AU2021290765B2 - Immunogenic composition against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - Google Patents

Abstract

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

Description

WO wo 2021/254473 PCT/CN2021/100826

Immunogenic Composition Against Severe Acute Respiratory Syndrome Coronavirus 2

(SARS-CoV-2)

CROSS-REFERENCE TO RELATED APPLICATIONS This

[0001] This application claims application claims priority prioritytoto and thethe and benefit of U.S. benefit Provisional of U.S. Application Provisional No. Application No.

63/040,696, filed on June 18, 2020 and PCT Application No. PCT/US21/020277, filed on March

01, 2021, the disclosure of which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION

[0002] 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 protein and S protein adjuvant. and adjuvant.

2. DESCRIPTION OF THE PRIOR ART

[0003] In In thethe endend of of 2019, 2019, a novel a novel coronavirus coronavirus emerged emerged andand waswas identified identified as as a cause a cause of of a a

cluster of respiratory infection cases. The viral pathogen did not match any other known virus

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, tiredness,

muscle or body aches, sore throat, diarrhea, conjunctivitis, headache, loss of taste or smell, a rash

on skin, and shortness of breath. While the majority of cases result in mild symptoms, some

progress to acute respiratory distress syndrome (ARDS), precipitated by cytokine storm,

multi-organ failure, septic shock, and blood clots. The first confirmed death from the coronavirus

infection occurred on January 9, and as of 13 June 2021, 175,306,598 confirmed cases of

COVID-19, including 3,792,777 deaths, have been reported to the WHO. The numbers are still

growing fast.

[0004] To reduce the risk of SARS-CoV-2 infection without curtailing everyday activities, a

COVID-19 vaccine is needed. In particular, a COVID-19 vaccine that is able to rapidly induce an

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immune response against SARS-CoV-2 is urgently needed needed.

SUMMARY OF THE INVENTION

[0005] In one aspect, the present invention provides an immunogenic composition against a

severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising an antigenic

recombinant protein and an adjuvant selected from the group consisting of an aluminum-containing adjuvant, an unmethylated cytosine-phosphate-guanosine (CpG) motif, and

a combination thereof, wherein the antigenic recombinant protein substantially consists of

residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987

and a "GSAS" substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization

domain.

[0006] In another aspect, the present invention provides a method for eliciting an immune

response against a 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 present invention.

[0007] In In anotheraspect, another aspect, the the present presentinvention inventionprovides a method provides for protecting a method a subject for protecting in a subject in

need thereof from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2),

comprising administering to the subject an effective amount of the immunogenic composition of

the present invention.

[0008] In another aspect, the present invention provides a method for preventing a subject in in

need thereof from contracting COVID-19 disease, comprising administering to the subject an

effective amount of the immunogenic composition of the present invention.

[0009] In In another another aspect, aspect, thethe present present invention invention provides provides useuse of of thethe immunogenic immunogenic composition composition

of the present invention for eliciting an immune response against a severe acute respiratory

syndrome coronavirus 2 (SARS-CoV-2) in a subject in need thereof.

[0010] In another aspect, the present invention provides use of the immunogenic composition

of the present invention for protecting a subject in need thereof from infection with severe acute

respiratory syndrome coronavirus 2 (SARS-CoV-2).

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[0011] In In another another aspect, aspect, thethe present present invention invention provides provides useuse of of thethe immunogenic immunogenic composition composition

of the present invention for preventing a subject in need thereof from contracting COVID-19

disease.

These

[0012] These andand other other aspects aspects will will become become apparent apparent from from thethe following following description description of of thethe

preferred embodiment taken in conjunction with the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] TheThe accompanying accompanying drawings drawings illustrate illustrate oneone or or more more embodiments embodiments of of thethe invention invention and, 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

like elements of an embodiment.

[0014] FIG. 1 shows the results from neutralization assays using sera from mice immunized

with the SARS-CoV-2 S-2P recombinant protein with or without aluminum phosphate adjuvant.

FIG.

[0015] FIG. 2 shows 2 shows thethe results results from from neutralization neutralization assays assays using using sera sera from from mice mice immunized immunized

with different formulations of the SARS-CoV-2 S-2P recombinant protein.

[0016] FIG. 3 shows induction of neutralizing antibodies by CpG 1018 and aluminum

hydroxide-adjuvanted SARS-CoV-2 S-2P at 2 weeks post-second injection. BALB/c mice (N = 6

per group) were immunized with 2 dose levels of Chinese hamster ovary (CHO) cell-expressed

SARS-CoV-2 S-2P adjuvanted with CpG 1018, aluminum hydroxide or a combination of both 3

weeks apart and the antisera were harvested at 2 weeks after the second injection. The antisera

were subjected to neutralization assay with pseudovirus expressing SARS-CoV-2 spike protein to

determine the ID50 (left) ID (left) and and IDID90 (right) (right) titers titers of neutralization of neutralization antibodies. antibodies. **p **p < 0.01, < 0.01, ***p< <

0.001.

[0017] FIG. 4 shows total anti-S IgG titers in mice immunized with S-2P with adjuvants. Sera

from BALB/c mice in FIG. 3 (N = 6 per group) immunized with 0, 1 or 5 ug µg of S-2P with CpG

1018, aluminum hydroxide or a combination of both were quantified for the total amount of anti-

S IgG in an enzyme linked immunosorbent assay (ELISA). < 0.001.

[0018] FIG. 5 shows neutralization of wild-type SARS-CoV-2 virus by antibodies induced by

SARS-CoV-2 S-2P adjuvanted with CpG 1018 and aluminum hydroxide. The antisera were

collected as described in FIG. 4 (N = 6 per group) and subjected to a neutralization assay with

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wild-type SARS-CoV-2 to determine neutralization antibody titers. **p<0.01,***p<0.001. **p<0.01, < ***p<0.001.

[0019] FIG. 6 shows inhibition of pseudoviruses carrying D614D (wild-type) or D614G

(variant) versions of the spike protein by mice immunized with S-2P with CpG 1018 and

aluminum hydroxide. The antisera of BALB/c mice immunized with 1 or 5 ug µg of S-2P with 10

ug µg CpG 1018 and 50 ug µg aluminum hydroxide as in Fig. 5 (N = 5 per group due to assay capacity)

were collected. Neutralization assays were performed with pseudoviruses with either D616D or

D614G spike proteins.

[0020] FIG. 7 shows IFN-y/IL-4, IFN-y/IL-5, IFN-/IL-4, IFN-/IL-5, and and IFN-y/IL-6 IFN-/IL-6 ratios. ratios. IFN-y, IFN-, IL-4, IL-4, IL-5, IL-5, and and

IL-6 values from the cytokine assays (N = 6 per group) were used to calculate ratios. Ratio values

greater than1 1indicate greater than indicate Th1 Th1 biasbias whereas whereas ratio ratio less less than than 1 indicate 1 indicate Th2 bias responses. Th2 bias responses. *p < 0.05, 0.05,

**p<0.01.

[0021] FIGS. 8A-8B show neutralizing antibody titers with pseudovirus assay in hamsters 2

weeks after second immunization. Hamsters (N=10 per group) were immunized twice at 3 weeks

apart with vehicle control (PBS), 1 ug µg (LD) or 5 ug µg (HD) of S-2P adjuvanted with 150 ug µg CpG

1018 and 75 ug µg aluminum hydroxide, or with adjuvant alone. The antisera were harvested at 2

weeks after the second injection and subjected to neutralization assay with pseudovirus

expressing SARS-CoV-2 spike protein to determine the ID90 titers ID titers ofof neutralization neutralization antibodies antibodies

(FIG. 8A) and total anti-S IgG antibody titers with ELISA (FIG. 8B). Results are presented as

geometric mean with error bars representing 95% confidence interval and statistical significance

calculated with Kruskal-Wallis with corrected Dunn's multiple comparisons test. Dotted lines

represent lower and upper limits of detection (40 and 5120 in ID90, 100 ID, 100 and and 1,638,400 1,638,400 inin IgG IgG

ELISA). ***p<0.001,****p<0.0001. ***p<0.001, ****p< 0.0001.

