TWI815130B - Immunogenic composition against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) - Google Patents

Abstract

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

Description

Anti-novel coronavirus immune composition

This case claims the priority and rights of U.S. Provisional Application No. 63/040,696 filed on June 18, 2020, and PCT International Application No. PCT/US21/20277 filed on March 1, 2021. The disclosed content is This article is incorporated by reference in its entirety.

The present invention relates to an immune composition against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, also known as novel coronavirus), in particular an immune composition comprising recombinant SARS-CoV-2 spike protein and Adjuvant immune compositions.

The World Health Organization (WHO) was alerted on December 31, 2019, that several cases of pneumonia were discovered in Wuhan, Hubei Province, China. The viral pathogen did not match any other known virus and was later officially named "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, also known as novel coronavirus)." 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, tiredness, muscle or body aches, sore throat, diarrhea, conjunctivitis, headache, loss of taste or smell, skin rash, and shortness of breath. Although most cases are mild, some patients develop acute respiratory distress syndrome (ARDS), which is caused by cytokine storms, multiple organ failure, septic shock, and blood clots. The first confirmed death from novel coronavirus infection occurred on January 9, 2020. As of June 13, 2021, WHO has been notified of 175,306,598 confirmed cases of COVID-19, including 3,792,777 deaths. And those numbers are still growing rapidly.

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

In one aspect, the present invention provides an anti-novel coronavirus (SARS-CoV-2) immunogenic composition, including an antigenic recombinant protein and an adjuvant, the adjuvant is selected from the group consisting of aluminum-containing adjuvants, unmethylated A group consisting of cytosine-phosphate-guanosine (CpG) motifs and combinations thereof, wherein the antigenic recombinant protein is basically composed of the third protein of the SARS-CoV-2 spike protein Composed of residues 14 to 1208, the 986th and 987th residues are replaced with proline, and the 682nd to 685th residues are replaced with "glycine-serine-propylamine" acid-serine (GSAS)" and has a T4 fibrin (fibritin) trimerization domain at the C terminus.

In another aspect, the present invention provides a method for inducing an immune response against novel coronavirus (SARS-CoV-2) in a subject in need thereof, comprising administering to the subject an effective amount of the present invention. immunogenic compositions.

In another aspect, the present invention provides a method for protecting a subject in need thereof from infection with a novel coronavirus (SARS-CoV-2), comprising administering to the subject an effective amount of the immunogen of the present invention. sexual compositions.

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

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

In another aspect, the invention provides a use of the immunogenic composition of the invention in the preparation of a medicament for protecting a subject in need thereof from infection with the novel coronavirus (SARS-CoV-2).

In another aspect, the invention provides a use of the immunogenic composition of the invention in the preparation of a medicament for preventing infection of COVID-19 disease in a subject in need thereof.

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

The present invention relates to an immunogenic composition against novel coronavirus (SARS-CoV-2). The immunogenic composition includes an antigenic recombinant protein and an adjuvant including an aluminum-containing adjuvant and/or an unmethylated cytosine-phosphate-guanosine (CpG) motif. The antigenic recombinant protein contains residues 14 to 1208 of the SARS-CoV-2 spike protein, of which residues 986 and 987 are replaced with proline, and residues 682 to 685 It is replaced by "glycine-serine-alanine-serine (GSAS)" and has a T4 fibrin trimerization domain at its C-terminus.

In certain embodiments, the T4 fibrin trimerization domain at the C-terminus includes the amino acid sequence shown in SEQ ID NO: 2 or is at least 90%, 91%, or 90% identical to SEQ ID NO: 2. Amino acid sequences with 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity.

In certain embodiments, residues 14 to 1208 of the SARS-CoV-2 spike protein, residues 986 and 987 are replaced with proline, and residues 682 to 1208 are substituted for proline. 685 residues were replaced with "glycine-serine-alanine-serine (GSAS)", including the amino acid sequence shown in SEQ ID NO: 1 or at least having the same amino acid sequence as SEQ ID NO: 1 Amino acid sequences with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity.

In certain embodiments, the antigenic recombinant protein comprises the amino acid sequence shown in SEQ ID NO: 5 or 6 or is at least 90%, 95%, 96%, or 97 identical to SEQ ID NO: 5 or 6. %, 98% or 99% similarity of amino acid sequences.

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

In certain embodiments, a 0.5 ml dose of the immunogenic composition includes about 250 to about 500 μg of Al ³⁺ , or about 375 μg of Al ³⁺ .

In certain embodiments, the unmethylated CpG motif includes SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, Synthetic oligodeoxynucleotide (ODN) shown in SEQ ID NO: 13 or a combination thereof.

In certain embodiments, a 0.5 ml dose of the immunogenic composition includes about 750 to about 3000 μg of oligonucleotide, or wherein the immunogenic composition includes about 750 μg, about 1500 μg, or Approximately 3000 μg of oligonucleotide.

The present invention also relates to a method for inducing an immune response against novel coronavirus (SARS-CoV-2) in a subject in need thereof, comprising administering to the subject an effective amount of the immunogenicity of the present invention composition.

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

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

The present invention also relates to the use of an immunogenic composition of the present invention in the preparation of a medicament for inducing an immune response against the novel coronavirus (SARS-CoV-2) in a subject in need thereof.

In certain embodiments, the immune response includes the production of neutralizing antibodies against SARS-CoV-2 and a Th1-biased immune response.

The present invention also relates to the use of the immunogenic composition of the present invention in the preparation of a medicament for protecting a subject in need thereof from infection with the novel coronavirus (SARS-CoV-2).

The present invention also relates to the use of the immunogenic composition of the present invention in the preparation of a medicament for preventing infection of COVID-19 disease in a subject in need thereof.

In certain embodiments, the immunogenic composition is administered via intramuscular injection.

Those of ordinary skill in the art will clearly understand the meaning of the technical and scientific terms described herein.

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

As used herein, the word "comprising" is open-ended, indicating that such embodiments may include additional elements. Conversely, the term "consisting of" is closed-form and indicates that such embodiments do not contain additional elements (other than trace impurities). The term "consisting essentially of" is partially closed-ended and indicates that such embodiments may also contain elements that do not materially alter the essential characteristics of such embodiments.

As used interchangeably herein, the terms "polynucleotide" and "oligonucleotide" include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (single- stranded RNA (ssRNA), as well as double-stranded RNA (dsRNA), modified oligonucleotides, and oligonucleotides or combinations thereof. Oligonucleotides can be in linear or circular configurations, or the oligonucleotides can contain linear and circular segments. Oligonucleotides are typically polymers of nucleosides linked by phosphodiester linkages, although alternative linkages, such as phosphorothioates, may also be used in oligonucleotides. Nucleosides are composed of purine (adenine (A) or guanine (G) or their derivatives) or pyrimidine (thymine (T), cytosine (C) or Composed of uracil (U) or its derivatives) bases. The four nucleoside units (or bases) in DNA are called deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine. Nucleotide is the phosphate ester of a nucleoside.

As used herein, the term "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, also known as novel coronavirus)" refers to the coronavirus that causes coronavirus disease 2019 (COVID-19). Virus strains. SARS-CoV-2 is a positive-stranded single-stranded RNA virus with a genome size of 29,903 bases. Each SARS-CoV-2 virus particle has a diameter of 50-200 nm and has four structural proteins, namely spine (S), outer membrane (E), membrane (Membrane, M), and nucleosheath (nucleocapsid, N) protein. The N protein contains the RNA genome, and the S, E, and M proteins together form the viral outer membrane. Spinin is the protein responsible for allowing the virus to attach to and fuse with the host cell membrane. Specifically, the S1 subunit of spike protein catalyzes attachment, that is, the fusion of the S2 subunit with the host cell membrane.

As used herein, the term "immunogenic composition against novel coronavirus (SARS-CoV-2)" refers to a composition used to stimulate or elicit an immune response against SARS-CoV-2. The immune response includes, but is not limited to, the production of neutralizing antibodies against SARS-CoV-2 and a Th1-biased immune response.

As used herein, the term "aluminum-containing adjuvant" refers to an adjuvant that contains aluminum. In certain embodiments, the aluminum-containing adjuvant includes, but is not limited to, 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 certain embodiments, the aluminum-containing adjuvant is an aluminum-containing adjuvant approved for use in humans by the U.S. Food and Drug Administration (FDA). In certain embodiments, the aluminum-containing adjuvant is an aluminum hydroxide adjuvant approved by the FDA for administration to humans. In certain 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 contributes to in vitro, Measurable immune responses in vivo and/or ex vivo. In certain embodiments, the CpG-containing oligonucleotide comprises a palindromic hexamer according to the following formula: 5'-purine-purine-CG-pyrimidine-pyrimidine-3'. In certain preferred embodiments, the unmethylated cytosine-phosphate-guanosine (CpG) motif has the structure shown in SEQ ID NO: 8 (5'-TGACTGTGAACGTTCGAGATGA-3') Oligonucleotides in which the Cs in the CGs are unmethylated. In certain embodiments, the CpG-containing oligonucleotide contains TCG, wherein C is unmethylated and is 8 to 100 nucleotides in length, preferably 8 to 50 nucleotides, or Preferably it is 8 to 25 nucleotides. In certain preferred embodiments, the unmethylated cytosine-phosphate-guanosine (CpG) motif has an oligosaccharide as shown in SEQ ID NO: 9 (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3') Nucleotides where the Cs in TCGs are unmethylated. Examples of the unmethylated cytosine-phosphate-guanosine (CpG) motif also include, but are not limited to, 5'-GGTGCATCGATGCAGGGGGGG-3' (SEQ ID NO: 10), 5'-TCCATGGACGTTCCTGAGCGTT-3 ' (SEQ ID NO: 11), 5'-TCGTCGTTCGAACGACGTTGAT-3' (SEQ ID NO: 12), and 5'-TCGTCGACGA TCGGCGCGCGCCG-3' (SEQ ID NO: 13). Unless otherwise stated, the CpG-containing oligonucleotides described herein exist as their pharmaceutically acceptable salts. In a preferred embodiment, the CpG-containing oligonucleotide is in the form of a sodium salt.

