutralizing antibodies by S-2P supplemented with CpG 1018 and aluminum hydroxide. To enable the establishment of stable clones for clinical research and commercial production, the ExpiCHO system was used as the expression system for the S-2P antigen. The S-2P protein and its structure produced in CHO cells showed typical spike trimers similar to the 293-expressed SARS-CoV-2 S protein under cryo-EM (Wrapp et al., Science, 367(6483): 1260 -1263, 2020), suggesting that CHO cells are feasible for S-2P production. Next, the possibility of clinical use of Th1-biased CpG 1018 was investigated. Because Alum has been characterized to enhance the efficacy of CpG adjuvants when used in combination while retaining the properties of inducing a Th1 response (Thomas et al., Hum. Vaccin., 5(2): 79-84). , 2009), and aluminum hydroxide (hereinafter abbreviated as Alum) were tested together with CpG 1018. Pseudovirus neutralization assay was performed with serum collected 3 weeks after the first injection or 2 weeks after the second injection. Neutralizing activity was already observed when mice were immunized with 1 μg and 5 μg of S-2P containing CpG 1018 and alum at 3 weeks after the primary injection. At 2 weeks after the second injection, immunization with 1 μg S-2P supplemented with CpG 1018, alum and both CpG 1018 and alum, respectively, resulted in mutually inhibitory dilution 50 (ID ₅₀ ) GMTs of 245, 3109 and 5120 ( FIG. 3 ). ). A similar trend was observed in both BALB/c (o Figure 3) and C57BL/6 mice at 5 μg of S-2P.

The serum from these mice was then examined for the amount of anti-S IgG. CpG 1018 in combination with alum produced significantly higher titers of anti-S IgG compared to CpG 1018 alone (FIG. 4). To confirm the activity of the antibody against the critical receptor-binding domain (RBD) of the S protein, the anti-RBD IgG was tested in immune sera and was similar to that of the anti-S IgG, and S in combination with both CpG 1018 and Alum. -2P induced the highest amount of IgG titer. There was a moderate correlation between anti-S IgG and anti-RBD IgG titers as evidenced by Spearman's rank correlation coefficient of 0.6486. Immune sera were further tested for their ability to neutralize against wild-type SARS-CoV-2 in a neutralization assay. S-2P was less potent than pseudovirus, but was able to inhibit SARS-CoV-2 at a concentration of 1 μg (FIG. 3, FIG. 5). The reverse ID ₅₀ GMT of 1 μg S-2P in the presence of CpG 1018, alum, and both CpG 1018 and alum were approximately 60, 250 and 1500, respectively ( FIG. 5 ). A pseudovirus carrying the now dominant D614G variant spike was also generated, and neutralizing antibodies from mice immunized with S-2P containing CpG 1018 and alum were directed against pseudovirus carrying both wild-type D614 and mutant D614G versions of the spike protein. was effective against (Fig. 6). Neutralizing titers and total anti-S IgG titers of wild-type virus and pseudovirus were both found to be highly correlated with Spearman's rank correlation coefficient greater than 0.8.

CpG 1018 induced Th1 immunity. To determine whether CpG 1018 can induce a Th1 response in a vaccine-adjuvant system, the Th1 and Th2 responses in splenocytes from mice immunized with S-2P together with alum, CpG 1018 or a combination of the two were examined. Involved cytokines were measured. As expected, S-2P supplemented with alum induced limited amounts of IFN-γ and IL-2, cytokines representative of the Th1 response. In contrast, significant increases in IFN-γ and IL-2 were most strongly detected at high antigen doses and CpG 1018 and Alum. In the case of the Th2 response, the levels of IL-4, IL-5 and IL-6 increased in the presence of alum and S-2P, whereas the levels of IL-5 and IL-6 were suppressed when CpG 1018 was added to alum. The IFN-γ/IL-4, IFN-γ/IL-5 and IFN-γ/IL-6 ratios show a strong Th1-biased response and are about 36-fold, respectively, in the presence of CpG 1018 and S-2P in combination with alum; 130-fold and 2-fold increase (FIG. 7). These results suggest that the effect of CpG 1018 is superior to alum in directing cell-mediated responses to Th1 responses while maintaining high antibody levels.

S-2P did not cause systemic side effects in rats. To elucidate the safety and potential toxicity of the vaccine candidate, single-dose and repeat-dose studies were conducted with 5 μg, 25 μg, or 50 μg of S-2P supplemented with 1500 μg CpG 1018 or 750 μg CpG 1018 combined with 375 μg alum. was administered to SD rats for No differences in mortality, abnormalities in clinical signs, weight changes, body temperature or food intake that could be attributed to S-2P (with or without adjuvant) were observed in either sex with a single dose administration. Elevated body temperature was found in both sexes in the single-dose study and the repeat-dose study at 4 or 24 hours post-administration; However, these temperature changes were moderate and recovered after 48 hours in both sexes of all treatment groups including the control group (PBS). No gross lesions were observed in the trachea of most male and female rats at single and double dose administration, except for one male rat, which was considered unrelated to vaccine treatment. In conclusion, as a result of intramuscular administration of CpG 1018 or S-2P protein using CpG 1018 and alum as an adjuvant to SD rats once or twice, no systemic side effects occurred.

From the results, two injections of a subunit vaccine consisting of CpG 1018 and alum-enhanced pre-fusion spike protein (S-2P) in mice killed both wild-type SARS-CoV-2 and pseudoviruses expressing wild-type and D614G mutant spike proteins. It has been shown to be effective in inducing strong neutralizing activity against The combination of S-2P with CpG 1018 and alum induced a Th1-dominant immune response with high neutralizing antibody levels in mice and no significant side effects in rats. Therefore, in this example, we found that S-2P in combination with the adjuvant CpG 1018 in combination with alum produced potent Th1-biased immunity to prevent wild-type virus infection while maintaining high antibody levels indicating cross-neutralization of the mutant virus. It has been proven to induce a reaction. Thus, the immunogenic composition against SARS-CoV-2 of the present invention serves as an ideal vaccine candidate to alleviate the burden of the global COVID-19 pandemic.