[0022] FIGS. 9A-9B show viral load in hamsters 3 or 6 days post infection (dpi) with SARS-

CoV-2. The hamsters were euthanized at 3 or 6 dpi and lung tissue samples were collected for

viral load determination by quantitative PCR of viral genome RNA (FIG. 9A), and TCID50 assay TCID assay

for virus titer (FIG. 9B). Results are presented as geometric mean with error bars representing

95% confidence interval and statistical significance calculated with Kruskal-Wallis with corrected

Dunn's multiple comparisons test. Dotted lines represent lower and limit of detection (100). *p <

0.05, **p<0.01.

FIG.

[0023] FIG. 1010shows showslung lung pathology pathology scoring scoringin in hamsters 3 or36 or hamsters days 6 post days infection (dpi) with post infection (dpi) with

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SARS-CoV-2. The hamsters were euthanized at 3 or 6 dpi and lung tissue samples were collected

for sectioning and staining. The histopathology sections were scored as outlined in the methods

and the results tabulated. Results are presented as mean of lung pathology scores with error bars

representing standard error and statistical significance calculated with one-way ANOVA with

Tukey's multiple Tukey's multiple comparisons comparisons test. test. p 0.0001. ****p< < 0.0001. <

[0024] FIG. 11 shows summary of solicited adverse events in a Phase I clinical trial.

Participants were asked to record solicited local and systemic adverse events in the participant's

diary card for up to 7 days after each vaccination. Solicited adverse events (AEs) were tabulated

and graded as mild, moderate, or severe.

[0025] FIGS. 12A-12C shows summary of humoral immune response in the Phase I clinical

trial. Sera of participants vaccinated with 5, 15, or 25 ug µg of MVC-COV1901 were measured for

anti-spike IgG by ELISA (FIG. 12A), and neutralization titers were measured by pseudovirus

neutralization assay (FIG. 12B) or live virus neutralization assay (FIG. 12C). Human

convalescent sera (HCS) from 35 recovered COVID-19 patients were analyzed by the same

assays for comparison and NIBSC 20/130 standard was used in the live virus neutralization assay

as a standard (asterisk in FIG. 12C). Bars indicate geometric mean titers and error bars indicate

95% confidence intervals.

FIG.

[0026] FIG. 1313shows showssummary summary of of cellular cellularimmune response immune in the response in Phase I clinical the Phase trial. Cells I clinical trial. Cells

were stimulated with a S1 peptides pool of peptides and incubated at 37°C for 24-48 hours. Cells

stimulated stimulatedwith CD3-2 with mAb mAb CD3-2 served as a as served positive control. a positive IFN-y (left control. IFN-panel) (leftorpanel) IL-4 (right panel) or IL-4 (right panel)

were detected using an ELISpot assay. The mean of spot-forming units (SFU) counted in peptide

pool stimulation triplicate was calculated and normalized by subtracting the mean of the negative

control replicates (control media). Results were expressed as SFU per million PBMC. Bars

indicate the mean values and error bars indicate standard deviations.

FIG.

[0027] FIG. 14 14 shows shows neutralization neutralization of of SARS-CoV-2 SARS-CoV-2 pseudovirus pseudovirus bearing bearing wildtype wildtype or or B.1.351 B.1.351

(Beta) variant spike proteins by antisera of rats vaccinated with adjuvanted S-2P. Rats were

immunized three times at 2 weeks apart with the indicated amounts of adjuvanted S-2P. Antisera

from five males were pooled into one sample and those of 5 females were pooled into another

sample. This resulted in two pooled samples (N=2) for each dose group. The antisera were

harvested two weeks after the second immunization (Day 29) or two weeks after the third

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immunization (Day 43), pooled as described above and subjected to neutralization assay with

pseudovirus expressing SARS-CoV-2 Wuhan wildtype or B.1.351 variant spike protein to

determine the ID50 and ID and IDID90 titers titers of neutralizing of neutralizing antibodies. antibodies. Results Results are are presented presented as bars as bars

representing geometric mean titers with symbols representing value of each sample.

[0028] FIGS. 15A-15F show neutralization of SARS-CoV-2 pseudovirus bearing wildtype or

variant spike proteins by antisera of clinical trial subjects vaccinated with different does of

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 from the first

immunization). ID50 neutralizing ID neutralizing titers titers for for low low dose dose (LD; (LD; FIG. FIG. 15A), 15A), mid-dose mid-dose (MD; (MD; FIG. FIG. 15B), 15B),

high dose (HD; FIG. 15C), ID90 neutralizing ID neutralizing titers titers for for low low dose dose (LD; (LD; FIG. FIG. 15D), 15D), mid-dose mid-dose (MD; (MD;

FIG. 15E), high dose (HD; FIG. 15F), and all dose groups were measured with pseudovirus

neutralization assays. Results are represented with each dot representing individual serum sample

neutralizing titer. Kruskal-Wallis with corrected Dunn's multiple comparisons test was performed

and statistical significance of variants relative to wildtype is shown above respective column. p **p

< 0.01, 0.01, <***, 0.001, ****p<< 0.0001. 0.001, 0.0001.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0029] TheThe present present invention invention relates relates to to an an immunogenic immunogenic composition composition against against SARS-CoV-2. SARS-CoV-2.

The immunogenic composition comprises an antigenic recombinant protein and an adjuvant

containing aluminum-containing adjuvant and/or an unmethylated an cytosine-phosphate-guanosine (CpG) motif. The antigenic recombinant protein comprises

residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987

and a "GSAS" substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization

domain.

[0030] In In some some embodiments, embodiments, thethe residues residues 14-1208 14-1208 of of SARS-CoV-2 SARS-CoV-2 S protein S protein with with proline proline

substitutions at residues 986 and 987 and a "GSAS" substitution at residues 682 - 685 comprise

an amino acid sequence of SEQ ID NO: 1 or the amino acid sequence at least 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, or 99% to SEQ ID NO: 1.

[0031] In some embodiments, the C-terminal T4 fibritin trimerization motif comprises an

amino acid sequence of SEQ ID NO: 2 or the amino acid sequence at least 90%, 91%, 92%, 93%,

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94%, 95%, 96%, 97%, 98%, or 99% to SEQ ID NO: 2.

[0032] In some embodiments, the antigenic recombinant protein comprises an amino acid

sequence of SEQ ID NO: 5 or 6 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98%,

or 99% to SEQ ID NO: 5 or 6.

[0033] In some embodiments, the aluminum-containing adjuvant comprises 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 a

combination thereof.

[0034] In In some some embodiments, embodiments, a 0.5 a 0.5 ml ml dose dose of of thethe immunogenic immunogenic composition composition comprises comprises from from

about 250 to about 500 ug µg A1 ³, or A1³, or about about 375 375 µg ug A1³.

[0035] 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.

[0036] In In some some embodiments, embodiments, a 0.5 a 0.5 ml ml dose dose of of thethe immunogenic immunogenic composition composition comprises comprises from from

about 750 to about 3000 ug µg of the oligonucleotide, or wherein the immunogenic composition

comprises about 750 ug, µg, about 1500 ug, µg, or about 3000 ug µg of the oligonucleotide.

[0037] TheThe presentinvention present invention also also relates relatesto to a method for for a method eliciting an immune eliciting responseresponse an immune against against

a 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

the present invention.

[0038] The present invention also relates to a method for protecting a subject in need thereof

from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising

administering to the subject an effective amount of the immunogenic composition of the present

invention.

[0039] TheThe presentinvention present invention also also relates relatestoto a method for for a method preventing a subject preventing in need in a subject thereof need thereof

from contracting COVID-19 disease, comprising administering to the subject an effective amount

of the immunogenic composition of the present invention.