An "effective amount" or a "sufficient amount" of a substance is an amount sufficient to produce a beneficial or desired result, including clinical results. Therefore, an "effective amount" depends on the context in which it is used. In the case of administering an immunogenic composition, an effective amount includes sufficient adjuvant and SARS-CoV-2 S-2P recombinant protein to elicit an immune response. One or more doses may be administered to achieve an effective amount.

The terms "individual" and "subject" refer to mammals. "Mammals" include, but are not limited to, humans, non-human primates (e.g., monkeys), farm animals, sporting animals, rodents (e.g., mice and rats), and pets (e.g., dogs and cats) ).

As used herein, the term "dose" with respect to an immunogenic composition refers to a measured portion of the immunogenic composition taken (given or received) by a subject at any time.

As used herein, the terms "isolated" and "purified" refer to a material that has been removed from at least one component with which it is naturally associated (eg, removed from its original environment). When used to refer to a recombinant protein, the term "isolated" refers to the protein that has been removed from the culture medium of the host cell in which it was produced.

"Stimulating" a response or parameter includes inducing and/or enhancing that response or parameter when compared to the same condition except for the target parameter, or alternatively, to another condition (e.g., compared to TLR signaling is increased in the presence of TLR agonists in the absence of TLR agonists). For example, "stimulation" of an immune response means an increase in that response. Depending on the parameter measured, the increase may be from 5-fold to 500-fold or more, or from 5, 10, 50 or 100-fold to 500, 1,000, 5,000 or 10,000-fold.

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

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

"Adjuvant" refers to a substance that, when added to a composition containing an antigen, non-specifically enhances or potentiates the immune response of a recipient to the antigen when exposed to the antigen.

The present invention is further illustrated by the following examples, which are provided for illustration and not limitation. Those skilled in the art should understand based on the disclosure of the present invention that various changes can be made to the disclosed specific embodiments without departing from the spirit and scope of the present invention, and similar or similar results can still be obtained.

Example

Example 1 anti- SARS-CoV-2 Preparation of immunogenic compositions

Plasmids with polynucleotides encoding the following protein sequence (SEQ ID NO: 14) were transfected into ExpiCHO-S cells (Thermo Fisher Scientific, Waltham, MA, USA): encoding SARS-CoV The 1st to 1208th residues of -2 spike protein (Wuhan-Hu-1 strain; GenBank number: MN908947), of which the 986th and 987th residues are replaced by proline, and furin ( furin) cleavage site (residues 682 to 685) was replaced with "glycine-serine-alanine-serine (GSAS)" and has a T4 fiber at the C-terminus of the protein Protein trimerization domain (SEQ ID NO: 2), an HRV3C protease cleavage site (SEQ ID NO: 3), an 8x His tag, and a Twin-Strep tag (SEQ ID NO: 4).

Cell cultures were harvested after 6 days and proteins were purified from the supernatants using Strep-Tactin resin (IBA Lifesciences, Göttingen, Germany). HRV3C protease (1% by weight) was added to the protein and the reaction was carried out at 4°C overnight. The cleaved protein was further purified using Superose 6 16/70 tubes (GE Healthcare Biosciences, Chicago, IL, USA) to obtain purified SARS-CoV-2 S-2P recombinant protein (or S-2P recombinant protein for short) ). The purified SARS-CoV-2 S-2P recombinant protein (SEQ ID NO: 5 or 6) is then combined with an unmethylated CpG motif (CpG 1018, SEQ ID NO: 8) and/or an aluminum-containing adjuvant, For example, aluminum hydroxide (Al(OH) ₃ ) or aluminum phosphate (AlPO ₄ ) can be used as an immunogenic composition against SARS-CoV-2.

Example 2 Immunogenic composition in mice against SARS-CoV-2 immunogenicity

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

A. preliminary trials 1 - Formulated with aluminum phosphate SARS-CoV-2 S-2P recombinant protein

Materials and methods

Immunization of mice . BALB/c mice (National Laboratory Animal Center, Taiwan) aged 6-8 weeks (N = 5/group) were inoculated with SARS-CoV-2 S-2P recombinant protein at weeks 0 and 3. Combine the SARS-CoV-2 S-2P recombinant protein diluted in phosphate-buffered saline (PBS) (final concentration: 1 μg/mL or 10 μg/mL) and aluminum phosphate (final concentration: 0.5 mg Aluminum/mL) mixed. Mice were inoculated with 100 μL of the above mixture by intramuscular injection (50 μL in each hind leg). Two weeks after the last immunization, sera were collected for measurement of antibody responses.

Production of fake viruses . The cDNA encoding Wuhan-Hu-1 virus strain spike protein (SEQ ID NO: 7) was synthesized using the QuikChange XL kit (Stratagene, San Diego, CA, USA) and then inserted into the CMV/R plasmid. Confirmation of CMV/R-SARS-CoV-2 spike proteosomes by sequencing. HEK293T cells were obtained from the American Type Culture Collection (ATCC) and maintained at 37°C, 5% _CO2 , supplemented with 10% fetal bovine serum (FBS), 2 cultured in DMEM medium containing 1% penicillin/streptomycin. To produce SARS-CoV-2 pseudovirus, CMV/R-SARS-CoV-2 spike proteosomes were combined with packaging plasmid pCMVDR8.2 using Fugene 6 transfection reagent (Promega, Madison, WI, USA) and Transduced plasmid pHR CMV-Luc was co-transfected into HEK293T cells. 72 hours after transfection, supernatants were collected, filtered, and frozen at -80°C.

Infectivity and neutralization assays of pseudoviruses . Huh7.5 cells (RRID: CVCL_7927) were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, and 1% penicillin/streptomycin at 37 °C and 5% _CO2 . . The infectivity of pseudoviruses was assessed on Huh7.5 cells cultured overnight in 96-well black/white isoplates (PerkinElmer, Waltham, MA, USA). Pseudovirus was added to static Huh7.5 cells after two-fold serial dilution, and triplicate experiments were performed. After 2 hours of incubation, fresh medium was added. After 72 hours of culture, the cells were lysed, and luciferase substrate (Promega Company) was added. Luciferase activity was measured as relative luciferase units (RLU) at a wavelength of 570 nm on a SpectramaxL microplate luminometer (Molecular Devices, Inc., San Jose, CA, USA). For neutralization experiments, serial dilutions of mouse serum (1:40, fourfold, eightfold dilutions) were mixed with various pseudoviral strains that had been previously quantified to a target concentration of 50,000 RLU. Take the average of the RLU readings of three replicate experiments for each dilution concentration to create a Sigmoids curve; uninfected cells are considered 100% neutralized, cells transduced with virus only are considered 0% neutralized, and 50% is calculated. Neutralizing power price (IC ₅₀ ).

result

The results of the neutralization test are shown in Figure 1. The SARS-CoV-2 S-2P recombinant protein formulated with aluminum phosphate (0.1 μg/mouse and 1 μg/mouse) was smaller than the recombinant protein alone (without aluminum phosphate) (0.1 μg/mouse and 1 μg/mouse). rats) caused greater neutralization. These data show that aluminum phosphate can significantly improve the immunogenicity of the SARS-CoV-2 S-2P recombinant protein as a coronavirus disease (COVID-19) vaccine antigen.

B. preliminary trials 2 - by CpG Formulated in combination with aluminum hydroxide SARS-CoV-2 S-2P recombinant protein

Materials and methods

Immunization of mice . BALB/c mice (National Laboratory Animal Center, Taiwan) aged 6-8 weeks (N = 6/group) were inoculated with SARS-CoV-2 S-2P recombinant protein at weeks 0 and 3. Combine SARS-CoV-2 S-2P recombinant protein diluted in PBS (final concentration: 10 μg or 50 μg/mL) with CpG 1018 (SEQ ID NO: 8) (final concentration: 0.1 mg/mL), hydrogenated Aluminum (final concentration 0.5 mg aluminum/mL), or a combination of CpG 1018 (final concentration 0.1 mg/mL) and aluminum hydroxide (final concentration 0.5 mg aluminum/mL) was mixed. Mice were inoculated with 100 μL of the above mixture by intramuscular injection (50 μL in each hind leg). Two weeks after the last immunization, sera were collected for measurement of antibody responses.

Pseudovirus generation, pseudovirus infectivity, and neutralization assays . Methods are as described in Part A.

result

The results of the neutralization test are shown in Figure 2. SARS-CoV-2 S-2P recombinant protein formulated with a combination of CpG and aluminum hydroxide elicited the highest neutralizing activity at both low dose (1 µg/mouse) and higher dose (5 µg/mouse). Furthermore, the SARS-CoV-2 S-2P recombinant protein formulated with aluminum hydroxide only (1 µg/mouse) elicited greater neutralizing effect. Recombinant proteins formulated with CpG only elicited neutralizing activity in a dose-dependent manner. These data show that CpG and/or aluminum hydroxide significantly improve the immunogenicity of SARS-CoV-2 S-2P recombinant protein as a coronavirus disease (COVID-19) vaccine antigen.