Example 3 Protection from SARS-CoV-2 challenge by an immunogenic composition against SARS-CoV-2 in hamsters

Materials and Methods

Pseudovirus-based neutralization assay and IgG ELISA. A lentivirus expressing the Wuhan-Hu-1 strain SARS-CoV-2 spike protein was constructed and a neutralization assay was performed as described in Example 2. Briefly, HEK293-hACE2 cells were seeded in 96-well white isoplates and incubated overnight. Serum from vaccinated and unvaccinated hamsters was heat inactivated, diluted in MEM supplemented with 2% FBS at an initial dilution factor of 20, followed by two-fold serial dilutions for a total of eight dilution steps. through a final dilution ratio of 1:5120. The diluted serum was mixed with an equal volume of pseudovirus (1,000 TU) and incubated for 1 hour at 37°C before adding to the plate with cells. Cells were lysed 72 hours after infection and relative luciferase units (RLU) were measured. 50% and 90% inhibition dilution titers (ID ₅₀ and ID ₉₀ ) were calculated with uninfected cells set to 100% neutralization and virus-only transduced cells set to 0% neutralization. Total serum anti-S IgG titers were detected by direct ELISA using custom 96-well plates coated with S-2P antigen.

Hamster immunization and challenge. Female golden Syrian hamsters, 6-9 weeks of age at the start of the study, were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Hamsters from different litters were randomly divided into 4 groups (n=10 in each group): hamsters were assigned vehicle control (PBS), S-2P protein supplemented with 150 μg CpG 1018 and 75 μg aluminum hydroxide (alum). Vaccination was given by intramuscular injection of 1 or 5 μg, or adjuvant alone, twice at 3-week intervals. Hamsters were bled via the mandibular vein 2 weeks after the second immunization to confirm the presence of neutralizing antibodies. Four weeks after the second immunization, hamsters were challenged intranasally with 1×10 ⁴ PFU of SARS-CoV-2 TCDC#4 (hCoV-19/Taiwan/4/2020, GISAID accession number EPI_ISL_411927) in a volume of 100 μL per hamster. . Hamsters were euthanized in 2 cohorts on days 3 and 6 post challenge for necropsy and tissue sampling. For each hamster, body weight and survival rate were recorded daily after infection. Hamsters were euthanized with carbon dioxide on days 3 and 6 post challenge. The right lung was collected for viral load determination (RNA titer and TCID ₅₀ assay). For histopathological examination, the left lung was fixed in 4% paraformaldehyde.

Viral titer quantification in lung tissue by cell culture infection assay (TCID ₅₀ ). Hamster middle-, inferior- and posterior vena cava lobes were homogenized in 600 μl of DMEM containing 2% FBS and 1% penicillin/streptomycin using a homogenizer. Tissue homogenates were centrifuged at 15,000 rpm for 5 minutes and supernatants were collected for live virus titration. Briefly, 10-fold serial dilutions of each sample were added in quadruplicate to Vero E6 cell monolayers and incubated for 4 days. Cells were then fixed with 10% formaldehyde and stained with 0.5% crystal violet for 20 minutes. Plates were washed with tap water and scored for infection. The 50% tissue culture infective dose (TCID ₅₀ )/mL was calculated by the Reed-Muench method (Reed and Muench, American Journal of Epidemiology, 27(3): 493-497, 1938).

Real-time RT-PCR for SARS-CoV-2 RNA quantification. To measure the RNA level of SARS-CoV-2, specific primers targeting regions 26,141 to 26,253 of the envelope (E) gene of the SARS-CoV-2 genome were used in the TaqMan real-time RT-PCR method (Corman et al. al., Eurosurveillance. 25(3): 2000045, 2020). In addition to probe E-Sarbeco-P1 5'-FAM-ACACTAGCCATCCTTACTGCGCTCG-BBQ-3' (SEQ ID NO: 17), forward primer E-Sarbeco-F1 5'-ACAGGTACGTTAATAGTTAATAGCGT-3' (SEQ ID NO: 15) and reverse primer E-Sarbeco- R2 5'-ATATTGCAGCAGTACGCACACA-3' (SEQ ID NO: 16) was used. A total of 30 μL of RNA solution was collected from each lung sample using the RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions. 5 μL of RNA sample was added to a total of 25 μL mixture of Superscript III One-Step RT-PCR System with Platinum Taq Polymerase (Thermo Fisher Scientific, USA). The final reaction mixture contained 400 nM forward and reverse primers, 200 nM probe, 1.6 mM deoxyribonucleoside triphosphate (dNTP), 4 mM magnesium sulfate, 50 nM ROX reference dye and 1 μL of enzyme mixture. Cycling conditions were performed using a one-step PCR protocol: 45 amplification cycles of 10 minutes at 55°C, followed by 3 minutes at 94°C, and 15 seconds at 94°C and 30 minutes at 58°C for first-strand cDNA synthesis. . Data were collected and calculated with an Applied Biosystems 7500 real-time PCR system (Thermo Fisher Scientific, USA). A synthetic 113-bp oligonucleotide fragment was used as a qPCR standard to estimate the copy number of the viral genome. Oligonucleotides were prepared by Genomics BioSci and Tech Co. Ltd. (Taipei, Taiwan).