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[0040] TheThe present present invention invention also also relates relates to to useuse of of thethe immunogenic immunogenic composition composition of of thethe

present invention for eliciting an immune response against a severe acute respiratory syndrome

coronavirus 2 (SARS-CoV-2) in a subject in need thereof, the method comprising administering

to the subject in need thereof an effective amount of the immunogenic composition.

[0041] In some embodiments, the immune response comprises production of neutralizing

antibodies against SARS-CoV-2 and Thl-skewed Th1-skewed immune response.

[0042] The present invention also relates to use of the immunogenic composition of the

present invention for protecting a subject in need thereof from infection with severe acute

respiratory syndrome coronavirus 2 (SARS-CoV-2), the method comprising administering to the

subject in need thereof an effective amount of the immunogenic composition.

[0043] The present invention also relates to use of the immunogenic composition of the

present invention for preventing a subject in need thereof from contracting COVID-19 disease,

the method comprising administering to the subject in need thereof an effective amount of the

immunogenic composition.

[0044] In In some some embodiments, embodiments, thethe immunogenic immunogenic composition composition is is administered administered by by intramuscular intramuscular

injection.

[0045] TheThe meaning meaning of of thethe technical technical andand scientific scientific terms terms as as described described herein herein cancan be be clearly clearly

understood by a person of ordinary skill in the art.

[0046] As used herein, the singular form "a", "an", and "the" includes plural references unless

indicated otherwise. For example, "an" excipient includes one or more excipients.

[0047]

[0047] Thephrase The phrase"comprising" "comprising"asasused usedherein hereinisisopen-ended, open-ended,indicating indicatingthat thatsuch such

embodiments may include additional elements. In contrast, the phrase "consisting of' is closed,

indicating that such embodiments do not include additional elements (except for trace impurities).

The phrase "consisting essentially of' is partially closed, indicating that such embodiments may

further comprise elements that do not materially change the basic characteristics of such

embodiments.

[0048] As used interchangeably herein, the terms "polynucleotide" and "oligonucleotide"

include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA

(ssRNA) and double-stranded RNA (dsRNA), modified oligonucleotides and oligonucleosides or

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combinations thereof. The oligonucleotide can be linearly or circularly configured, or the

oligonucleotide can contain both linear and circular segments. Oligonucleotides are polymers of

nucleosides joined, generally, through phosphodiester linkages, although alternate linkages, such

as phosphorothioate esters may also be used in oligonucleotides. A nucleoside consists of a

purine (adenine (A) or guanine (G) or derivative thereof) or pyrimidine (thymine (T), cytosine (C) (C)

or uracil (U), or derivative thereof) base bonded to a sugar. The four nucleoside units (or bases) in in

DNA are called deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine. A nucleotide is

a phosphate ester of a nucleoside.

[0049] As used herein, the terms "severe acute respiratory syndrome coronavirus 2

(SARS-CoV-2)" refers to the strain of coronavirus 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 50-200 nanometres in diameter, with 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 create the viral

envelope. The spike protein is the protein responsible for allowing the virus to attach to and fuse

with the membrane of a host cell; specifically, its S1 subunit catalyzes attachment, the S2 subunit

fusion.

[0050] As As used used herein, herein, thethe terms terms "immunogenic "immunogenic composition composition against against severe severe acute acute respiratory respiratory

syndrome coronavirus 2 (SARS-CoV-2)" refers to a composition for stimulating or eliciting an

immune response against a SARS-CoV-2. The immune response includes, but not limited to,

production of neutralizing antibodies against SARS-CoV-2 and Thl-skewed Th1-skewed immune response.

[0051] As used herein, the terms "aluminum-containing adjuvant" refers to an adjuvant

including aluminum. In some embodiments, the aluminum-containing adjuvant includes, but 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 a combination thereof. In some embodiments, the aluminum-containing adjuvant

is an aluminum-containing adjuvant approved for administration to humans by the FDA. In some

embodiments, the aluminum-containing adjuvant is an aluminum hydroxide adjuvant approved

for administration to humans by the FDA. In some embodiments, the aluminum-containing

adjuvant is an aluminum phosphate adjuvant approved for administration to humans by the FDA.

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[0052] As As used used herein, herein, thethe terms terms "unmethylated "unmethylated cytosine-phosphate-guanosine cytosine-phosphate-guanosine (CpG) (CpG) motif" motif"

refers to a CpG- containing oligonucleotide in which the C is unmethylated, and which

contributes to a measurable immune response as measured in vitro, in vivo, and/or ex vivo. In

some embodiments, the CpG-containing oligonucleotide contains palindromic hexamers

following the general formula of: 5'-purine-purine- CG-pyrimidine-pyrimidine-3' CG-pyrimidine-pyrimidine-3'.In Insome some

preferred embodiments, the unmethylated cytosine-phosphate-guanosine (CpG) motif has an

oligonucleotide of SEQ ID NO: 8 (5'-TGACTGTGAACGTTCGAGATGA-3') inwhich (5'-TGACTGTGAACGTTCGAGATGA-3) in whichthe theCs Cs

of the CGs are unmethylated. In some embodiments, the CpG-containing oligonucleotide

contains TCG in which the C is unmethylated, and which is from 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") (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3') in which the Cs of the TCGs are unmethylated. Examples of the unmethylated cytosine-phosphate-guanosine (CpG) motif further

includes, but not limited to, 5'-GGTGCATCGATGCAGGGG GG-3' (SEQ ID NO: 10), 11), 11), 5'-TCCATGGACGTTCCTGAGCGTT-31 5'-TCCATGGACGTTCCTGAGCGTT-3' (SEQ ID NO: 5'-TCGTCGTTCGAACGACGTTGAT-3' (SEQ ID NO: 12), and 5'-TCGTCGACGATCGGC GCGCGCCG-3' (SEQ ID NO: 13). The CpG-containing oligonucleotide described herein are in

their pharmaceutically acceptable salt form unless otherwise indicated. In one preferred

embodiment, the CpG-containing oligonucleotides are in the sodium salt form.

[0053] An "effective amount" or a "sufficient amount" of a substance is that amount sufficient

to effect beneficial or desired results, including clinical results, and, as such, an "effective amount"

depends upon the context in which it is being applied. In the context of administering an

immunogenic composition, an effective amount contains sufficient adjuvant and SARS-CoV-2

S-2P recombinant protein to elicit an immune response. An effective amount can be administered

in one or more doses.

[0054] The terms "individual" and "subject" refer to mammals. "Mammals" include, but are

not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals,

rodents (e.g., mice and rats) and pets (e.g., dogs and cats).

[0055] The term "dose" as used herein in reference to an immunogenic composition refers to a

measured portion of the immunogenic composition taken by (administered to or received by) a

subject at any one time.

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[0056] TheThe terms"isolated" terms "isolated" and and "purified" "purified"as as used herein used refers herein to a material refers that is that to a material removed is removed

from at least one component with which it is naturally associated (e.g., 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"

[0057] "Stimulation" of of a response a response or or parameter parameter includes includes eliciting eliciting and/or and/or enhancing enhancing that that

response or parameter when compared to otherwise same conditions except for a parameter of

interest, or alternatively, as compared to another condition (e.g., increase in TLR-signaling in the

presence of a TLR agonist as compared to the absence of the TLR agonist). For example,

"stimulation" of an immune response means an increase in the response. Depending upon the

parameter measured, the increase may be from 5-fold to 500-fold or over, or from 5, 10, 50, or

100-fold to 500, 1,000, 5,000, or 10,000-fold.

[0058] As As used used herein herein thethe term term "immunization" "immunization" refers refers to to a process a process that that increases increases a mammalian a mammalian

subject's reaction to antigen and therefore improves its ability to resist or overcome infection.

[0059] The term "vaccination" as used herein refers to the introduction of vaccine into a body

of a mammalian subject.

[0060] "Adjuvant" refers to a substance which, when added to a composition comprising an

antigen, nonspecifically enhances or potentiates an immune response to the antigen in the

recipient upon exposure.