C. Adjuvanted stable prefusion SARS-CoV-2 Development of spike protein antigen

Materials and methods

Production and quantification of pseudoviruses . In order to produce SARS-CoV-2 pseudovirus, TransIT-LT1 transfection reagent (Mirus Bio Company) was used to express the full-length wild-type Wuhan-Hu-1 strain SARS-CoV-2 spike protein (SEQ ID NO: 7). pCMV 8.91 and reporter plasmid pLAS2w.FLuc.Ppuro (RNA Technology Platform, Academia Sinica, Taiwan) were co-transfected into HEK293T cells. The D614G mutant strain was generated by site-directed mutagenesis by changing nucleotide 23403 on the Wuhan-Hu-1 reference strain from A to G. A simulated pseudovirus was generated by omitting the p2019-nCoV spike protein (wild type). 72 hours after transfection, the supernatant was collected, filtered, and frozen at -80 °C. The transduction unit (TU) of SARS-CoV-2 pseudotyped lentivirus was estimated by cell viability analysis based on the cell response to the limiting dilution of lentivirus. Briefly, HEK-293 T cells stably expressing the human ACE2 gene were seeded on a 96-well plate 1 day before lentiviral transduction. To quantify pseudoviruses, varying amounts of pseudoviruses were added to polybrene-containing culture medium. Centrifuge the 96-well plate at 1100 xg for 30 minutes at 37°C. After culturing the cells for 16 hours at 37°C, the medium containing virus and polybrene was removed and replaced with fresh complete DMEM medium containing 2.5 µg/ml puromycin. After 48 hours of puromycin treatment, the culture medium was removed and cell viability was detected using 10% Alarma Blue reagent according to the manufacturer's instructions. The viability of uninfected cells (not treated with puromycin) was set as 100%. Viral titers (transduction units) were determined by plotting viable cells versus diluted virus dose.

Pseudovirus-based neutralization assay . HEK293-hAce2 cells (2 x 10 cells/ ^well ) were seeded in 96-well white cell culture plates and cultured overnight. Heat serum at 56°C for 30 minutes to inactivate complement and dilute serum in MEM medium supplemented with 2% FBS, starting with a dilution factor of 20, followed by two-fold serial dilutions (8 dilution steps in total, final dilution concentration is 1:5120). The diluted serum was mixed with an equal volume of pseudovirus (1000 TU) and incubated at 37°C for 1 hour before being added to the culture plate with cells. After 1 hour of incubation, the medium was replaced with 50 µL of fresh medium. The medium was replaced with 100 µL of fresh medium the next day. Cells were lysed 72 hours post-infection and relative luciferase units (RLU) were measured. Luciferase activity was detected with Tecan i-control (Infinite 500). Consider uninfected cells as 100% neutralization, and cells transduced with virus only as 0% neutralization. Calculate the dilution multiples to achieve 50% and 90% inhibition (i.e., ID ₅₀ and ID ₉₀ ). Confirm the geometric mean force price (GMT) of ID ₅₀ and ID ₉₀ , because when the ID ₅₀ force price has been at the upper detection limit and reaches saturation, the ID ₉₀ force price can be used to judge.

Neutralization of wild-type SARS-CoV-2 virus. The neutralization assay of 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 x ¹⁰ cells/well) were seeded in 96-well plates and cultured overnight. Heat the serum at 56°C for 30 minutes to inactivate complement, and dilute the serum in serum-free MEM medium with an initial dilution factor of 20, and then perform two-fold serial dilutions for a total of 11 dilution steps, with a final dilution concentration of 1 :40,960. The diluted serum was mixed with an equal volume of 100 TCID _50/50 µL of SARS-CoV-2 virus (hCoV-19/Taiwan/CGMH-CGU-01/2020 strain, GenBank accession number MT192759), and incubated at 37° C for 2 hours. This serum virus mixture was then added to a 96-well plate containing Vero E6 cells and cultured in MEM medium containing 2% FBS at 37°C for 5 days. After incubation, 4% formaldehyde was added to each well to fix the cells for 10 min and stained with 0.1% crystal violet for visualization. Calculate its NT ₅₀ power price using the Reed-Muench formula.

Immunization of mice . Female BALB/c and C57BL/6 mice were obtained from the National Laboratory Animal Center, Academia Sinica (Taiwan) and Lesko Biotechnology Co., Ltd. (Taiwan). For antigen preparations, SARS-CoV-2 S-2P protein was mixed with equal volumes of CpG 1018 (SEQ ID NO: 8), aluminum hydroxide, PBS, or CpG 1018 plus aluminum hydroxide. Mice aged 6-9 weeks were immunized twice, with each 3 weeks apart (50 µL injected intramuscularly into the left and right quadriceps muscles of each mouse). Using a 96-well plate coated with S-2P recombinant protein antigen, direct ELISA was used to detect the IgG antibody titer of total serum anti-spike protein, and the use of a 96-well plate coated with the region containing the receptor binding domain (RBD) of the spike protein. E. coli expressing fragments in 96-well plates were used to detect total serum anti-RBD IgG antibody titers by direct ELISA.

Cytokine analysis . Two weeks after the second injection, mice were euthanized, and splenocytes were isolated and treated with S-2P recombinant protein as previously described (Lu et al., Immunology, 130(2):254–261, 2010). (2 µg/well) stimulation. To detect IFN- , IL-2, IL-4, and IL-5, collect the culture supernatant in a 96-well microplate, and use mouse IFN- Quantikine ELISA Kit, Mouse IL-2 Quantikine ELISA Kit, Mouse IL-4 Quantikine ELISA Kit, and Mouse IL-5 Quantikine ELISA Kit (R&D System Company) were used to analyze the contents of these cytokines by ELISA. The OD450 value was read with a Multiskan GO analyzer (Thermo Fisher Scientific).

Sprague Dawley (SD) rats were used to study the dose range of single and double intramuscular injection ( IM) . Crl:CD Sprague Dawley (SD) rats were obtained from Resco Biotechnology Co., Ltd. (Taiwan). Animal experiments were performed at the biosafety testing facility of Qihong Biotechnology Co., Ltd. (Taiwan). S-2P recombinant protein at 5 µg, 25 µg or 50 µg adjuvanted with 1500 µg CpG 1018 alone or 750 µg CpG 1018 in combination with 375 µg aluminum hydroxide in SD rats aged 6-8 weeks Immunize. On the first day of the experiment (for single-dose and double-dose studies) and on the 15th day (for double-dose studies), each rat was injected intramuscularly (0.25 mL/site, a total of 2 sites in the left and right quadriceps muscles) of the test product. or vehicle control. The observation periods were 14 days (for single-dose studies) and 28 days (for double-dose studies). Parameters assessed included clinical signs during survival, local provocation testing, morbidity/mortality, body temperature, body weight, and food intake. Blood samples are collected for hematology testing, including coagulation testing and serum chemistry. All animals were euthanized and necropsied for gross lesion examination, organ weights, and histopathological evaluation of the injection site as well as the lungs.

Statistical analysis . For neutralization experiments, the bar height represents the geometric mean valence, and the error bars represent the 95% confidence interval. For cytokine and rat data, bar heights or symbols represent the mean, and error bars represent the standard deviation (SD). The dashed lines indicate the lower and upper limits of detection. Statistical analysis was performed using Prism 6.01 (GraphPad Inc.) analysis suite software. Data were compared at the same S-2P recombinant protein dose with different adjuvants or the same adjuvant system with different antigen doses. Nonparametric tests between more than 2 experimental groups were calculated using Kruskal-Wallis with corrected Dunn's multiple comparisons test. The Mann-Whitney U test was used to compare the two experimental groups. Correlation between antibody titers and neutralizing titers was calculated using Spearman's rank correlation coefficient. * p < 0.05, ** p < 0.01, *** p < 0.001.

result

Effective neutralizing antibodies were induced with S-2P recombinant protein containing CpG 1018 and aluminum hydroxide adjuvant . The ExpiCHO system is used as the expression system for S-2P recombinant protein antigen to establish stable clones for clinical research and commercial production. Under cryo-electron microscopy, the structure of the S-2P recombinant protein produced in CHO cells showed a typical spine trimer, similar to the SARS-CoV-2 spine protein expressed in 293 cells (Wrapp et al., Science, 367( 6483): 1260–1263, 2020), which means that CHO cells can be used to produce S-2P recombinant protein. Next, we examined the clinical potential of CpG 1018 in inducing Th1-biased responses. and tested aluminum hydroxide with CpG 1018, as aluminum hydroxide has been found to enhance the potency of CpG adjuvants when used in combination with CpG while retaining the property of CpG to induce a biased Th1 response (Thomas et al., Hum. Vaccin ., 5(2):79–84, 2009). Pseudovirus neutralization test was conducted with serum drawn 3 weeks after the first injection or 2 weeks after the second injection. Neutralizing activity was already observed 3 weeks after the first injection when mice were immunized with 1 µg and 5 µg of S-2P recombinant protein containing CpG 1018 adjuvanted with aluminum hydroxide. Dilutions at which 50% inhibition was achieved 2 weeks after the second injection with 1 µg of S-2P recombinant protein adjuvanted with CpG 1018, aluminum hydroxide, or a combination of CpG 1018 and aluminum hydroxide. The geometric mean valence (GMT) of concentrations (ID ₅₀ ) were 245, 3109, and 5120, respectively (Figure 3). Similar trends were observed in BALB/c (Figure 3) and C57BL/6 mice immunized with 5 μg of S-2P recombinant protein.