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Hamster as a model for SARS-CoV-2 viral challenge. To develop a SARS-CoV-2 virus challenge model for the S2-P vaccine in hamsters, an initial study was conducted to determine the optimal virus dose for challenge experiments. Inoculate unvaccinated hamsters with 10 ³ , 10 ⁴ or 10 ⁵ PFU of SARS-CoV-2 They were euthanized on day 3 or 6 post infection for tissue sampling. After infection with 10 ³ to 10 ⁵ PFU of SARS-CoV-2, the hamsters showed a dose-dependent weight loss. Hamsters infected with 10 ³ PFU gained weight, whereas hamsters infected with 10 ⁴ and 10 ⁵ PFU suffered progressively severe weight loss at 6 days post infection (dpi). However, there was no significant difference between viral genomic RNA levels and viral titers measured in hamsters infected with 10 ³ to 10 ⁵ PFU of SARS-CoV-2 at 3 and 6 dpi. All doses of virus increased lung pathology even at 10 ³ PFU where animals did not undergo weight loss. There were also no dose-dependent effects of viral inoculation on lung pathology scores and lung viral load. Therefore, 10 ⁴ PFU of virus was used for virus palinge studies as it provides an appropriate balance between viral titers for inoculation and clinical signs.

Administration of S-2P supplemented with CpG 1018 and aluminum hydroxide to hamsters induced high levels of neutralizing antibodies. Hamsters were treated with either low-dose (LD) or high-dose (HD) S-2P (S-2P+CpG 1018+Alum) in combination with vehicle control (PBS alone), adjuvant alone, CpG 1018 and aluminum hydroxide 2 times every 21 days. It was divided into 4 groups that received twice immunization. No difference in body weight change was observed between the four groups after vaccination. 14 days after the second immunization, high levels of neutralizing antibody titers were found in both the LD and HD groups with 90% inhibitory dilution (ID ₉₀ ) geometric mean titers (GMTs) of 2,226 and 1,783, respectively (FIG. 8A). Anti-S IgG antibody levels were high enough for several individual samples to reach the upper limit of detection, with GMTs of 1,492,959 and 1,198,315 for the LD and HD groups, respectively (FIG. 8B). In general, S-2P+CpG 1018+alum induced strong levels of immunogenicity in hamsters even at low doses.

S-2P supplemented with adjuvant protected hamsters from clinical signs and viral load after SARS-CoV-2 challenge. Four weeks after the second immunization, hamsters were challenged with 10 ⁴ PFU of SARS-CoV-2 virus and body weights were followed up to 3 or 6 days post infection (dpi). Groups of animals were sacrificed at 3 or 6 dpi for viral load and histopathology analysis. The LD and HD vaccinated groups did not show any weight loss until up to 3 or 6 days after virus challenge, instead increasing their average body weight by 5 g and 3.8 g, respectively, at 6 dpi. The protective effect was most pronounced at 6 dpi in both vaccinated groups, whereas the vehicle control and adjuvant only groups suffered significant weight loss. Viral RNA and TCID ₅₀ analysis Pulmonary viral load measured by vaccinated hamsters showed that both viral RNA and viral titer significantly decreased at 3 dpi and fell below the lower limit of detection at 6 dpi (FIGS. 9A-9B). It is noticeable that the viral load, especially the viral titer measured by TCID ₅₀ , dropped significantly at 6 dpi in the control and adjuvant only groups due to the hamster's natural immune response (Figs. 9A to 9B). Lung sections were analyzed and pathology scores tabulated (FIG. 10). There was no difference between control and experimental groups at 3 dpi; However, at 6 dpi, the vehicle control and adjuvant only groups had significantly increased lung pathology including extensive immune cell infiltration and diffuse alveolar damage compared to the HD antigen/adjuvant immunization group (FIG. 10). These results indicate that S-2P+CpG 1018+alum inhibits viral load in the lung and induces a strong immune response that can prevent weight loss and lung pathology in infected hamsters.

In the control group at 6 dpi, all hamsters in the S-2P+CpG 1018+alum-immunized group, in contrast to virus-induced diffuse alveolar damage in the hamster lungs (graded moderate to severe, mean score 4.09 in vehicle and adjuvant controls) It was protected by significantly reduced lung pathology (generally minimal to mild grade, mean score 1.72 in LD and HD groups). The significance of this study lies in demonstrating safety as well as efficacy in vivo. Viral challenge studies have allowed us to assess the disease-enhancing risk of vaccine candidates. The histopathological scores of the immunized groups did not differ from non-challenged animals, indicating no vaccine-enhanced pathology. The findings of this Example provide more data to support the progress of vaccine candidates in clinical development.

Example 4 Safety and Immunogenicity of CpG-Enhanced S-2P Subunit Vaccine "MVC-COV1901" in Humans

This example provides a Phase I study conducted in healthy human subjects to evaluate the safety and immunogenicity of a SARS-CoV-2 subunit vaccine (ie, the immunogenic composition of the present invention). The SARS-CoV-2 subunit vaccine, referred to herein as "S-2P+CpG 1018+Alum" or "MVC-COV1901", is described in more detail in Example 1.

vaccine. MVC-COV1901 was formulated with three different doses of SARS-CoV-2 Spike (S) protein with CpG 1018 and aluminum hydroxide as adjuvants. Each MVC-COV1901 vaccine contained 750 μg of CpG 1018 and 5, 15 or 25 μg of S-2P supplemented with 375 μg (Al equivalent weight) of aluminum hydroxide and was administered as a single 0.5 ml intramuscular (IM) injection. .

Participant. copy The study aimed to enroll 45 subjects. Eligible participants were healthy adults between the ages of 20 and 49 years. Eligibility was determined based on medical history, physical examination, laboratory tests, and clinical judgment of the investigator. Exclusion criteria included history of known potential exposure to SARS CoV-1 or 2 virus, other COVID-19 vaccinations, impaired immune function, history of autoimmune disease, unresolved HIV, HBV or HCV infection, abnormal autoantibody test, first Fever or acute illness within 2 days of dosing and acute respiratory illness within 14 days of first dosing were included.

study design. copy The study is a phase 1 prospective, unblinded, single-site study to evaluate the safety and immunogenicity of the SARS-CoV-2 vaccine MVC-COV1901. This study was a dose-escalation study in three separate groups of participants aged 20 to 49 years. Each sub-stage consisted of 15 participants. The three different dose levels used were 5, 15 and 25 μg of S-2P protein for cohorts 1a, 1b and 1c, respectively. The vaccination schedule consisted of two doses administered by IM injection into the deltoid muscle of the non-dominant arm on days 1 and 29, 28 days apart.