[0061] The present invention is further illustrated by the following examples, which are

provided for the purpose of demonstration rather than limitation. Those of skill in the art should,

in light of the present disclosure, appreciate that many changes can be made in the specific

embodiments which are disclosed and still obtain a like or similar result without departing from

the spirit and scope of the invention.

[0062]

[0062] EXAMPLES EXAMPLES

[0063] Example1 1Preparation

[0063] Example PreparationofofImmunogenic ImmunogenicCompositions CompositionsAgainst AgainstSARS-CoV-2 SARS-CoV-2

[0064] A plasmid A plasmid having having a polynucleotide a polynucleotide encoding encoding thethe residues residues 1-1208 1-1208 of of SARS-CoV-2 SARS-CoV-2 S S

protein (Wuhan-Hu-1 strain; GenBank: MN908947) with proline substitutions at residues 986

and 987, a "GSAS" substitution at the furin cleavage site (residues 682 - 685) (SEQ ID NO: 14)

and a C-terminal T4 fibritin trimerization domain (SEQ ID NO: 2), an HRV3C protease cleavage

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site (SEQ ID NO: 3), an 8x His Tag, and a Twin-Strep Tag (SEQ ID NO: 4) was transfected into

ExpiCHO-S cells (Thermo Fisher Scientific, Waltham, MA, USA).

Cell

[0065] Cell culturewas culture was harvested harvested after after6 6days, andand days, protein was purified protein from the was purified supernatant from the supernatant

using Strep-Tactin resin (IBA Lifesciences, Göttingen, Germany). 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). The purified SARS-CoV-2 S-2P recombinant protein (SEQ ID NO: 5 or 6) was then

formulated with an unmethylated CpG motif (CpG 1018, SEQ ID NO: 8) and/or aluminum-containing adjuvant, such as aluminum hydroxide (A1(OH)3) or aluminum (A1(OH)) or aluminum phosphate phosphate

(AIPO4) as the (AIPO) as the immunogenic immunogenic compositions compositions against against SARS-CoV-2. SARS-CoV-2.

[0066] Example 2 Immunogenicity of the Immunogenic Compositions Against SARS-CoV-2 in Mice

This

[0067] This exampleprovides example provides a a description description of of preclinical studies preclinical to assess studies the immunogenicity to assess the immunogenicity

of the immunogenic compositions against SARS-CoV-2 obtained from Example 1 in mice.

[0068] A. Preliminary test 1- SARS-CoV-2 S-2P recombinant protein formulated with

aluminum phosphate

[0069] Materials and

[0069] Materials and Methods Methods

[0070] Mouse immunizations. BALB/c mice aged 6-8 weeks (The Laboratory Animal Center,

Taiwan) (N = 5/group) were vaccinated with the SARS-CoV-2 S-2P recombinant protein at 0 and

3rd week. SARS-CoV-2 S-2P recombinant protein (a final concentration of 1 ug µg or 10 ug/mL) µg/mL)

diluted in PBS was mixed with aluminum phosphate (to a final concentration of 0.5 mg

aluminum/mL). Mice were inoculated with 100 uL µL intramuscularly (50 uL µL into each hind leg).

Two weeks after the final immunization, sera were collected for measurement of antibody

responses.

[0071] Pseudovirus production. cDNA encoding spike protein of Wuhan-Hu-1 strain (SEQ ID

NO: 7) was synthesized using the QuikChange XL kit (Stratagene, San Diego, CA, USA) and

then inserted into CMV/R plasmid. The CMV/R-SARS-CoV-2 spike plasmid was confirmed

using sequencing. 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%

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CO2. To produce CO. To produce SARS-CoV-2 SARS-CoV-2 pseudoviruses, pseudoviruses, CMV/R-SARS-CoV-2 CMV/R-SARS-CoV-2 spike spike plasmid plasmid was was co-transfected into HEK293T cells with packaging plasmid pCMVDR8.2 and transducing

plasmid pHR CMV-Luc, using Fugene 6 transfection reagent (Promega, Madison, WI, USA).

Seventy-two (72) hours post-transfection, supernatant was collected, filtered, and frozen at -80

°C. °C.

[0072] Pseudovirus infectivity and neutralization assay. 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. Pseudovirusinfectivity CO. Pseudovirus infectivitywas wasassessed assessedin inHuh7.5 Huh7.5

cells plated overnight in 96-well black/white isoplates (PerkinElmer, Waltham, MA, USA).

Twofold serial dilutions of pseudoviruses were added to resting Huh7.5 cells, in triplicate. After a

2-hour 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 of mouse sera (1:40, fourfold, eight dilutions) were mixed with

various pseudovirus strains, which were previously titered to target 50,000 RLU. Sigmoidal

curves, taking averages of triplicates at each dilution, were generated from RLU readings; 50%

neutralization neutralization titers (IC50) titers (IC)were calculated were considering calculated uninfected considering cells ascells uninfected 100% neutralization as 100% neutralization

and cells transduced with only virus as 0% neutralization.

[0073] Results

[0073] Results

[0074] The results of neutralization assay are shown in FIG. 1. SARS-CoV-2 S-2P

recombinant protein (0.1 ug/mouse µg/mouse and 1 ug/mouse) µg/mouse) formulated with aluminum phosphate elicited greater neutralization than the recombinant protein (0.1 ug/mouse µg/mouse and 1 ug/mouse) µg/mouse) alone.

These data demonstrate that aluminum phosphate significantly increase the immunogenicity of

the SARS-CoV-2 S-2P recombinant protein as an antigen of a vaccine against coronavirus disease

(COVID-19).

[0075] B. Preliminary test 2- SARS-CoV-2 S-2P recombinant protein formulated with the

combination of CpG and aluminum hydroxide

[0076] Materials and

[0076] Materials and Methods Methods

Mouse

[0077] Mouse immunizations. immunizations. BALB/c BALB/c mice mice aged aged 6-86-8 weeks weeks (The (The Laboratory Laboratory Animal Animal Center, Center,

Taiwan) (N = 6/group) were vaccinated with the SARS-CoV-2 S-2P recombinant protein at 0 and

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3rd week. SARS-CoV-2 S-2P recombinant protein (a final concentration of 10 ug µg or 50 ug/mL) µg/mL)

diluted in PBS was mixed with CpG 1018 (SEQ ID NO: 8) (to a final concentration of 0.1

mg/mL), aluminum hydroxide (to a final concentration of 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 with 100 uL µL

intramuscularly (50 uL µL into each hind leg). Two weeks after the final immunization, sera were

collected for measurement of antibody responses.

[0078] Pseudovirus production, pseudovirus infectivity, and neutralization assay. The methods

are described in section A.

[0079] Results

[0079] Results

[0080] The results of neutralization test are shown in FIG 2. SARS-CoV-2 S-2P recombinant

protein formulated with the combination of CpG and aluminum hydroxide elicited the highest

neutralizing activity both at a low dose (1 ug/mouse) µg/mouse) and a higher dose (5 ug/mouse). µg/mouse). In addition,

SARS-CoV-2 S-2P recombinant protein (1 ug/mouse) µg/mouse) formulated with aluminum hydroxide

alone elicited greater neutralization than the recombinant protein (1 ug/mouse µg/mouse and 5 ug/mouse) µg/mouse)

formulated with CpG alone. The recombinant protein formulated with CpG alone elicited

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 of a vaccine against coronavirus disease (COVID-19).

[0081] C. C. Development Development of of adjuvanted adjuvanted stable stable prefusion prefusion SARS-CoV-2 SARS-CoV-2 spike spike protein protein antigen antigen

[0082] Materials and Methods

[0083] Pseudovirus

[0083] Pseudovirusproduction productionand andtitration. titration.ToToproduce produceSARS-CoV-2 SARS-CoV-2pseudoviruses, pseudoviruses,a a

plasmid expressing full-length wild-type Wuhan-Hu-1 strain SARS-CoV-2 spike protein (SEQ

ID NO: 7) was co-transfected into HEK293T cells with packaging and reporter plasmids

pCMVA8.91 and pLAS2w.FLuc.Ppuro (RNAi Core, Academia Sinica), using TransIT-LT1

transfection reagent (Mirus Bio). Site-directed mutagenesis was used to generate the D614G

variant by changing nucleotide at position 23403 (Wuhan-Hu-1 reference strain) from A to G.