Sera from these mice were then examined for levels of IgG antibodies against spike protein. Compared with the group using CpG 1018 alone as an adjuvant, the group using a combination of CpG 1018 and aluminum hydroxide as an adjuvant had significantly higher anti-spike IgG antibody titers (Figure 4). In order to confirm the activity of the antibody against the key receptor binding domain (RBD) in spike protein, the anti-RBD IgG antibodies in the immune serum were examined. The results were similar to those of the anti-thorn protein IgG antibodies, using CpG 1018 and aluminum hydroxide. The combination of S-2P recombinant protein with adjuvant induced the highest IgG antibody titer. There was a moderate correlation between the IgG antibody titers against Spinin and the IgG antibody titers against RBD, as shown by the Spearman's rank correlation coefficient of 0.6486. The neutralizing ability of the immune serum against the wild-type SARS-CoV-2 strain was further tested with a neutralization test. S-2P recombinant protein at a concentration of 1 µg can induce neutralizing antibodies in mice that inhibit SARS-CoV-2 strains, although its inhibitory efficacy against SARS-CoV-2 strains is lower than that against pseudoviruses ( Figure 3, Figure 5). The geometric mean valence (GMT) of the ID ₅₀ antibody induced by 1 μg of S-2P recombinant protein adjuvanted with CpG 1018, aluminum hydroxide and the combination of CpG 1018 and aluminum hydroxide is approximately 60 and 250, respectively. and 1500 (Figure 5). In addition, pseudoviruses with the current dominant mutation D614G spike protein were also generated. Neutralizing antibodies produced by mice immunized with S-2P recombinant protein containing a combination of CpG 1018 and aluminum hydroxide as an adjuvant were all effective against the virus. There are pseudoviruses with wild-type D614 and mutant D614G spike proteins (Figure 6). It was found that the neutralizing potency of wild-type virus and pseudovirus and the overall anti-spike protein IgG antibody potency were highly correlated, and their Spearman's rank correlation coefficient was greater than 0.8.

CpG 1018 induces Th1 immune response . In order to confirm whether CpG 1018 can induce Th1 response in the vaccine adjuvant system, mice were immunized with S-2P recombinant protein and aluminum hydroxide, CpG 1018 or a combination of the two as adjuvants, and their splenocytes were removed. Measures the cytokines involved in Th1 and Th2 responses. As expected, IFN- induced by S-2P recombinant protein adjuvanted with aluminum hydroxide and IL-2 are limited in quantity, while IFN- and IL-2 are representative cytokines of Th1 response. In contrast, in the group with high dose antigen plus CpG 1018 and aluminum hydroxide as adjuvant, IFN- and IL-2 increased most significantly. Regarding the Th2 response, although an increase in the levels of IL-4, IL-5 and IL-6 was observed in the S-2P recombinant protein plus aluminum hydroxide as adjuvant group, CpG 1018 and aluminum hydroxide as adjuvant were observed. In the drug group, it was observed that the contents of IL-5 and IL-6 were inhibited. In the group of S-2P recombinant protein plus CpG 1018 and aluminum hydroxide as adjuvant, IFN- /IL-4, IFN- /IL-5, and IFN- /IL-6 ratios increased by approximately 36, 130, and 2-fold, respectively, a result that strongly indicates a Th1-biased immune response (Figure 7). These results show that CpG 1018 is more effective than aluminum hydroxide in directing cell-mediated immune responses toward a Th1 response while maintaining high antibody content.

S-2P recombinant protein will not cause systemic adverse reactions in rats . To illustrate the safety and potential toxicity of the candidate vaccine, SD rats were administered 5 µg, 25 µg, or 50 µg of S-2P adjuvanted with 1500 µg CpG 1018 or 750 µg CpG 1018 in combination with 375 µg aluminum hydroxide. Recombinant proteins for single-dose and double-dose studies. No death, abnormal clinical symptoms, changes in body weight, body temperature, food intake, etc. caused by administration of a single dose of S-2P recombinant protein (with or without adjuvant) were observed in both female and male rats. . In both single-dose and double-dose studies, rats of both sexes were found to have elevated body temperatures within 4 hours or 24 hours after vaccination; however, these temperature changes were modest and included the control group (PBS) at In all treatment groups, the body temperature of rats of both sexes returned to normal within 48 hours after vaccination. No obvious organ damage was observed in most male and female rats after single and double doses, except for one male rat, but the organ damage in this male rat was not considered to be related to the vaccine. . In conclusion, intramuscular injection of S-2P recombinant protein adjuvanted with CpG 1018 or a combination of CpG 1018 and aluminum hydroxide once or twice in SD rats did not cause any systemic adverse reactions.

The results showed that two injections of a subunit vaccine composed of CpG 1018 with aluminum hydroxide adjuvant and fusion pre-spike protein (S-2P recombinant protein) into mice can effectively induce the production of neutralizing antibodies against wild-type expression in mice. As well as pseudoviruses with D614G mutant spike protein and wild-type SARS-CoV-2 viruses. The combination of S-2P recombinant protein with CpG 1018 and aluminum hydroxide induced a Th1-dominated immune response with moderate neutralizing antibody content in mice and showed no major adverse effects in rats. Therefore, in this example, the inventors of the present case demonstrated that the S-2P recombinant protein combined with the adjuvant of CpG 1018 and aluminum hydroxide induced an effective Th1-biased immune response to prevent wild-type virus infection while maintaining high antibody content. , these antibodies showed cross-neutralizing responses to virus variants. Therefore, the immunogenic composition against SARS-CoV-2 of the present invention can be used as an ideal vaccine candidate to reduce the burden of the global COVID-19 pandemic.

Example 3 anti- SARS-CoV-2 Immunogenic compositions provided for hamsters SARS-CoV-2 The protective effect of virus attack

Materials and methods

Pseudovirus-based neutralization assay and IgG ELISA . A lentivirus expressing the SARS-CoV-2 spike protein of the Wuhan-Hu-1 strain was constructed, and a neutralization test was performed as described in Example 2. Briefly, HEK293-hACE2 cells were seeded in 96-well white cell culture plates and cultured overnight. Sera from vaccinated and unvaccinated hamsters were heat-inactivated and diluted in MEM medium supplemented with 2% FBS, with an initial dilution factor of 20, and then 2-fold serial dilutions for a total of 8 dilution steps. Final dilution to 1:5120. The diluted serum was mixed with an equal volume of pseudovirus (1,000 TU) and incubated at 37°C for 1 hour before being added to the above-mentioned culture plate containing cells. Cells were lysed 72 hours post-infection and relative luciferase units (RLU) were measured. Uninfected cells were considered 100% neutralized, and cells transduced with virus alone were considered 0% neutralized to calculate the dilution multiples required to achieve 50% and 90% inhibition (i.e., ID ₅₀ and ID ₉₀ ). Total serum anti-Spin IgG antibody titers were measured by direct ELISA using a 96-well plate coated with S-2P recombinant protein antigen.

Immunity and challenge experiments in hamsters. Female golden Syrian hamsters aged 6-9 weeks were obtained from the National Laboratory Animal Center (Taipei, Taiwan) before the start of the study. Hamsters from different litters were randomly divided into four groups (N=10 per group): vehicle control (PBS), 1 or 5 µg of S- adjuvanted with 150 µg CpG 1018 and 75 µg aluminum hydroxide. 2P recombinant protein, or adjuvant alone, was injected intramuscularly into hamsters twice, each time 3 weeks apart. Two weeks after the second immunization, hamsters were bled via the submandibular vein to confirm the presence of neutralizing antibodies. Hamsters were challenged intranasally with 1 x 10 ⁴ PFU of SARS-CoV-2 TCDC#4 strain (hCoV-19/Taiwan/4/2020, GISAID registration number: EPI_ISL_411927) 4 weeks after the second immunization, each time Challenge hamsters with 100 μL. The hamsters were divided into two groups and euthanized on days 3 and 6 after challenge for necropsy and tissue sampling. The weight and survival rate of each hamster were recorded every day after infection. On days 3 and 6 after challenge, hamsters were euthanized with carbon dioxide. The right lung was collected for viral load determination (RNA titration and TCID ₅₀ analysis). The left lung was fixed in 4% paraformaldehyde for histopathological examination.

Viral titers in lung tissue were quantified using a cell culture infection assay (TCID ₅₀ ) . Use a homogenizer to homogenize the middle, lower and postcavitary lung lobes of the hamster in 600 μl of DMEM medium containing 2% FBS and 1% penicillin/streptomycin. The tissue homogenate was centrifuged at 15,000 rpm for 5 minutes, and the supernatant was collected for viable virus quantification. Briefly, 10-fold serial dilutions of each sample were added to Vero E6 cell monolayers and cultured for 4 days, and each sample was tested in quadruplicate. The cells were then fixed with 10% formaldehyde and stained with 0.5% crystal violet for 20 minutes. Cell culture plates were washed with tap water and scored for infection. The 50% tissue culture infectious dose (TCID ₅₀ )/mL was calculated according to the Reed and Muench method (Reed and Muench, American Journal of Epidemiology, 27(3): 493-497, 1938).