Cohort 1a: Four Sentinel participants were recruited to receive the vaccine with 5 μg of S-2P to evaluate preliminary safety data of the vaccine. If no grade 3 adverse events (AEs) or serious adverse events (SAEs) occurred within 7 days of the first administration in 4 Sentinel participants, proceed with administration of the remaining participants in phases 1a and 1b.

Cohort 1b: Another 4 Sentinel participants enrolled to receive vaccination with 15 μg of S-2P. If ≥ Grade 3 AE or SAE does not occur within 7 days of the first dose in 4 Sentinel participants, proceed with the remaining participants in Phases 1b and 1c.

Cohort 1c: Another 4 Sentinel participants may be enrolled to receive the vaccine with 25 μg S-2P. If ≥ Grade 3 AEs or SAEs do not occur within 7 days of the first dose in 4 Sentinel participants, dosing of the remaining participants in Phase 1c may proceed.

Vital signs and electrocardiogram (ECG) were performed before and after vaccination. To identify immediate AEs, participants were observed for at least 30 minutes after each dose and local (pain, erythema, swelling/induration) and systemic (fever, myalgia, malaise/induration) as requested on participant's diary card for up to 7 days after each dose. fatigue, nausea/vomiting, diarrhea) AEs were required to be recorded. Any AEs were recorded for 28 days after each dose; All other AEs, SAEs and adverse events of particular interest (AESI) were recorded during the study period (approximately 7 months). Serum samples were collected for hematology, biochemistry and immunology evaluation.

Immunogenicity endpoints were neutralizing antibody titers at 14 (Day 15) and 28 (Day 29) after the first dose and 14 (Day 43) and 28 (Day 57) after the second dose and 90 and 180 days after the second dose and It was to evaluate the binding antibody titer. Convalescent serum samples from 35 recovered COVID-19 patients (Mitek COVID-19 Panel 1.1 and COVID-19 Panel 1.4 obtained from Access Biologicals LLC (Vista, CA, USA)) were also examined. At 28 days after the second administration, cellular immune responses were evaluated by IFN-γ ELISpot and IL-4 ELISpot.

SARS-CoV-2 spike-specific immunoglobulin G (IgG) : detection of total serum anti-spike IgG titers by direct enzyme-linked immunosorbent assay (ELISA) using custom 96-well plates coated with S-2P antigen did

SARS-CoV-2 Pseudovirus Neutralization Assay : Serial dilutions of the samples to be tested were performed (initial 1:20 dilution followed by 2-fold dilution to a final 1:2560 dilution). Diluted serum was mixed with an equal volume of pseudovirus (1000 TU) and incubated with HEK293-hAce2 cells (1×10 ⁴ cells/well) before adding to the plate. Cells were lysed and relative luciferase units (RLUs) were measured to calculate the amount of pseudovirus entering the cells. Calculate the 50% inhibitory dilution (concentration) titer (ID _{50 ) with uninfected cells considered 100% neutral and virus-transduced cells considered 0% neutral, and reverse ID 50 geometric} mean titer (GMT) of all decided.

Wild-type SARS-CoV-2 neutralization assay. SARS-CoV-2 virus (hCoV-19/Taiwan/CGMH-CGU-01/2020, GenBank accession MT192759) was titrated to obtain TCID ₅₀ Vero E6 cells (2.5 × 10 ⁴ cells/well) were seeded in 96-well plates and incubated. Serum was diluted 2-fold to a final dilution of 1:8192, and the diluted serum was mixed with an equal volume of virus solution containing 100 TCID ₅₀ . After incubation the serum-virus mixture was added to plates containing Vero E6 cells and further incubated. of the highest dilution capable of inhibiting the cytopathic effect by 50% (CPE NT ₅₀ ), calculated using the Rett-Münch method. Neutralization titers were defined as reciprocal numbers. 20/130 of the NIBSC (National Institute for Biological Standards and Control; Potters Bar, UK) reference serum samples were analyzed using the same validated assay as a comparator.

cellular immune response. The number of antigen-specific IFN-γ or IL-4 secreted spot forming units (SFUs) was determined by ELISpot assay. Cryopreserved peripheral blood mononuclear cells (PBMCs) were rapidly thawed and allowed to rest overnight. Cells were seeded at 1 × 10 ⁵ cells per well for the IFN-γ ELISpot assay (Human IFN-γ ELISpot Kit, Mabtech, Stockholm, Sweden), or For the IL-4 ELISpot assay (Human IFN-γ ELISpot Kit, Mabtech, Stockholm, Sweden), 2 × 10 ⁵ cells were distributed per well. Cells were stimulated with a peptide pool (PepTivator SARS-CoV-2 Prot_S1, Miltenyi Biotec) consisting mainly of 15-mer sequences with 11 amino acid overlaps containing the N-terminal S1 domain of the S protein of SARS-CoV-2; Incubate at 37° C. for 24-48 hours. Cells stimulated with CD3-2 mAb served as a positive control. IFN-γ or IL-4 release was detected according to instructions and spots were counted using a CTL automated ELISpot reader. Average SFU calculated with triple peptide pool stimulation was calculated and normalized by subtracting the average of negative control replicates (control medium). Results were expressed as SFU per million PBMCs.

statistical analysis. A safety analysis was performed on the Total Vaccination Group (TVG) population who received at least one dose of the vaccine. Immunogenicity endpoints included antigen-specific immunoglobulin and geometric mean titers (GMT) and seroconversion rates (SCR) of wild-type virus and pseudovirus neutralizing antibody titers. SCR is defined as the percentage of participants with a ≥4-fold increase in titer from half the lower limit of detection (LoD) at baseline or if undetectable at baseline. GMT and SCR are presented as two-tailed 95% CIs. Antigen-specific cellular immune responses were presented as means determined by IFN-γ ELISpot and IL-4 ELISpot.