Mock pseudoviruses were produced by omitting the p2019-nCoV spike (WT). Seventy-two hours

post-transfection, supernatants were collected, filtered, and frozen at -80 °C. The transduction

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unit (TU) of SARS- CoV-2 pseudotyped lentivirus was estimated by using cell viability assay in

response to the limited dilution of lentivirus. In brief, HEK-293 T cells stably expressing human

ACE2 gene were plated on 96-well plate 1 day before lentivirus transduction. For the titering of

pseudovirus, different amounts of pseudovirus were added into the culture medium containing

polybrene. Spin infection was carried out at 1100xg in 96-well plate for 30 min at 37 °C. After

incubating cells at 37 °C for 16 hours, the culture media containing virus and polybrene were

removed and replaced with fresh complete DMEM containing 2.5 1g/ml µg/ml puromycin. After treating with puromycin for 48 h, the culture media were removed and cell viability was detected

by using 10% AlarmaBlue reagents according to manufacturer's instruction. The survival rate of

uninfected cells (without puromycin treatment) was set as 100%. The virus titer (transduction

units) was determined by plotting the survival cells versus diluted viral dose.

[0084] Pseudovirus-based neutralization assay. HEK293-hAce2 cells (2 104 X 10cells/well) cells/well)were were

seeded in 96-well white isoplates and incubated for overnight. Sera were 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, and then twofold serial dilutions were carried out (for a total of 8 dilution

steps to a final dilution of 1:5120). The diluted sera were mixed with an equal volume of

pseudovirus (1000 TU) and incubated at 37 °C for 1 h before adding to the plates with cells.

After the 1 h incubation, the culture medium was replaced with 50 uL µL of fresh medium. On the

following day, the culture medium was replaced with 100 uL µL of fresh medium. Cells were lysed

at 72 h post infections and relative luciferase units (RLU) were measured. The luciferase activity

was was detected detectedbyby Tecan i-control Tecan (Infinite i-control 500). The (Infinite 50%The 500). and 50% 90% inhibition dilution titers and 90% inhibition (ID50titers (ID dilution

and ID90) were calculated ID9) were calculated considering considering uninfected uninfected cells cells as as 100% 100% neutralization neutralization and and cells cells

transduced with only virus as 0% neutralization. Reciprocal ID50 and ID and IDID90 geometric geometric meanmean titers titers

(GMT) were both determined as ID90 titers are ID9 titers are useful useful when when ID ID50 titer titer levels levels areare consistently consistently

saturating at the upper limit of detection.

[0085] Wild-type SARS-CoV-2 neutralization, neutralization. The neutralization assay with SARS-CoV-2

virus was conducted as previously reported (Huang et al., J. Clin. Microbiol. 58(8): e01068-

e1120, 2020). Vero E6 cells (2.5 X 104 cells/well) were 10 cells/well) were seeded seeded in in 96-well 96-well plates plates and and incubated incubated

overnight. Sera were heated at 56 °C for 30 min to inactivate complement and diluted in

serum-free MEM at an initial dilution factor of 20, and then further twofold serial dilutions were

performed for a total of 11 dilution steps to a final dilution of 1:40,960. The diluted sera were

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mixed with an equal volume of SARS-CoV-2 virus at 100 TCID50/50 TCID/50 µLuL (hCoV- (hCoV- 19/Taiwan/CGMH-CGU-01/2020, GenBank 19/Taiwan/CGMH-CGU-01/2020, GenBank accession accession MT192759) MT192759) and and incubated incubated at at 37 37 °C °C for for 22

h. The sera-virus mixture was then added to 96-well plate with Vero E6 cells and incubated in

MEM with 2% FBS at 37°C for 5 days. After incubation, cells were fixed by adding 4% formalin

to each of the wells for 10 min and stained with 0.1% crystal violet for visualization. Results

were calculated with the Reed-Muench method for log 50% end point for ID50 and ID and log log 90% 90% end end

point point for forID90 ID titers. titers.

[0086] Immunization of mice. Female BALB/c and C57BL/6 mice were obtained from the

Laboratory Animal Center, Academia Sinica, Taiwan and BioLASCO Taiwan Co. Ltd. For

antigen formulation, SARS-CoV-2 S-2P protein was mixed with either an equal volume of CpG

1018 (SEQ ID NO: 8), aluminum hydroxide, PBS, or CpG 1018 plus aluminum hydroxide. Mice

aged 6-9 weeks were immunized twice (50 uL µL intramuscularly in each of the left and right

quadriceps femoris muscles per mouse) at 3 weeks apart as previously described (Pallesen et al.,

Proc. Natl. Acad. Sci. USA, 114(35): E7348-E7357, 2017). Total serum anti-S IgG and

anti-RBD IgG titers were detected with direct ELISA using custom 96-well plates coated with

S-2P antigen and an E. coli-expressed fragment of the S protein containing RBD region,

respectively.

[0087] Cytokine assays. Two weeks after the second injection, mice were euthanized and

splenocytes were isolated and stimulated with S-2P protein (2 ug/well) µg/well) as previously described

(Lu (Lu et et al. al.Immunology, 130(2): Immunology, 254-261, 130(2): 2010).2010). 254-261, For detection of IFN-y,of For detection IL-2, IL-4, IFN-, and IL-4, IL-2, IL-5, the and IL-5, the

culture supernatant from the 96-well microplates was harvested to analyze the levels of cytokines

by ELISA using Mouse IFN-y 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). The OD450

values were read by Multiskan GO (Thermo Fisher Scientific).

[0088] Dose range finding study for single- and repeat-dose intramuscular injection (IM) in

Sprague Dawley (SD) rats. Crl:CD Sprague Dawley (SD) rats were obtained from BioLASCO

Taiwan Co. Ltd. Animal studies were conducted in the Testing Facility for Biological Safety,

TFBS Bioscience Inc., Taiwan. SD rats aged 6-8 weeks were immunized with 5 ug, µg, 25 ug µg or 50

ug µg of S-2P adjuvanted with either 1500 ug µg CpG 1018 alone or 750 ug µg CpG 1018 combined with

375 ug µg aluminum hydroxide. The test article or vehicle control was administered intramuscularly

(0.25 mL/site, 2 sites of quadriceps femoris muscle) to each rat on Day 1 (for single-dose study)

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and and Day Day 15 15 (for (for repeat-dose repeat-dose study). study). The The observation observation period period was was 14 14 days days (for (for single-dose single-dose study) study)

and 28 days (for repeat-dose study). Parameters evaluated included clinical signs, local irritation

examination, moribundity/mortality, body temperature, body weights, and food consumption

during the in-life period. Blood samples were taken for hematology, including coagulation tests

and serum chemistry. All animals were euthanized and necropsied for gross lesion examination,

organ weights, and histopathology evaluation on injection sites and lungs.

Statistical

[0089] Statistical

[0089] analysis. analysis. For For neutralization neutralization assays, assays, geometric geometric meanmean titers titers are are represented represented by by

the heights of bars with 95% confidence intervals represented by the error bars. For cytokine and

rat data, heights of bars or symbols represent means with SD represented by error bars. Dotted

lines represent lower and upper limits of detection. Analysis package in Prism 6.01 (GraphPad)

was used for statistical analysis. The data were compared at the same S-2P dose level with

different adjuvant or at the same adjuvant system with varying antigen dose. Kruskal-Wallis with

corrected Dunn's multiple comparisons test was used for non-parametric test between more than

2 experimental groups. Mann-Whitney U-test was used to compare two experimental groups. For

correlation between antibody titers and neutralization titers, Spearman's rank correlation

coefficient waswas coefficient used. *p <0.05, used. < **p<0.01, <0.05, < 0.01,<< 0.001. < 0.001.