Quantification of SARS-CoV-2 viral RNA by real-time RT-PCR . The TaqMan real-time RT-PCR method (Corman et al., Eurosurveillance. 25( 3): 2000045, 2020) to measure the RNA content of the SARS-CoV-2 virus. In addition to the forward primer E-Sarbeco-F1 5'-ACAGGTACGTTAATAGTTAATAGCGT-3' (SEQ ID NO: 15) and the reverse primer E-Sarbeco-R2 5'-ATATTGCAGCAGTACGCACACA-3' (SEQ ID NO: 16), and Probe E-Sarbeco-P1 5'-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BBQ-3' (SEQ ID NO: 17) was used. A total of 30 µL 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 25 µL of a mixture containing Superscript III one-step RT-PCR system and Platinum Taq polymerase (Thermo Fisher Scientific, USA). The final reaction mixture contains 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 enzyme mixture. PCR was performed using the cycling conditions for one-step PCR: 55°C for 10 min to synthesize first strand cDNA, followed by 94°C for 3 min, followed by 45 amplification cycles: 94°C for 15 sec and 58°C for 30 Second. Data were collected and calculated using 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 assess viral genome copy number. Oligonucleotides were synthesized by Keelon Mix Biotechnology Co., Ltd. (Taipei, Taiwan).

result

Hamster as SARS-CoV-2 virus challenge model . In order to develop the SARS-CoV-2 virus hamster challenge model of the S2-P recombinant protein vaccine, a preliminary study was first conducted to determine the optimal virus dose for the challenge experiment. Unvaccinated hamsters were vaccinated with 10 ³ , 10 ⁴ or 10 ⁵ PFU of SARS-CoV-2 virus and euthanized on day 3 or 6 post-infection for tissue sampling. Hamsters experienced dose-dependent weight loss after infection with 10 ³ to 10 ⁵ PFU of SARS-CoV-2 virus. The weight of hamsters infected with 10 ³ PFU virus increased, while the weight loss of hamsters infected with 10 ⁴ and 10 ⁵ PFU viruses gradually became more severe at 6 days post-infection (dpi). However, there was no significant difference in viral genomic RNA content and virality measured between hamsters infected with 10 ³ to 10 ⁵ PFU of SARS-CoV-2 virus at 3 and 6 days post-infection. All doses of virus resulted in increased lung lesions, even in hamsters infected with ¹⁰ PFU of virus that did not lose weight. Pulmonary pathology scores and pulmonary viral load also did not have a dose-dependent effect on virus inoculation. Therefore, virus challenge studies were performed with 10 ⁴ PFU of virus because this concentration provides a sufficient balance between clinical symptoms and virality for vaccination.

Administration of S-2P recombinant protein adjuvanted with CpG 1018 and aluminum hydroxide to hamsters induced high levels of neutralizing antibodies . Hamsters that received two immunizations 21 days apart were divided into four groups and vaccinated respectively: vehicle control (PBS only), adjuvant group, low dose (LD) or high dose (HD) of S-2P recombinant protein, and CpG 1018 With aluminum hydroxide (S-2P+CpG 1018+aluminum hydroxide). No differences in body weight changes were observed among the four groups of hamsters after vaccination. Fourteen days after the second immunization, high levels of neutralizing antibody titers were found in hamsters in the low-dose (LD) and high-dose (HD) groups, and the geometric mean of their 90% inhibitory dilutions (ID ₉₀ ) The force price (GMT) is 2,226 and 1,783 respectively (Figure 8A). The levels of anti-Sphin IgG antibodies in the low-dose (LD) and high-dose (HD) groups were very high, and even several individual samples reached the detection limit. The geometric mean price (GMT) of anti-Spike protein IgG antibodies in these two groups was ) were 1,492,959 and 1,198,315 respectively (Figure 8B). In summary, S-2P+CpG 1018+aluminum hydroxide was able to induce an effective degree of immunogenicity in hamsters even at low doses.

Adjuvanted S-2P recombinant protein can protect hamsters from clinical symptoms and viral load after challenge with SARS-CoV-2 virus. Hamsters were challenged with 10 ⁴ PFU of SARS-CoV-2 virus 4 weeks after the second immunization, and body weights were followed at 3 or 6 days post-infection (dpi). Animals were sacrificed 3 or 6 days postinfection for viral load and histopathological analysis. The low-dose (LD) and high-dose (HD) vaccination groups did not lose weight 3 or 6 days after virus challenge. Instead, the average weight increased by 5 g and 3.8 g respectively 6 days after infection. Both the two dose groups had the most significant protective effect 6 days after infection, while the weight of the vehicle control group and the adjuvant group were significantly reduced. The viral RNA and TCID ₅₀ analysis were used to measure the viral load in the lungs. The results showed that the viral RNA and viral titer of hamsters in the low-dose (LD) and high-dose (HD) vaccination groups were significantly reduced 3 days after infection. It dropped below the lower limit of detection 6 days after infection (Figures 9A-9B). Please note that due to the natural immune response of hamsters, the viral load of hamsters in the control and adjuvant groups, especially the viral titer measured by TCID ₅₀ , decreased significantly 6 days after infection (Figure 9A-9B). Lung sections were analyzed and pathology scores were graphed (Figure 10). There was no difference between the control and experimental groups at 3 days post-infection; however, at 6 days post-infection, the vehicle control group and the adjuvant group had significantly increased lung cancer compared with the high-dose (HD) antigen/adjuvant immunization group. The lesions contained extensive immune cell infiltration and diffuse alveolar damage (Figure 10). These results show that S-2P+CpG 1018+aluminum hydroxide induces a robust immune response that suppresses viral load in the lungs and prevents weight loss and lung lesions in infected hamsters.

Compared with the diffuse alveolar damage caused by the virus in the lungs of hamsters in the control group 6 days after infection (the average score of the vehicle control group and adjuvant group was 4.09, rated as moderate to severe), S-2P+ All hamsters in the CpG 1018 + aluminum hydroxide immunized group were protected and had significantly reduced lung lesions [average score for low-dose (LD) and high-dose (HD) vaccinated groups was 1.72, with a general score of very mild to mild Spend]. The significance of this study is not only to prove the efficacy of the vaccine in live animals, but also its safety. Viral challenge studies can assess the risk of increased disease from vaccine candidates. The histopathological scores of the immunized group were indistinguishable from those of the non-challenged animals, indicating the absence of increased lesions due to the vaccine. The findings in this example provide additional data supporting the progress of clinical development of this vaccine candidate.

Example 4 by CpG as an adjuvant S-2P Recombinant protein subunit vaccine "MVC-COV1901" In vivo safety and immunogenicity in humans

This Example provides a Phase I clinical trial study in healthy human subjects to evaluate the safety and immunogenicity of a SARS-CoV-2 subunit vaccine (i.e., the immunogenic composition of the invention) . The SARS-CoV-2 subunit vaccine is referred to as "S-2P+CpG 1018+aluminum hydroxide" or "MVC-COV1901" herein. Please refer to Example 1 for a detailed description of the vaccine.

Vaccine . MVC-COV1901 is formulated to contain three different doses of SARS-CoV-2 recombinant spike (S) protein and is adjuvanted with CpG 1018 and aluminum hydroxide. Each dose of the MVC-COV1901 vaccine contains 5, 15 or 25 μg of S-2P recombinant protein adjuvanted with 750 μg of CpG 1018 and 375 μg (Al ³⁺ content equivalent to 375 μg) of aluminum hydroxide as a single dose Administer 0.5 mL intramuscularly (IM).

subjects . The goal of this study is to recruit 45 subjects. Eligible subjects were healthy adults aged 20 to 49 years. Eligibility is determined based on medical history, physical examination, laboratory testing, and the investigator's clinical judgment. Exclusion criteria included known history of possible exposure to SARS CoV-1 or SARS CoV-2 viruses, receipt of any other COVID-19 vaccine, compromised immune function, history of autoimmune disease, uncontrolled HIV, HBV, or HCV infection, abnormal autoantibody test, fever, or acute illness within 2 days of first dose, and acute respiratory illness within 14 days of first dose.

Research design . This study is a Phase I preliminary, open-label (subjects and investigators know the drugs being tested), single-center clinical study, aiming to evaluate the safety of the SARS-CoV-2 vaccine MVC-COV1901 and immunogenicity. This study was a dose-escalation study involving three different groups of subjects aged 20 to 49 years. Each sub-phase contains 15 subjects. Groups 1a, 1b and 1c were administered three different doses (5, 15 and 25 μg) of S-2P recombinant protein respectively. The vaccination plan consisted of two doses, administered intramuscularly into the deltoid muscle of the non-dominant arm on day 1 and day 29 of the experiment, 28 days apart.

Cohort 1a: Four sentinel subjects were recruited to receive a vaccine containing 5 μg of S-2P recombinant protein to assess the preliminary safety of the vaccine. If the four sentinel subjects do not have grade ≥ 3 adverse events (AEs) or serious adverse events (SAEs) within 7 days after the first vaccination, the remaining subjects in Phase 1a will continue to be treated. Subjects were vaccinated.

Cohort 1b: An additional 4 sentinel subjects were recruited to receive a vaccine containing 15 μg of S-2P recombinant protein. If the four sentinel subjects do not experience grade ≥ 3 adverse events (AEs) or serious adverse events (SAEs) within 7 days after the first vaccination, the remaining subjects in Phase 1b will continue to be vaccinated.

Cohort 1c: An additional 4 sentinel subjects were recruited to receive a vaccine containing 25 μg of S-2P recombinant protein. If the four sentinel subjects do not experience grade ≥ 3 adverse events (AEs) or serious adverse events (SAEs) within 7 days after the first vaccination, the remaining subjects in Phase 1c will continue to be vaccinated.