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safety. No SAE or AESI occurred at this data cutoff point. The study intervention was not modified or discontinued. The occurrence of requested AEs is summarized in FIG. 11 . The most commonly reported local AE was pain/tenderness (80.0%), while the most commonly reported systemic AE among all treatment groups was malaise/fatigue (28.9%). All local and systemic AEs were mild except for one malaise/fatigue in the 25 μg dose group. None of the participants had a fever. AEs requested after the first and second doses were similar. Evaluation of safety laboratory values, ECG interpretation, and other random adverse events did not raise any particular concerns.

humoral immune response. Humoral immunogenicity results are summarized in Figures 12A-12C. As shown in Figure 12A, the titer of IgG binding to S protein increased rapidly after the second dose, and seroconverted by day 43 and day 57 in all participants. GMT was 7178.2 (95% CI: 4240.3 to 12151.7), 7746.1 (95% CI: 5530.2 to 10849.8), and 11220.6 (95% CI: 8592.293 to 14652.84) at 43 days in the 5 μg, 15 μg, and 25 μg dose groups, respectively. peaked at At day 43, the GMT levels of the 5 μg, 15 μg, and 25 μg dose groups ranged from 3.3 to 5.1 times the GMT of convalescent serum samples (2179.6, [95% CI: 1240.9 - 3828.4]).

As shown in Figure 12B. At baseline, the detectable pseudovirus neutralizing titer (ID ₅₀ ) at the lower limit (1:20 dilution) of the serum concentration tested in the assay was calculated. There were no subjects with At day 43, pseudovirus neutralizing titers (ID ₅₀ ) were 538.5 (95% CI: 261.9 - 1107.0), 993.1 (95% CI: 655.0 - 1505.7), and 1905.8 (95% CI: 655.0 - 1505.7) in the 5 μg, 15 μg, and 25 μg dose groups, respectively. % CI: 1601.7 - 2267.8) of peak GMT. All participants (100%) were seroconverted after the second dose. At day 43, GMT levels in the 5 μg, 15 μg, and 25 μg dose groups ranged from 1.25 to 4.4 times the GMT of convalescent serum samples (430.5, [95% CI: 274.9 - 674.0]).

The results of wild-type SARS-CoV-2 neutralizing antibody titers are summarized in FIG. 12C. Prior to vaccination, none of the subjects had a detectable wild-type virus neutralizing titer (NT ₅₀ ) at the lower limit of the serum concentration tested in the assay (1:8 dilution). After the second dose, neutralizing responses were confirmed in the serum samples of all participants in the 15 μg and 25 μg dose groups. At day 43, GMT was 33.3 (95% CI: 18.5 to 59.9), 76.3 (95% CI: 53.7 to 108.3), and 167.4 (95% CI: 122.1 to 229.6) in the 5 μg, 15 μg, and 25 μg dose groups, respectively. was GMTs on day 57 were similar for the 15 μg and 25 μg dose groups: 52.2 (95% CI: 37.9 - 71.8) and 81.9 (95% CI: 55.8 - 120.2), respectively. At day 43, the GMT levels of the 5 μg, 15 μg, and 25 μg dose groups were 0.8-, 1.8-, and 3.9-fold the GMT of the convalescent serum samples (42.7, [95% CI: 26.4 - 69.0]; titers ranged from undetected to 631.0). range up to). All participants in the 15 μg and 25 μg dose groups were seroconverted on days 43 and 57; Some were similar to the NIBSC reference serum 20/130 (281.8).

cellular immune response. The results of the cellular immune response are summarized in FIG. 13 . All participants had minimal IFN-γ secreting T cells at baseline. By day 57, an average of 161.3, 85.5, and 94.9 IFN-γ secreting T cells per million cells were observed in participants vaccinated with 5 μg, 15 μg, and 25 μg, respectively. Prior to vaccination, all participants had minimal IL-4 secreting T cells. By day 57, an average of 24.1, 16.0, and 31.3 IL-4 secreting T cells per million cells were observed in participants vaccinated with 5 μg, 15 μg, and 25 μg, respectively. Cellular immune responses induced by MVC-COV1901 demonstrated substantially higher numbers of IFN-γ producing cells, suggesting a Th1 biased immune response.

In conclusion, the side effects requested were mostly mild and similar. None of the subjects experienced fever. After the second dose, all participants at both the 15 μg and 25 μg doses of the three doses evaluated elicited a high neutralizing antibody response with seroconversion and a Th1-biased T cell immune response. Therefore, 15 μg S-2P in combination with CpG 1018 and aluminum hydroxide is considered appropriate to induce a significant humoral immune response. The results also indicate that the MVC-COV1901 vaccine was well tolerated, induced a strong immune response and is suitable for further development.

Example 5 Evaluation of neutralizing ability of CpG-enhanced S-2P subunit vaccine “MVC-COV1901” against SARS-CoV-2 variant of concern (VoC)

Mutations have been detected periodically since the start of the COVID-19 pandemic. Many of these, termed variants of concern (VoCs), have been found to carry mutations in the critical receptor binding domain (RBD), a key target for antibody recognition and neutralization. The most representative of these VoCs, all with N501Y mutations in spike RBD, are B.1.1.1.7 (alpha variant), B.1.351 (beta variant) and P1 (gamma variant). VoCs with these mutations have been shown to reduce the neutralizing ability of monoclonal and vaccine-derived antibodies, potentially making current therapeutics and vaccines ineffective (Garcia-Beltran et al., Cell, 184(9) :2372-2383.e9, 2021). This Example presents a study investigating the neutralizing ability of the MVC-COV1901 vaccine against SARS-CoV-2 VoC using sera from two sources: rat sera from animal toxicology studies and human sera from phase 1 clinical trials. to provide.