[0090] Results

[0090] Results

[0091] Induction of potent neutralizing antibodies by CpG 1018 and aluminum hydroxide-adjuvanted S-2P. To facilitate establishment of stable clones for clinical studies and

commercial production, the ExpiCHO system was used as the expression system of S-2P antigen.

The S-2P proteins produced in CHO cells and their structure displayed typical spike trimers

under cryo-EM, resembling that of 293-expressed SARS-CoV-2 S protein (Wrapp et al., Science,

367(6483): 1260-1263, 2020), suggesting that CHO cells are feasible in production of S-2P. Next,

the potential of Thl-biasing Th1-biasing CpG 1018 for clinical use was examined. Aluminum hydroxide

(hereafter abbreviated as alum) was tested along with CpG 1018 since alum has been

characterized to enhance the potency of CpG adjuvant when used in combination while also

retaining the property of inducing Thl Th1 responses (Thomas et al., Hum. Vaccin., 5(2): 79-84,

2009). The pseudovirus neutralization assay was performed with sera drawn either 3 weeks after

the first injection or 2 weeks after the second injection. At 3 weeks after the first injection,

neutralizing activities were already observed when mice were immunized with both 1 and 5 ug µg of

S-2P with CpG 1018 and alum. At 2 weeks after the second injection, reciprocal inhibition

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dilution 50 (ID50) GMT (ID) GMT ofof 245, 245, 3109, 3109, and and 5120 5120 were were obtained obtained with with immunization immunization ofof 1 1 µgug S-2P S-2P

adjuvanted with CpG 1018, alum, and with both CpG 1018 and alum, respectively (FIG. 3).

Similar trends were observed at 5 ug µg of S-2P in both BALB/c (FIG. 3) and C57BL/6 mice.

Sera

[0092] Sera from from these these mice mice were were then then examined examined forfor thethe amount amount of of anti-S anti-S IgG. IgG. CpGCpG 1018 1018 in in

combination with alum produced significantly higher titers of anti-S IgG compared to CpG 1018

alone (FIG. 4). To confirm the activities of the antibodies against the critical receptor-binding

domain (RBD) of the S protein, immune sera were examined for anti-RBD IgG and the results

were similar to that of the anti-S IgG with S-2P in combination with both CpG 1018 and alum

induced the highest amount of IgG titer. There was a moderate correlation between anti-S IgG

and anti-RBD IgG titers as shown by Spearman's rank correlation coefficient of 0.6486. The

immune sera were further tested for their neutralization capabilities against wild-type SARS-

CoV-2 in a neutralization assay. S-2P was able to inhibit SARS-CoV-2 at a concentration of 1 ug, µg,

although at lower potency than that of pseudovirus (FIG. 3, FIG. 5). The reciprocal ID50 GMT ID GMT ofof

1 ug µg S-2P in the presence of CpG 1018, alum, and with both CpG 1018 and alum were

approximately 60, 250, and 1500, respectively (FIG. 5). Pseudovirus carrying the current

dominant D614G variant spike was also generated and neutralizing antibodies from mice

immunized with S-2P with CpG 1018 and alum were effective against both pseudoviruses

carrying the wild-type D614 and mutant D614G versions of spike proteins (FIG. 6).

Neutralization titers of wild-type virus and pseudovirus and total anti-S IgG titers were all found

to be highly correlated with Spearman's rank correlation coefficients greater than 0.8.

[0093] CpG 1018 induced Th1 Thl immunity. To identify whether CpG 1018 could induce Th1

responses in the vaccine-adjuvant system, cytokines involved in Th1 and Th2 responses were

measured in splenocytes from mice immunized with S-2P with alum, CpG 1018, or combination

of the two. As expected, S-2P adjuvanted with alum induced limited amounts of IFN-y and IL-2, IFN- and IL-2,

the representative cytokines of Th1 response. In contrast, significant increases in IFN-y and IL-2 IFN- and IL-2

were detected most strongly in high antigen dose plus CpG 1018 and alum. For Th2 response,

while the levels of IL-4, IL-5 and IL-6 increased in the presence of alum and S-2P, addition of

CpG 1018 to alum suppressed the levels of IL-5 and IL-6. IFN-y/IL-4, IFN-y/IL-5, and IFN-/IL-4, IFN-y/IL-5, and IFN-/IL- IFN-y/IL-

6 ratios are strongly indicative of a Th1-biased Thl-biased response and were increased by approximately

36-, 130-, and two-fold, respectively, in the presence of S-2P combined with CpG 1018 and alum

(FIG. 7). These results suggested that the effect of CpG 1018 is dominant over alum in directing

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the cell-mediated response towards Th1 response, while retaining high antibody levels.

S-2P

[0094] S-2P diddid notnot result result in in systemic systemic adverse adverse effects effects in in rats. rats. To To elucidate elucidate thethe safety safety andand

potential toxicity of the vaccine candidate, 5 ug, µg, 25 ug µg or 50 ug µg of S-2P adjuvanted with 1500 ug µg

CpG 1018 or 750 ug µg CpG 1018 combined with 375 ug µg alum were administered to SD rats for

single-dose and repeat-dose studies. No mortality, abnormality of clinical signs, differences in

body weight changes, body temperature, nor food consumption were observed in either gender

that could be attributed to S-2P (with or without adjuvant) with single dose administration.

Increased body temperature at 4-h or 24-h after dosing was found in both genders of single-dose

study and repeat-dose study; however, these temperature changes were moderate and were

recovered after 48-h in both genders of all treated groups including controls (PBS). No gross

lesions were observed in organs of most of the male and female rats with single-dose and

two-dose administration, except for one male rat which was deemed to be non-vaccine-related. In

conclusion, S-2P protein, with CpG 1018 or CpG 1018 with alum as adjuvants administrated

intramuscularly once or twice to SD rats did not induce any systemic adverse effect.

[0095] The results show that in mice, two injections of a subunit vaccine consisting of the

prefusion spike protein (S-2P) adjuvanted with CpG 1018 and alum were effective in inducing

potent neutralization activity against both pseudovirus expressing wild-type and D614G variant

spike proteins, and wild-type SARS-CoV-2. The combination of S-2P with CpG 1018 and alum

elicited Th1-dominant Thl-dominant immune responses with high neutralizing antibody levels in mice and

showed no major adverse effects in rats. Therefore, the inventors have demonstrated in this

Example that the S-2P combined with adjuvant CpG 1018 in combination with alum induced

potent Thl-biased Th1-biased immune responses to prevent wild-type virus infections while retaining high

antibody levels that show cross-neutralization of variant viruses. Therefore, the immunogenic

compositions against SARS-CoV-2 of the present invention serves as an ideal vaccine candidate

in alleviating the burden of the global COVID-19 pandemic.

[0096] Example 3 Protection from SARS-CoV-2 Challenge by the Immunogenic Compositions Against SARS-CoV-2 in Hamster

[0097] Materials and

[0097] Materials and Methods Methods

[0098] Pseudovirus-based neutralization assay and IgG ELISA. Lentivirus expressing the

Wuhan-Hu-1 strain SARS-CoV-2 spike protein was constructed and the neutralization assay

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performed as described in Example 2. Briefly, HEK293-hACE2 cells were seeded in 96-well

white isoplates and incubated overnight. Sera from vaccinated and unvaccinated hamsters were

heat-inactivated and diluted in MEM supplemented with 2% FBS at an initial dilution factor of

20, and then 2-fold serial dilutions were carried out for a total of 8 dilution steps to a final

dilution of 1:5120. The diluted sera were mixed with an equal volume of pseudovirus (1,000 TU)

and incubated at 37°C for 1 hour before adding to the plates with cells. Cells were lysed at 72

hours post-infection and relative luciferase units (RLU) was measured. The 50% and 90%

inhibition dilution titers (ID50 and (ID and ID90) ID9) were were calculated calculated referencing referencing uninfected uninfected cells cells as as 100% 100%

neutralization and cells transduced with only virus as 0% neutralization. Total serum anti-S IgG

titers were detected with direct ELISA using custom 96-well plates coated with S-2P antigen.