Vital signs and electrocardiogram (ECG) examinations were performed before and after vaccination. Subjects were observed for at least 30 minutes after each vaccination to confirm any immediate adverse events (AEs), and were asked to report preset local (pain, erythema, swelling/induration) symptoms within 7 days after each vaccination. and systemic (fever, myalgia, malaise/fatigue, nausea/vomiting, diarrhea) adverse events (AEs) were recorded in the subjects' daily record cards. Non-prespecified adverse events (AEs) were recorded within 28 days after each vaccination; all other adverse events (AEs), serious adverse events (SAEs), and adverse events of special interest were recorded throughout the study period (approximately 7 months) (adverse events of special interest, AESIs). Serum samples were collected for hematological, biochemical, and immunological evaluation.

Immunogenicity endpoints were 14 days (Day 15) and 28 days (Day 29) after the first vaccination, and 14 days (Day 43) and 28 days (Day 57) after the second vaccination , and to evaluate neutralizing antibody titers and binding antibody titers 90 and 180 days after the second vaccination. Convalescent serum samples from 35 recovered COVID-19 patients were also tested (Mitek COVID-19 Panel 1.1 and COVID-19 Panel 1.4, obtained from Access Biologicals, Inc., Vista, CA, USA). Cellular immune responses were assessed 28 days after the second vaccination with IFN-γ ELISpot and IL-4 ELISpot.

SARS-CoV-2 spike protein specific immunoglobulin G (IgG) : Total serum anti-thorn protein was detected by direct enzyme-linked immunosorbent assay (ELISA) using a custom-made 96-well plate coated with S-2P recombinant protein antigen. The IgG antibody titer of the protein.

SARS-CoV-2 pseudovirus neutralization test : perform serial dilutions of the test sample (initial dilution factor is 1:20, followed by two-fold dilution, and the final dilution factor is 1:2560). The diluted serum was mixed and incubated with an equal volume of pseudovirus (1000 TU), and then added to a cell culture plate containing HEK293-hACE2 cells (1 x ¹⁰ cells/well). The amount of pseudovirus entering cells was calculated by lysing cells and measuring relative luciferase units (RLU). Treat uninfected cells as 100% neutralization and cells transduced with virus only as 0% neutralization. Calculate the dilution factor (ID ₅₀ ) required to achieve 50% inhibition and determine the geometry of ID ₅₀ Average power price (GMT).

Wild-type SARS-CoV-2 neutralization assay . The SARS-CoV-2 virus (hCoV-19/Taiwan/CGMH-CGU-01/2020, GenBank accession number MT192759) was titrated to obtain its TCID ₅₀ , and Vero E6 cells (2.5 x 10 ⁴ cells/well) were inoculated and culture in 96-well plates. The serum was diluted twofold to a final dilution factor of 1:8192, and the diluted serum was mixed with an equal volume of virus solution containing 100 TCID ₅₀ . The serum-virus mixture was cultured and then added to culture plates containing Vero E6 cells and further cultured. Neutralizing potency was defined as the highest dilution factor capable of inhibiting 50% of the cytopathic effect (CPE NT ₅₀ ) and was calculated using the Reed-Muench method. Analyzes were performed using the same validation tests using a reference serum sample 20/130 from the National Institute for Biological Standards and Control (NIBSC; Potters Bar, UK) as a comparator.

Cellular immune response . The number of antigen-specific IFN-γ or IL-4 secreted spot forming units (SFU) was determined by ELISpot analysis. Cryopreserved peripheral blood mononuclear cells (PBMC) were quickly thawed and allowed to stand overnight. 1 x ¹⁰ cells per well for IFN-γ ELISpot assay (Human IFN-γ ELISpot Kit, Mabtech, Stockholm, Sweden) or ² x 10 cells per well for IL-4 ELISpot assay (Human IFN-γ ELISpot kit, Mabtech, Stockholm, Sweden). Cells were stimulated with a peptide library (PepTivator SARS-CoV-2 Prot_S1, Miltenyi Biotec). This peptide library mainly consists of a 15-mer sequence covering the S1 domain sequence at the N-terminus of the SARS-CoV-2 spike protein. There was 11 amino acid overlap between the polymer sequences and the stimulated cells were cultured at 37°C for 24-48 hours. Cells stimulated with CD3-2 monoclonal antibody (mAb) were used as positive control. Detect IFN-γ or IL-4 release according to the user manual, and count spots using a CTL automated ELISpot reader. The average spot-forming units (SFU) calculated from three replicates of stimulation in the peptide library were calculated and normalized by subtracting the average of the negative control replicates (control medium). Results are expressed as spot-forming units (SFU) per million peripheral blood mononuclear cells (PBMC).

Statistical analysis . Safety analysis was performed on the total vaccinated group (TVG) population who received at least 1 dose of vaccine. Immunogenicity endpoints include the geometric mean potency (GMT) and seroconversion rate (SCR) of antigen-specific immunoglobulins, wild-type virus and pseudovirus neutralizing antibody titers. Seroconversion rate (SCR) is defined as the increase in antibody titer in the sample ≥ 4 times from the baseline or half of the lower limit of detection (LoD) (if the baseline cannot be measured). Percentage of subjects. The geometric mean titer (GMT) and seroconversion rate (SCR) are presented with two-sided 95% CI. The antigen-specific cellular immune response was expressed as the average value determined by IFN-γ ELISpot and IL-4 ELISpot.

result

Security . No serious adverse events (SAEs) or adverse events of special interest (AESIs) had occurred at the time of data cutoff in this study. This study was not modified or interrupted. Figure 11 summarizes the occurrence of prespecified adverse events (AEs). The most common local adverse event (AEs) was pain/tenderness (80.0%), while malaise/fatigue (28.9%) was the most common systemic AEs across all treatment groups. All local and systemic adverse events (AEs) were minor, except for one case of discomfort/fatigue in the 25 μg dose group. None of the subjects developed fever. Prespecified adverse events (AEs) were similar after the first and second vaccinations. Evaluation of safety laboratory values, ECG interpretation, and other nonprespecified adverse events did not reveal areas of particular concern.

Humoral immune response . The humoral immunogenicity results are summarized in Figures 12A to 12C. As shown in Figure 12A, the titer of IgG antibodies bound to spike protein increased rapidly after the second vaccination, and all subjects experienced seroconversion on days 43 and 57. The geometric mean titer (GMT) of the antibody reached its peak on day 43, with the peak values of the 5 μg, 15 μg and 25 μg dose groups being 7178.2 (95% CI: 4240.3 - 12151.7) and 7746.1 (95% CI: 5530.2 - respectively). 10849.8), 11220.6 (95% CI: 8325.7). On day 43, the GMT of antibodies in the 5 μg, 15 μg, and 25 μg dose groups ranged from 3.3 to 5.1 times the GMT of the convalescent serum samples (2179.6, [95% CI: 1240.9 - 3828.4]).

As shown in Figure 12B, before vaccination, no subject had detectable pseudovirus neutralizing potency ( _ID50 ) at the lower limit of the serum concentration tested (1:20 dilution). On day 43, the pseudovirus neutralizing potency (ID ₅₀ ) of the 5 μg, 15 μg and 25 μg dose groups reached the peak, and their geometric mean potency (GMT) were 538.5 (95% CI: 261.9 - 1107.0), respectively. 993.1 (95% CI: 655.0 - 1505.7), and 1905.8 (95% CI: 1601.7 - 2267.8). All subjects (100%) seroconverted after the second vaccination. On day 43, the GMT of antibodies in the 5 μg, 15 μg, and 25 μg dose groups ranged from 1.25 to 4.4 times the GMT of the convalescent serum samples (430.5, [95% CI : 274.9 - 674.0]).

The results of neutralizing antibody titers for wild-type SARS-CoV-2 virus are summarized in Figure 12C. Prior to vaccination, no subject had detectable wild-type virus neutralizing titers ( _NT50 ) at the lower limit of serum concentration tested in this assay (1:8 dilution). After the second vaccination, neutralizing responses were confirmed in serum samples from all subjects in the 15 μg and 25 μg dose groups. On day 43, the antibody geometric mean titers (GMT) for the 5 μg, 15 μg, and 25 μg dose groups were 33.3 (95% CI: 18.5 - 59.9), 76.3 (95% CI: 53.7 - 108.3), and 167.4, respectively. (95% CI: 122.1 - 229.6). On day 57, the antibody geometric mean titers (GMT) were similar between 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. On day 43, the GMT values for the 5 μg, 15 μg, and 25 μg dose groups ranged from 0.8, 1.8, and 3.9 times (42.7, [ 95%CI: 26.4 - 69.0]; price range from undetected to 631.0). All subjects in the 15 μg and 25 μg dose groups seroconverted on days 43 and 57; the neutralizing titers of some subjects were even similar to NIBSC reference serum 20/130 (281.8).

Cellular immune response . The results of the cellular immune response are summarized in Figure 13. All subjects had the fewest IFN-γ-secreting T cells before vaccination. By day 57, an average of 161.3, 85.5, and 94.9 IFN-γ-secreting T cells per million cells were observed in subjects vaccinated with 5 μg, 15 μg, and 25 μg of vaccine, respectively. Before vaccination, all subjects had the fewest 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 subjects vaccinated with 5 μg, 15 μg, and 25 μg of vaccine, respectively. The cellular immune response induced by MVC-COV1901 showed a significant increase in the number of IFN-γ-producing cells, indicating the existence of a Th1-biased immune response.

Overall, the predicted adverse events were mostly mild and similar. None of the subjects developed fever. After the second dose, all three doses evaluated, 15 μg and 25 μg, elicited moderately neutralizing antibody responses, and all subjects experienced seroconversion and Th1-biased T cell immune responses. Therefore, 15 μg of S-2P recombinant protein and the combination of CpG 1018 and aluminum hydroxide were considered sufficient to elicit a significant humoral immune response. The results also show that the MVC-COV1901 vaccine is well tolerated and elicits a strong immune response, making it suitable for further development.