A. Neutralizing ability of MVC-COV1901 vaccine against SARS-CoV-2 VoC in rats .

Materials and Methods

animal studies. Crl:CD Sprague Dawley (SD) rats were purchased from BioLASCO Taiwan Co., Ltd. Ltd. (Taipei, Taiwan) and studies were performed at the biosafety testing facility of TFBS Bioscience Inc. (New Taipei City, Taiwan). Immunization of SD rats was performed as described in Example 2, Section C. Briefly, rats were immunized three times at 2-week intervals with 5, 25 or 50 μg of S-2P protein supplemented with 1,500 μg of CpG 1018 and 750 μg of aluminum hydroxide. Serum was collected 2 weeks after the secondary immunization (day 29) or 2 weeks after the 3rd immunization (day 43), and pseudotypes expressing SARS-CoV-2 Wuhan wild type (WT) or B.1.351 variant (beta variant) spike proteins were collected. Neutralization assays were performed with viruses.

Pseudovirus neutralization assay. A lentivirus expressing the SARS-CoV-2 spike protein of the Wuhan-Hu-1 wild-type strain (WT) was constructed and a neutralization assay was performed as described in Example 2, Section C. A lentivirus expressing the B.1.351 variant (beta variant) spike protein was constructed in the same manner except that the wild type spike protein sequence was replaced with the variant sequence (GenBank Accession No. MZ314998.1).

statistical analysis. Prism 6.01 (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis. Significance was calculated using the two-way ANOVA test with Tukey multiple comparisons and the Kruskal-Wallis test with modified Dunn multiple comparisons as indicated in the respective figure legends. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001.

result

In rats, the MVC-COV1901-derived antibody effectively neutralized the variant comparable to the wild-type . As shown in Figure 14, on days 29 and 43 the antibody retained potency against B.1.351 (beta variant) but the titer decreased. In particular, the sera sampled 2 weeks after the third immunization (day 43) had higher ID ₅₀ and ID ₉₀ geometric mean titers (GMT) than the serum sampled 2 weeks after the second immunization (day 29), indicating that the VoC with the third immunization It suggests a trend toward improved neutralizing activity for The effect was particularly pronounced at a low dose (5 μg). By day 43, all dose groups achieved similar levels of GMT for B.1.351 at ID ₅₀ (FIG. 14, left panel) and ID ₉₀ (FIG. 14, right panel).

In summary, rat studies indicate that similar levels of neutralizing titers can be induced across the 3 dose groups by using a 3-dose regimen. Given that the three-dose regimen showed high immunogenicity for the variants, these results can be applied to humans as boost immunization could be a strategy to increase immunity to VoC.

B. Neutralizing ability of MVC-COV1901 vaccine against SARS-CoV-2 VoC in humans.

Materials and Methods

clinical trial. Forty-five (45) human subjects between the ages of 20 and 49 years were enrolled in a prospective, unblinded, single-site dose-escalation phase 1 study with three separate subphases for participants aged 20 to <50 years. Each sub-stage included 15 participants. The three different dose levels used in this clinical trial were low dose (LD; 5 μg), medium dose (MD; 15 μg) supplemented with 750 μg of CpG 1018 and 375 μg of aluminum hydroxide for phases 1a, 1b and 1c, respectively. ) and high dose (HD; 25 μg) of S-2P protein. The vaccination schedule consisted of two doses administered by intramuscular (IM) injection of 0.5 mL into the deltoid region of the non-dominant arm on days 1 and 29, preferably spaced 28 days apart. On day 57 (4 weeks after the second dose), a serum sample was taken for pseudovirus neutralization assay. The clinical trial is described in more detail in Example 4.

Pseudovirus neutralization assay. A lentivirus expressing the SARS-CoV-2 spike protein of the Wuhan-Hu-1 wild-type strain (WT) was constructed and a neutralization assay was performed as described in Example 2, Section C. D614G, B.1.1.7 (alpha variant; GenBank accession number MZ314997.1), B.1.351 (beta variant; GenBank accession number MZ314998.1), P1 (gamma variant; GenBank accession number LR963075), and B.1.429 ( Epsilon variant; GenBank Accession No. MW591579) lentiviruses expressing the Spike protein were constructed in the same manner except that the wild-type Spike protein sequence was replaced with the respective variant sequence.

statistical analysis. Statistical analysis methods were performed as described in the previous section.

result

Human antisera vaccinated with MVC-COV1901 neutralized the D614G, B.1.1.7 (alpha), and P1 (gamma) variants, but decreased neutralization with the B.1.351 (beta) and B.1.429 (epsilon) variants. Figures 15A-15C show data from pseudovirus neutralization assays of human serum using the WT, D614G, B.1.1.7 (alpha), B.1.351 (beta), P1 (gamma) and B.1.429 (epsilon) variant panels. presents The titers of neutralizing antibodies in all groups (LD, MD and HD groups) against D614G and B.1.1.7 (alpha variant) fell compared to those against WT (Figures 15A to 15C), but the decrease was not statistically significant. did not However, when comparing B.1.351 (beta variant) and WT, neutralizing antibody titers were significantly decreased in all groups (LD, MD, HD groups). In contrast, neutralizing antibody titers of all groups (LD, MD and HD groups) against P1 (gamma variant) were higher than those against WT. The titer of neutralizing antibody to B.1.429 (epsilon variant) was significantly decreased in the LD and MD groups when compared to WT (Fig. 15A to 15B), but the titer of neutralizing antibody to B.1.429 (epsilon variant) in the HD group. There was no significant difference in (Fig. 15C). A dose-dependent effect could be observed when the neutralization titer of each dose group was plotted for the variants (Figs. 15A to 15C). Neutralizing titers against the B.1.351 (beta), P1 (gamma) and B.1.429 (epsilon) variants can be increased by using higher doses of antigen.