[0099] Immunization and challenge of hamsters. Female golden Syrian hamsters aged 6-9

weeks old on study initiation were obtained from the Laboratory Animal Center (Taipei, Taiwan).

The hamsters were randomized from different litters into four groups (n=10 for each group):

hamsters were vaccinated intramuscularly with 2 injections of vehicle control (PBS), 1 or 5 ug µg of

S-2P protein adjuvanted with 150 ug µg CpG 1018 and 75 ug µg aluminum hydroxide (alum), or

adjuvant alone at 3 weeks apart. The hamsters were bled at 2 weeks after the second

immunization via submandibular vein to confirm presence of neutralizing antibodies. Hamsters

were challenged at 4 weeks after the second immunization with X 1 104 X 10PFU PFUof ofSARS- SARS-CoV-2 CoV-2

TCDC#4 (hCoV-19/Taiwan/4/2020, GISAID Accession ID: EPI_ISL_411927) intranasally in a

volume of 100 uL µL per hamster. The hamsters were divided into two cohorts to be euthanized on 3

and 6 days after challenge for necropsy and tissue sampling. Body weight and survival rate for

each hamster were recorded daily after infection. On days 3 and 6 after challenge, hamsters were

euthanized by carbon dioxide. The right lung was collected for viral load determination (RNA

titer and TCID5 assay). The TCID assay). The left left lung lung was was fixed fixed in in 4% 4% paraformaldehyde paraformaldehyde for for histopathological histopathological

examination.

[0100] Quantification of viral titer in lung tissue by cell culture infectious assay (TCID50). The (TCID). The

middle, inferior, and post-caval lung lobes of hamsters were homogenized in 600 ul µl of DMEM

with 2% FBS and 1% penicillin/streptomycin using a homogenizer. Tissue homogenate was

centrifuged at 15,000 rpm for 5 minutes and the supernatant was collected for live virus titration.

Briefly, 10-fold serial dilutions of each sample were added onto Vero E6 cell monolayer in

quadruplicate and incubated for 4 days. Cells were then fixed with 10% formaldehyde and

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stained with 0.5% crystal violet for 20 minutes. The plates were washed with tap water and

scored scored for forinfection. The The infection. fifty-percent tissuetissue fifty-percent cultureculture infectious dose (TCID50)/mL infectious was calculated dose (TCID)/mL was calculated

by the Reed and Muench method (Reed and Muench, American Journal of Epidemiology, 27(3):

493-497, 1938).

[0101] Real-time RT-PCR for SARS-CoV-2 RNA quantification. To measure the RNA levels of

SARS-CoV-2, specific primers targeting 26,141 to 26,253 region of the envelope (E) gene of

SARS-CoV-2 genome were used in a TaqMan real-time RT-PCR method (Corman et al.,

Eurosurveillance. 25(3): 2000045, 2020). Forward primer E-Sarbeco-F1 5'-

ACAGGTACGTTAATAGTTAATAGCGT-3' (SEQ ID ACAGGTACGTTAATAGTTAATAGCGT-3 (SEQ ID NO: NO: 15) 15) and and the the reverse reverse primer primer E- E-

Sarbeco-R2 5'-ATATTGCAGCAGTACGCACACA-3' (SEQID 5'-ATATTGCAGCAGTACGCACACA-3 (SEQ IDNO: NO:16), 16),in inaddition additionto tothe the

probe E-Sarbeco-P1 5'-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BBQ-3' (SEQ ID 5'-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BBQ-3 (SEQ ID NO: NO: 17) were used. A total of 30 uL µL RNA solution was collected from each lung sample using

RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions. Five (5) uL µL

of RNA sample was added into a total 25 uL µL mixture of the Superscript III one-step RT-PCR

system with Platinum Taq Polymerase (Thermo Fisher Scientific, USA). The final reaction mix

contained 400 nM forward and reverse primers, 200 nM probe, 1.6 mM of deoxyribonucleoside

triphosphate (dNTP), 4 mM magnesium sulfate, 50 nM ROX reference dye, and 1 uL µL of enzyme

mixture. Cycling conditions were performed using a one-step PCR protocol: 55°C for 10 min for

first-strand cDNA synthesis, followed by 3 min at 94°C and 45 amplification cycles at 94°C for

15 sec and 58°C for 30 sec. Data was collected and calculated by 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 copy numbers of the viral genome. The

oligonucleotides were synthesized by Genomics BioSci and Tech Co. Ltd. (Taipei, Taiwan).

[0102] Results

[0103] Hamsters as SARS-CoV-2 virus challenge model. To develop a SARS-CoV-2 virus

challenge model in hamsters for the S2-P vaccine, an initial study was conducted to determine the

optimal dose of virus for the challenge experiments. Unvaccinated hamsters were inoculated with

10 , 104, 10³, or 10 10, or 105 PFU PFU ofof SARS-CoV-2 SARS-CoV-2 and and euthanized euthanized onon Day Day 3 3 oror 6 6 after after infection infection for for tissue tissue

sampling. sampling.Following Followinginfection of 10³ infection of to 10³105 toPFU 10 of PFUSARS-CoV-2, the hamsters of SARS-CoV-2, exhibitedexhibited the hamsters

dose-dependent weight loss. Hamsters infected with 103 10³ PFU gained weight while 104 and 10 10 and 105

PFU- PFU- infected infected hamsters hamsters experienced experienced progressively progressively severe severe weight weight loss loss at at 66 days days post-infection post-infection

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(dpi). However, there were no significant differences between levels of viral genome RNA and

viral titer measured in 103 10³ to 105 PFU of 10 PFU of SARS-CoV-2-infected SARS-CoV-2-infected hamsters hamsters at at 33 and and 66 dpi. dpi. All All

dosages of virus resulted in elevated lung pathology, even at 103 10³ PFU where the animals did not

experience weight loss. There was also no virus inoculation dose-dependent effect on lung

pathology scores and lung viral load. Therefore 104 PFUof 10 PFU ofvirus viruswas wasused usedfor forvirus viruschallenge challenge

studies as it provides an adequate balance between clinical signs and virus titer for inoculation.

[0104] Administration of S-2P adjuvanted with CpG 1018 and aluminum hydroxide to

hamsters induced high levels of neutralizing antibodies. Hamsters were divided into four groups

receiving two immunizations at 21 days apart of either vehicle control (PBS only), adjuvant alone,

low dose (LD) or high dose (HD) of S-2P in combination with CpG 1018 and aluminum

hydroxide (S-2P+CpG 1018+alum). No differences in body weight changes were observed after

vaccination among the four groups. Fourteen days after the second immunization, high level of

neutralizing antibody titers were found in both LD and HD groups with ninety-percent inhibition

dilution (ID90) geometric (ID) geometric mean mean titer titer (GMT) (GMT) ofof 2,226 2,226 and and 1,783, 1,783, respectively respectively (FIG. (FIG. 8A). 8A). Anti-S Anti-S

IgG antibody levels were high enough that several individual samples reached the upper

threshold of detection, with GMTs of LD and HD groups of 1,492,959 and 1,198,315,

respectively (FIG. 8B). In general, even at a low dose, S-2P+CpG 1018+alum induced potent

levels of immunogenicity in hamsters.

Adjuvanted

[0105] Adjuvanted S-2P S-2P protected protected hamsters hamsters from from clinical clinical signs signs andand viral viral load load after after SARS- SARS-

CoV-2 challenge. Four (4) weeks after the second immunization, hamsters were challenged with

104 PFU of 10 PFU of SARS-CoV-2 SARS-CoV-2 virus virus and and body body weights weights were were tracked tracked up up to to 33 or or 66 days days post post infection infection

(dpi). Groups of animals were sacrificed on 3 or 6 dpi for viral load and histopathology analyses.