Example 5 by CpG as an adjuvant S-2P Recombinant protein subunit vaccine "MVC-COV1901" right SARS-CoV-2 Variants of high concern ( Variants of Concern, VoCs) Assessment of the neutralizing ability of

Mutated strains have been detected at regular intervals since the COVID-19 pandemic began. Some of them, called variants of high concern (VoCs), were found to have mutations in the key receptor-binding domain (RBD). The primary target for antibody recognition and neutralization. The most representative of these variants of high concern (VoCs) are B.1.1.7 (Alpha variant), B.1.351 (Bata variant), and P1 (Gamma variant). The proteins all carry the N501Y mutation in the receptor binding domain (RBD). Variants of high concern (VoCs) harboring these mutations have been found to reduce the neutralizing capacity of monoclonal and vaccine-induced antibodies, which may render current treatments as well as vaccines ineffective (Garcia-Beltran et al., Cell, 184(9) ):2372-2383.e9, 2021). This example provides studies involving the use of sera from two sources to investigate the neutralizing capacity of the MVC-COV1901 vaccine against SARS-CoV-2 variants of high concern (VoCs): rat sera from animal toxicology studies, and Human sera from Phase I clinical trials.

A.MVC-COV1901 Vaccine triggers resistance in rats SARS-CoV-2 Variants of High Concern (VoCs) neutralizing ability.

Materials and methods

Animal research . Crl:CD Sprague Dawley (SD) rats were obtained from Resco Biotechnology Co., Ltd. (Taipei, Taiwan) and studied at the biosafety testing facility of Qihong Biotechnology Co., Ltd. (New Taipei City, Taiwan) . Immunization of SD rats was performed as described in Example 2, Part C. Briefly, rats were immunized three times at 2-week intervals with 5, 25, or 50 µg of S-2P recombinant protein adjuvanted with 1,500 µg of CpG 1018 and 750 µg of aluminum hydroxide. Serum was collected two weeks after the second immunization (day 29) or two weeks after the third immunization (day 43) and analyzed to express SARS-CoV-2 Wuhan wild-type (WT) or B.1.351 variant strains ( Beta mutant strain) spike protein pseudovirus for neutralization test.

Pseudovirus neutralization assay . Lentiviruses expressing the spike protein of the SARS-CoV-2 Wuhan-Hu-1 wild-type strain (WT) were constructed and neutralization assays were performed as described in Example 2, Part C. A lentivirus expressing the spike protein of the B.1.351 variant (Beta variant; GenBank accession number MZ314998.1) was constructed in the same manner, but the wild-type spike protein sequence was replaced with the sequence of the mutant.

Statistical analysis . Statistical analysis was performed with Prism 6.01 (GraphPad Software, Inc., San Diego, CA, USA). Significance was calculated using two-way ANOVA with Tukey's multiple comparisons test and the Kruskal-Wallis method with corrected Dunn's multiple comparisons test, as described in the respective figure brief descriptions. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

result

Antibodies induced by MVC-COV1901 in rats effectively neutralized the mutant strain and were as effective as neutralizing the wild-type virus . As shown in Figure 14, on days 29 and 43, the antibodies remained effective against the B.1.351 (Beta variant) virus, although the potency was slightly lower than against the wild-type virus. It is worth noting that the geometric mean price (GMT) of the ID ₅₀ and ID ₉₀ serum sampled two weeks after the third immunization (day 43) was higher than that of the serum sampled two weeks after the second immunization (day 29). serum, indicating that the neutralizing activity of the third immunization against this variant strain of high concern (VoCs) has a tendency to increase. This effect was particularly evident in the low dose (5 µg) group. By day 43, all dose groups achieved similar levels of ID ₅₀ (Figure 14, left panel) and ID ₉₀ (Figure 14, right panel) against the B.1.351 variant.

In conclusion, the rat study showed that by administering three doses of the vaccine, the inventors were able to induce similar levels of neutralizing titers in the three dose groups. Given that a three-dose regimen produced high immunogenicity against this variant, these results can be extrapolated to humans, as additional doses of immunization may be a strategy to increase immunity against variants of high concern (VoCs).

B.MVC-COV1901 Vaccines trigger resistance in humans SARS-CoV-2 Variants of High Concern (VoCs) neutralizing ability.

Materials and methods

clinical trials . Forty-five human subjects aged 20 to 49 years participated in a pilot, open-label, single-center dose-escalation Phase I clinical study. The study offers three separate sub-phases for subjects aged 20 to 49 years: Phase 1a, Phase 1b, and Phase 1c. There were 15 subjects in each sub-phase. The clinical trial used three different doses in three separate sub-phases: low dose (LD; 5 µg) in Phase 1a, mid-dose (MD; 15 µg) in Phase 1b, and high dose in Phase 1c (HD; 25 µg) of S-2P recombinant protein in each phase and adjuvanted with 750 µg CpG 1018 and 375 µg aluminum hydroxide (containing approximately 375 µg Al ³⁺ ). The vaccination plan included two doses, with 0.5 mL of vaccine injected intramuscularly into the deltoid muscle of the non-dominant arm on the 1st day and 29th day of the experiment, 28 days apart. On day 57 (4 weeks after the second vaccination), serum samples were collected for pseudovirus neutralization tests. Please see Example 4 for details of this clinical trial.

Pseudovirus neutralization assay . Lentiviruses expressing the spike protein of the SARS-CoV-2 Wuhan-Hu-1 wild-type strain (WT) were constructed and neutralization assays were performed as described in Example 2, Part C. The expression D614G, B.1.1.7 (Alpha variant; GenBank accession number MZ314997.1), B.1.351 (Beta variant; GenBank accession number MZ314998.1), and P1 (Gamma variant; GenBank accession number) were constructed in the same way. LR963075), and the lentivirus of the spike protein of B.1.429 (Epsilon variant; GenBank accession number MW591579), but the wild-type spike protein sequence was replaced with the sequence of each variant.

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

result

Antiserum from humans vaccinated with MVC-COV1901 can effectively neutralize the D614G , B.1.1.7 (Alpha) , and P1 (Gamma) mutant strains, but is less effective against the B.1.351 (Beta) and B.1.429 (Epsilon) mutant strains. and the effect is relatively weakened . Figure 15A-C shows data from pseudovirus neutralization assays with human sera containing wild type, D614G, B.1.1.7 (Alpha), B.1.351 (Beta), P1 (Gamma), and B. 1.429 (Epsilon) mutant strain. Although the neutralizing antibody potency against D614G and B.1.1.7 (Alpha variant) in all dose groups (LD, MD and HD groups) was slightly lower than that against wild-type virus (Figure 15A-C), However, the degree of reduction is not statistically significant. However, when comparing wild-type virus and B.1.351 (Beta variant), the neutralizing antibody titers against B.1.351 (Beta variant) were significantly lower in all dose groups (LD, MD, and HD groups). In comparison, the neutralizing antibody titers against P1 (Gamma variant) in all dose groups (LD, MD, and HD groups) were higher than those against the wild-type virus. Although when comparing the wild-type virus strain with B.1.429 (Epsilon variant), the neutralizing antibody potency against B.1.429 (Epsilon variant) was significantly reduced in the low-dose (LD) and mid-dose (MD) groups (Figure 15A -B), but the neutralizing antibody titer against B.1.429 (Epsilon variant) in the high-dose (HD) group was not significantly different from that against the wild-type virus strain (Figure 15C). When the neutralizing antibody titers for each dose group were plotted against these variants, a dose-dependent effect was observed (Figure 15A-C). By using higher doses of antigen, the neutralizing antibody potency against B.1.351 (Beta variant), P1 (Gamma variant), and B.1.429 (Epsilon variant) can be increased.

In conclusion, vaccinated human subjects in the Phase I clinical trial showed reduced but still significant neutralizing ability against B.1.351 (Beta variant), P1 (Gamma variant), and B.1.429 (Epsilon variant) (ID ₅₀ ), especially the higher dose group. The results showed that vaccination with two doses of MVC-COV1901 can induce neutralizing antibodies against SARS-CoV-2 variants in a dose-dependent manner.

Of course, many changes and modifications can be made to the above-described embodiments of the invention without departing from the scope of the invention. Accordingly, in order to promote the advancement of science and useful fields, this invention is disclosed and is intended to be limited only by the scope of the appended claims.

The 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 refer to the same or similar elements of specific embodiments.

Figure 1 shows the results of a neutralization test using sera from mice immunized with SARS-CoV-2 S-2P recombinant protein with or without aluminum phosphate adjuvant.

Figure 2 shows the results of a neutralization test using sera from mice immunized with different formulations of SARS-CoV-2 S-2P recombinant protein.

Figure 3 shows the neutralizing antibodies induced by SARS-CoV-2 S-2P recombinant protein adjuvanted with CpG 1018 and aluminum hydroxide 2 weeks after the second injection. BALB/c mice (N = 6 per group) were immunized with 2 doses of SARS-CoV-2 S-2P recombinant protein, separated by 3 weeks between doses. Antisera expressed on Chinese hamster ovary (CHO) cells and adjuvanted with CpG 1018, aluminum hydroxide, or a combination of both were harvested 2 weeks after the second injection. Neutralization tests were conducted with antisera against pseudoviruses expressing SARS-CoV-2 spike protein to determine the potency of neutralizing antibodies with ID ₅₀ (left) and ID ₉₀ (right). ** p ＜ 0.01, *** p ＜ 0.001.