In conclusion, vaccinated phase 1 human subjects showed a further reduced but still significant neutralizing capacity for the ID ₉₀ , particularly for the B.1.351 (beta), P1 (gamma) and B.1.429 (epsilon) variants at higher doses. . Results suggest that both doses of MVC-COV1901 can induce neutralizing antibodies against SARS-CoV-2 variants in a dose-dependent manner.

Naturally, many changes and modifications may be made to the foregoing embodiments of the present invention without departing from the scope of the present invention. Therefore, in order to encourage the progress of science and useful arts, it is intended that this invention be limited only by the scope of the claims disclosed and followed.

SEQUENCE LISTING <110> Medigen Vaccine Biologics Corporation Dynavax Technologies Corporation <120> Immunogenic Composition Against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) <130> OP211002 <150> US 63/040,696 <151> 2020-06-18 <150> PCT/US21/020277 <151> 2021-03-01 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 1195 <212> PRT <213> Artificial Sequence <220> <223> Recombinant coronavirus spike protein <400> 1 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 305 310 315 320 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 325 330 335 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 370 375 380 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 385 390 395 400 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 420 425 430 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 435 440 445 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 450 455 460 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 465 470 475 480 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 485 490 495 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 500 505 510 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 515 520 525 Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys 530 535 540 Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr 545 550 555 560 Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro 565 570 575 Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser 580 585 590 Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro 595 600 605 Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser 610 615 620 Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala 625 630 635 640 Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly 645 650 655 Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Gly Ser Ala Ser 660 665 670 Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala 675 680 685 Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn 690 695 700 Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys 705 710 715 720 Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys 725 730 735 Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg 740 745 750 Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val 755 760 765 Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe 770 775 780 Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser 785 790 795 800 Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala 805 810 815 Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala 820 825 830 Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu 835 840 845 Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu 850 855 860 Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala 865 870 875 880 Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile 885 890 895 Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn 900 905 910 Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr 915 920 925 Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln 930 935 940 Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile 945 950 955 960 Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala 965 970 975 Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln 980 985 990 Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser 995 1000 1005 Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln 1010 1015 1020 Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser 1025 1030 1035 Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr 1040 1045 1050 Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile 1055 1060 1065 Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val 1070 1075 1080 Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu 1085 1090 1095 Pro Gln Ile Ile Thr Thr Asp Asp Thr Phe Val Ser Gly Asn Cys 1100 1105 1110 Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu 1115 1120 1125 Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe 1130 1135 1140 Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly 1145 1150 1155 Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu 1160 1165 1170 Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln 1175 1180 1185 Glu Leu Gly Lys Tyr Glu Gln 1190 1195 <210> 2 <211> 32 < 212> PRT <213> Artificial Sequence <220> <223> T4 fibritin trimerization domain <400> 2 Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val 1 5 10 15 Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Arg Ser 20 25 30 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HRV3C protease cleavage site <400> 3 Leu Glu Val Leu Phe Gln Gly Pro Gly 1 5 <210> 4 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Twin-Strep Tag <400> 4 Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly 1 5 10 15 Gly Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys 20 25 30 <210> 5 <211> 1227 <212> PRT <213> Artificial Sequence <220> <223> Recombinant coronavirus protein spike < 400> 5 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 305 310 315 320 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 325 330 335 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 370 375 380 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 385 390 395 400 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 420 425 430 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 435 440 445 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 450 455 460 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 465 470 475 480 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 485 490 495 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 500 505 510 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 515 520 525 Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys 530 535 540 Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr 545 550 555 560 Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro 565 570 575 Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser 580 585 590 Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro 595 600 605 Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser 610 615 620 Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala 625 630 635 640 Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly 645 650 655 Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Gly Ser Ala Ser 660 665 670 Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala 675 680 685 Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn 690 695 700 Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys 705 710 715 720 Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys 725 730 735 Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg 740 745 750 Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val 755 760 765 Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe 770 775 780 Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser 785 790 795 800 Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala 805 810 815 Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala 820 825 830 Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu 835 840 845 Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu 850 855 860 Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala 865 870 875 880 Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile 885 890 895 Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn 900 905 910 Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr 915 920 925 Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln 930 935 940 Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile 945 950 955 960 Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala 965 970 975 Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln 980 985 990 Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser 995 1000 1005 Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln 1010 1015 1020 Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser 1025 1030 1035 Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr 1040 1045 1050 Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile 1055 1060 1065 Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val 1070 1075 1080 Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu 1085 1090 1095 Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys 1100 1105 1110 Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu 1115 1120 1125 Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe 1130 1135 1140 Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly 1145 1150 1155 Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu 1160 1165 1170 Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln 1175 1180 1185 Glu Leu Gly Lys Tyr Glu Gln Gly Ser Gly Tyr Ile Pro Glu Ala 1190 1195 1200 Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val 1205 1210 1215 Leu Leu Ser Thr Phe Leu Gly Arg Ser 1220 1225 <210> 6 <211> 1233 <212> PRT < 213> Artificial Sequence <220> <223> Recombinant coronavirus spike protein <400> 6 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe A sp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 305 310 315 320 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 325 330 335 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 370 375 380 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 385 390 395 400 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 420 425 430 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 435 440 445 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 450 455 460 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 465 470 475 480 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 485 490 495 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 500 505 510 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 515 520 525 Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys 530 535 540 Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr 545 550 555 560 Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro 565 570 575 Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser 580 585 590 Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro 595 600 605 Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser 610 615 620 Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala 625 630 635 640 Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly 645 650 655 Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Gly Ser Ala Ser 660 665 670 Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala 675 680 685 Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn 690 695 700 Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys 705 710 715 720 Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys 725 730 735 Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg 740 745 750 Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val 755 760 765 Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe 770 775 780 Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser 785 790 795 800 Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala 805 810 815 Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala 820 825 830 Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu 835 840 845 Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu 850 855 860 Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala 865 870 875 880 Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile 885 890 895 Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn 900 905 910 Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr 915 920 925 Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln 930 935 940 Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile 945 950 955 960 Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala 965 970 975 Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln 980 985 990 Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser 995 1000 1005 Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln 1010 1015 1020 Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser 1025 1030 1035 Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr 1040 1045 1050 Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile 1055 1060 1065 Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val 1070 1075 1080 Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu 1085 1090 1095 Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys 1100 1105 1110 Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu 1115 1120 1125 Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe 1130 1135 1140 Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly 1145 1150 1155 Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu 1160 1165 1170 Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln 1175 1180 1185 Glu Leu Gly Lys Tyr Glu Gln Gly Ser Gly Tyr Ile Pro Glu Ala 1190 1195 1200 Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val 1205 1210 1215 Leu Leu Ser Thr Phe Leu Gly Arg Ser Leu Glu Val Leu Phe Gln 1220 1225 1230 <210> 7 <211> 1273 <212> PRT <213> Severe Acute Respiratory Syndrome Coronavirus 2 <400> 7 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 21 0 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Se r Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pr o Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gl y Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Il e Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gl n Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Ty r Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Sers Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260 Val Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CpG motif <400> 8 tgactgtgaa cgttcgagat ga 22 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CpG motif <400> 9 tcgtcgtttt gtcgttttgt cgtt 24 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CpG motif <400> 10 ggtgcatcga tgcagggggg 20 <210> 11 <211> 22 <212> DNA <213 > Artificial Sequence <220> <223> CpG motif <400> 11 tccatg gacg ttcctgagcg tt 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CpG motif <400> 12 tcgtcgttcg aacgacgttg at 22 <210> 13 <211> 23 <212> DNA < 213> Artificial Sequence <220> <223> CpG motif <400> 13 tcgtcgacga tcggcgcgcg ccg 23 <210> 14 <211> 1208 <212> PRT <213> Artificial Sequence <220> <223> Recombinant coronavirus protein spike <400> 14 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Gly Ser Ala Ser Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser V al Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln 1205 <210> 15 <211> 26 <212 > DNA <213> Artificial Sequence <220> <223> Forward primer <400> 15 acaggtacgt taatagttaa tagcgt 26 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400 > 16 atattgcagc agtacgcaca ca 22 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Probe<400> 17 acactagcca tccttactgc gcttcg 26