Figure 4 shows the potency of total anti-Spin IgG antibodies in mice immunized with S-2P recombinant protein containing adjuvant. The total amount of IgG antibodies against spinin in the serum of BALB/c mice (N = 6 per group) sampled from the description of Figure 3 was quantified using enzyme linked immunosorbent assay (ELISA). BALB/c mice were immunized with 0, 1, or 5 µg of S-2P recombinant protein along with CpG 1018, aluminum hydroxide, or a combination of both. *** p < 0.001.

Figure 5 shows the neutralizing effect of antibodies induced by the SARS-CoV-2 S-2P recombinant protein using CpG 1018 and aluminum hydroxide as adjuvants against the wild-type SARS-CoV-2 virus. Antisera were collected as described in the legend to Figure 4 (N = 6 per group), and neutralization assays were performed with wild-type SARS-CoV-2 to determine the potency of neutralizing antibodies. ** p < 0.01, *** p < 0.001.

Figure 6 shows the antibodies produced by mice immunized with SARS-CoV-2 S-2P recombinant protein adjuvanted with CpG 1018 and aluminum hydroxide, against the D614D (wild type) or D614G (variant) form. The inhibitory effect of spike protein on pseudoviruses. Antisera were collected from BALB/c mice immunized with 1 or 5 µg of S-2P recombinant protein and 10 µg of CpG 1018 and 50 µg of aluminum hydroxide as described in the legend to Figure 5 (Due to limitations in detection capabilities, N = 5 per group). Neutralization tests were conducted with pseudoviruses carrying spike proteins of D614D or D614G.

Figure 7 shows IFN- /IL-4, IFN- /IL-5, and IFN- /IL-6 ratio. Using IFN- derived from cytokine analysis , IL-4, IL-5, and IL-6 values (N = 6 per group) to calculate ratios. A ratio greater than 1 indicates a bias toward Th1 responses, while a ratio less than 1 indicates a bias toward Th2 responses. * p < 0.05, ** p < 0.01.

Figures 8A-8B show the determination of neutralizing antibody titers using pseudovirus in hamsters 2 weeks after the second immunization. Hamsters (N = 10 per group) were immunized twice with an interval of 3 weeks. The hamsters were immunized with vehicle control (PBS), 1 μg (low dose group, LD) or 5 μg (high dose group, HD) of S-2P recombinant protein. Immunization was performed with 150 μg CpG 1018 and 75 μg aluminum hydroxide as adjuvant, or with the adjuvant alone. Antisera were collected 2 weeks after the second injection, and neutralization assays were performed using pseudoviruses expressing the SARS-CoV-2 spike protein to determine the ID values of _neutralizing antibodies (Figure 8A), and overall antiserum was analyzed by ELISA. Valence of IgG antibodies against spikein (Fig. 8B). Results are expressed as geometric means, error bars represent 95% confidence intervals, and statistical significance was calculated using Kruskal-Wallis with corrected Dunn's multiple comparisons test. The dashed lines indicate the lower and upper limits of detection (40 and 5120, respectively, in ID ₉₀ and 100 and 1,638,400, respectively, in IgG ELISA). *** p < 0.001, **** p < 0.0001.

Figures 9A-9B show the viral load of hamsters 3 or 6 days post infection (dpi) after infection with SARS-CoV-2 virus. Hamsters were euthanized at 3 or 6 days post-infection, and lung tissue samples were collected for quantitative PCR of viral genomic RNA to determine viral load (Fig. 9A) and viral titer by TCID ₅₀ assay (Fig. 9B). Results are expressed as geometric means, error bars represent 95% confidence intervals, and statistical significance was calculated using Kruskal-Wallis with corrected Dunn's multiple comparisons test. The dashed line indicates the lower limit of detection (100). * p < 0.05, ** p < 0.01.

Figure 10 shows the lung pathology scores of hamsters 3 or 6 days after infection with SARS-CoV-2 virus. Hamsters were euthanized 3 or 6 days after infection, and lung tissue samples were collected for sectioning and staining. Histopathology sections were scored as described in the Example Methods and results were tabulated. The results are presented as the mean value of lung pathology scores, and error bars represent standard errors. Statistical significance was calculated through one-way ANOVA and Tukey's multiple comparison test. **** p < 0.0001.

Figure 11 shows a summary of adverse events reported in Phase I clinical trials. Subjects were required to record local and systemic adverse events in their daily record cards for up to 7 days after each vaccination. Adverse events (AEs) were tabulated and graded as mild, moderate, or severe.

Figures 12A-12C show a summary of humoral immune responses in Phase I clinical trials. IgG antibodies against spike protein were measured by ELISA in sera from subjects vaccinated with 5, 15, or 25 μg of MVC-COV1901 (Fig. 12A) and by pseudovirus neutralization assay (Fig. 12B) or live virus neutralization The assay (Figure 12C) measures neutralizing antibody titers. Human convalescent sera (HCS) from 35 recovered COVID-19 patients were compared through the same assay, and the NIBSC 20/130 international serum standard was used as a standard in a live virus neutralization assay (Figure 12C asterisk in ). The horizontal bar represents the geometric mean price, and the error bars represent the 95% confidence interval.

Figure 13 shows a summary of cellular immune responses in Phase I clinical trials. Cells were stimulated with peptides from the S1 peptide library and cultured at 37°C for 24-48 hours. Cells stimulated with CD3-2 monoclonal antibody (mAb) were used as positive controls. ELISpot analysis was used to detect IFN-γ (left) or IL-4 (right). The average spot-forming units (SFU) calculated from three replicates of stimulation in the peptide library were calculated and normalized by subtracting the average of the negative control replicates (control medium). Results are expressed as SFU per million peripheral blood mononuclear cells (PBMCs). The horizontal bar represents the mean and the error bars represent the standard deviation.

Figure 14 shows the neutralization of SARS-CoV-2 pseudoviruses carrying the spike protein of wild-type or B.1.351 (Beta) mutant strains using antisera from rats vaccinated with adjuvanted S-2P recombinant protein. and function. Rats were immunized three times with the specified amount of S-2P recombinant protein containing adjuvant, with an interval of 2 weeks each time. Antisera from five male rats were pooled into one sample, and antisera from five female rats were pooled into another sample. Thus two pooled samples were generated for each dose group (N=2). Antisera were collected two weeks after the second immunization (day 29) or two weeks after the third immunization (day 43), samples were pooled as above, and expressed as SARS-CoV-2 Wuhan wild-type or B .1.351 mutant strain spike protein pseudovirus was subjected to a neutralization test to determine the ID ₅₀ and ID ₉₀ valence of neutralizing antibodies. The results are represented as bars representing the geometric mean force valence, while the symbols represent values for each sample.

Figures 15A-15C show the neutralizing effect of antisera from clinical trial subjects vaccinated with different doses of MVC-COV1901 vaccine on SARS-CoV-2 pseudoviruses carrying wild-type or mutant spike proteins. Serum samples from the Phase I clinical trial of MVC-COV1901 subjects were collected 4 weeks after the second immunization (56 days after the first immunization). The ID ₅₀ neutralizing potency of all dose groups, including low dose (LD; Figure 15A), medium dose (MD; Figure 15B), and high dose (HD; Figure 15C), was measured in a pseudovirus neutralization assay. Each point in the graph represents the neutralizing titer of an individual serum sample. Kruskal-Wallis and corrected Dunn's multiple comparison tests were performed, and the statistical significance of the mutant strains compared with the wild type is shown above the corresponding column. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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Claims (10)

An immunogenic composition against novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), comprising an antigenic recombinant protein and an adjuvant, the adjuvant comprising an aluminum-containing adjuvant and an Synthetic oligodeoxynucleotide (ODN) of unmethylated cytosine-phosphate-guanosine (CpG) motif, wherein the antigenic recombinant protein has a SEQ ID NO: amino acid sequence shown in 5 or 6. The immunogenic composition of claim 1, wherein the aluminum-containing adjuvant includes aluminum hydroxide, aluminum oxyhydroxide, aluminum hydroxide gel, aluminum phosphate, aluminum phosphate gel, aluminum hydroxyphosphate, and aluminum hydroxyphosphate sulfate. , amorphous aluminum hydroxyphosphate sulfate, potassium aluminum sulfate, aluminum monostearate or combinations thereof. The immunogenic composition of claim 2, wherein a 0.5 ml dose of the immunogenic composition contains about 250 to about 500 μg of Al ³⁺ , or about 375 μg of Al ³⁺ . The immunogenic composition of claim 1, wherein the synthetic oligodeoxynucleotide (ODN) comprising an unmethylated CpG motif comprises SEQ ID NO: 8, SEQ ID NO: 9, Sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or combinations thereof. The immunogenic composition of claim 4, wherein a 0.5 ml dose of the immunogenic composition contains about 750 to about 3000 μg of oligodeoxynucleotides, or wherein the immunogenic composition contains about 750 μg , about 1500 μg, or about 3000 μg of oligodeoxynucleotide. The use of an immunogenic composition as described in claim 1 in the preparation of a medicament for inducing an immune response against a novel coronavirus (SARS-CoV-2) in a subject in need thereof. The use as described in claim 6, wherein the immune response includes the production of neutralizing antibodies against SARS-CoV-2 and a Th1-biased immune response. The use of an immunogenic composition as described in claim 1 in preparing a medicament for protecting a subject in need from infection with a novel coronavirus (SARS-CoV-2). The use of an immunogenic composition as described in claim 1 in preparing a medicament for preventing a subject in need of COVID-19 infection. The use according to any one of claims 6 to 9, wherein the immunogenic composition is administered by intramuscular injection.