Claims (15)

An immunogenic composition against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising an antigenic recombinant protein, and an aluminum containing adjuvant, an unmethylated cytosine-phosphate-guanosine (CpG) motif, and these wherein the antigenic recombinant protein is substantially residue 14 of a SARS-CoV-2 S protein having a proline substitution at residues 986 and 987 and a "GSAS" substitution at residues 682 to 685; to 1208 and the C-terminal T4 fibritin trimerization domain. The amino acid sequence of claim 1, wherein residues 14 to 1208 of the SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and "GSAS" substitutions at residues 682 to 685 are relative to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 1 An immunogenic composition comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%. 3. The method of claim 1 or 2, wherein the C-terminal T4 fibritin trimerization motif is at least 90%, 95%, 96%, 97%, 98%, or relative to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2, or An immunogenic composition comprising 99% amino acid sequence. 4. The antigenic recombinant protein according to any one of claims 1 to 3, wherein the antigenic recombinant protein is at least 90%, 95%, 96%, 97%, 98% relative to the amino acid sequence of SEQ ID NO: 5 or 6 or to SEQ ID NO: 5 or 6 , or 99% amino acid sequence. 5. The aluminum-containing adjuvant according to any one of claims 1 to 4, wherein the aluminum-containing adjuvant is aluminum hydroxide, aluminum oxyhydroxide, aluminum hydroxide gel, aluminum phosphate, aluminum phosphate gel, aluminum hydroxyphosphate, aluminum hydroxyphosphate sulfate, amorphous An immunogenic composition comprising aluminum hydroxyphosphate sulfate, potassium aluminum sulfate, aluminum monostearate or a combination thereof. 6. The immunogenic composition of any preceding claim, wherein a 0.5 ml dose of the immunogenic composition comprises about 250 to about 500 μg Al ³⁺ or about 375 μg Al ³⁺ . 7. The method according to any one of claims 1 to 6, wherein the unmethylated CpG motif is a synthetic oligodeoxy of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or a combination thereof. An immunogenic composition comprising nucleotides (ODNs). 8. The method of any one of claims 1 to 7, wherein the 0.5 ml dose of the immunogenic composition comprises about 750 to about 3000 μg of oligonucleotides, or the immunogenic composition contains about 750 μg, about 1500 μg, or about 3000 μg An immunogenic composition comprising μg of oligonucleotide. An immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a subject in need thereof, comprising administering to the subject an effective amount of the immunogenic composition of any one of claims 1 to 8. How to induce. 10. The method of claim 9, wherein the immune response comprises the production of neutralizing antibodies to SARS-CoV-2 and a Th1 biased immune response. A first step for inducing an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a subject in need thereof. Use of the immunogenic composition of any one of claims 1 to 8, wherein the method comprises administering to a subject in need thereof an effective amount of the immunogenic composition. 12. The use of claim 11, wherein the immune response comprises the production of neutralizing antibodies to SARS-CoV-2 and a Th1 biased immune response. Claims 1 to 8 for protecting a subject in need of protection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection A use of the immunogenic composition of any one of claims wherein the method comprises administering an effective amount of the immunogenic composition to a subject in need thereof. Use of the immunogenic composition of any one of claims 1 to 8 for preventing COVID-19 disease in a subject in need thereof, wherein the method comprises administering an effective amount of the immunogenic composition to A use comprising administering to a subject in need thereof. 15. Use according to any one of claims 11 to 14, wherein the immunogenic composition is administered by intramuscular injection.

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