LD and HD vaccinated groups did not show weight loss up to 3 or 6 days after virus challenge

and instead gained 5 and 3.8 g of mean weight at 6 dpi, respectively. The protective effect was

most significant at 6 dpi in both vaccinated groups, while vehicle control and adjuvant only

groups experience significant weight loss. Lung viral load measured by viral RNA and TCID, TCID

assays showed that both viral RNA and viral titer decreased significantly at 3 dpi in vaccinated

hamsters and dropped to below the lower limit of detection at 6 dpi (FIGS. 9A-9B). Note that

viral viral load, load,especially viral especially titertiter viral measured by TCID50 measured dropped by TCID noticeably dropped at 6 dpi at noticeably in 6control dpi inand control and

adjuvant only groups due to hamsters' natural immune response (FIGS. 9A-9B). Lung sections

were analyzed and pathology scoring was tabulated (FIG. 10). There were no differences at 3 dpi

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between control and experimental groups; 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 immunized groups

(FIG. 10). These results showed that S-2P+CpG 1018+alum induced a robust immune response

that was able to suppress viral load in lungs and prevent weight loss and lung pathology in

infected hamsters.

[0106] All of the hamsters in the S-2P+CpG 1018+alum-immunized groups were protected

with significantly reduced lung pathology (generally graded minimal to mild, with a mean score

of 1.72 in LD and HD groups), in contrast to diffuse alveolar damage (graded moderate to severe,

with a mean score of 4.09 in vehicle and adjuvant control groups) caused by the virus in the lungs

of hamsters, in the control groups at 6 dpi. The significance of this study lies not only in the

demonstration of in vivo efficacy, but also in safety. The viral challenge study allowed for the

assessment of risk of disease enhancement with the vaccine candidate. The histopathology scores

of the immunized groups have not differed from the non-challenged animals, indicative of a lack

of vaccine-enhanced pathology. The result of the study in this Example provides more data that

supports progression of the vaccine candidate's clinical development.

[0107] Example 4 Safety and Immunogenicity of a CpG-adjuvanted S-2P Subunit

Vaccine "MVC-COV1901" in Humans

This

[0108] This Example Example provides provides a Phase a Phase I study I study conducted conducted in in healthy, healthy, human human subjects subjects to to assess assess

safety and immunogenicity of a SARS-CoV-2 subunit vaccine (i.e., the immunogenic

composition of the present invention). The SARS-CoV-2 subunit vaccine, which is referred to

herein as "S-2P+CpG 1018+alum" or "MVC-COV1901", is described in greater detail in

Example 1.

[0109] Vaccines. MVC-COV1901 is formulated in with three different dosages of SARS-

CoV-2 Spike (S) protein with CpG 1018 and aluminum hydroxide as adjuvants. Each

ug of S-2P adjuvanted with 750 µg MVC-COV1901 vaccine contains 5, 15, or 25 µg ug of CpG 1018

and 375 ug µg (Al equivalent to weight) of aluminum hydroxide, administered as a single 0.5 mL

intramuscular (IM) injection.

[0110] Participant. The study aimed to enroll 45 subjects. Eligible participants were healthy

adults 20 to 49 years of age. Eligibility was determined based on medical history, physical

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examination, laboratory tests, and investigators' clinical judgment. Exclusion criteria included a

history of known potential exposure to SARS CoV-1 or 2 viruses, having received any other

COVID-19 vaccine, impaired immune function, history of autoimmune disease, uncontrolled

HIV, HBV, or HCV infection, abnormal autoantibody tests, febrile or acute illness within 2 days

of first dose, and acute respiratory illness within 14 days of first dose.

[0111] Study Design. This study is a phase I prospective, open-labeled, single-center study to

evaluate the safety and immunogenicity of the SARS-CoV-2 vaccine MVC-COV1901. This study was a dose escalation study with three separate groups of participants 20 to 49 years of age.

Each sub-phase consisted of 15 participants. The three different dose levels employed were 5, 15,

and 25 ug µg of S-2P protein for cohort 1a, 1b, and 1c, respectively. The vaccination schedule

consisted of two doses, administered by IM injection in the deltoid muscle of the non-dominant

arm 28 days apart, on Day 1 and Day 29.

[0112] Cohort 1a: Four sentinel participants were to be recruited to receive vaccine with 5 ug µg

of S-2P totoevaluate of S-2P evaluatethethe preliminary preliminary safety safety data data of the of the vaccine. vaccine. If no If no Grade Grade 3event 3 adverse adverse (AE) event (AE)

or serious adverse event (SAE) occurred within 7 days after the first dose in the 4 sentinel

participants, dosing of the remaining participants in Phase la and Phase 1b would proceed.

[0113] Cohort 1b: Another 4 sentinel participants were to be enrolled to receive vaccine with

15 ug µg of S-2P. If no > Grade Grade 33 AE AE or or SAE SAE occurred occurred within within 77 days days after after the the first first dose dose in in the the 44

sentinel participants, dosing of the remaining participants in Phase 1b and Phase 1c would

proceed.

[0114] Cohort 1c: Another 4 sentinel participants would be enrolled to receive vaccine with 25

ug µg of S-2P. If no Grade Grade3 3AE AEor orSAE SAEoccurred occurredwithin within7 7days daysafter afterthe thefirst firstdose dosein inthe the4 4

sentinel participants, dosing of the remaining participants in Phase 1c would proceed.

Vital

[0115] Vital signs signs andand electrocardiogram electrocardiogram (ECG) (ECG) were were performed performed before before andand after after vaccination. vaccination.

Participants were observed for at least 30 min after each dose to identify any immediate AEs, and

were asked to record solicited local (pain, erythema, swelling/induration) and systemic (fever,

myalgia, malaise/fatigue, nausea/vomiting, diarrhea) AEs in the participant's diary card for up to

7 days after each dose. Unsolicited AEs were recorded for 28 days following each dose; all other

AEs, SAEs and adverse events of special interest (AESIs) were recorded throughout the study

period (approximately 7 months). Serum samples were collected for hematology, biochemistry

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and immunology evaluation.

[0116] TheThe immunogenicity immunogenicity endpoints endpoints were were to to evaluate evaluate neutralizing neutralizing antibody antibody titers titers andand

binding antibody titers at 14 days (Day 15) and 28 days (Day 29) after first and at 14 days (Day

43) and 28 days (Day 57) after second dose, as well as 90 days and 180 days after the second

dose. Convalescent serum specimens 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 tested. Cellular immune responses were evaluated at 28 days after the second dose by

IFN-y ELISpot and IL-4 ELISpot.

[0117] SARS-CoV-2 Spike-Specific Immunoglobulin G (IgG): Total serum anti-Spike IgG titers

were detected with direct enzyme-linked immunosorbent assay (ELISA) using customized

96-well plates coated with S-2P antigen.

[0118] SARS-CoV-2 Pseudovirus Neutralization Assay: Serial dilutions of the samples to be

tested were performed (initial dilution of 1:20 followed by two-fold dilutions to a final dilution of

1:2560). The diluted serum was mixed with an equal volume of pseudovirus (1000 TU) and

incubated before adding to the plates with HEK293-hAce2 cells (1 X 104 cells/well). The 10 cells/well). The amount amount

of pseudovirus entering the cells was calculated by lysing and measuring the relative luciferase

units (RLU). Fifty percent inhibition dilution (concentration) titers (ID50) were (ID) were calculated calculated

considering uninfected cells as 100% neutralization and cells transduced with virus as 0%

neutralization and reciprocal ID50 geometric ID geometric mean mean titers titers (GMT) (GMT) were were both both determined determined.

[0119] Wild-Type SARS-CoV-2 Neutralization Assay. virus SARS-CoV-2 (hCoV-19/Taiwan/CGMH-CGU-01/2020, GenBank (hCoV-19/Taiwan/CGMH-CGU-01/2020), GenBank accession accession MT192759) MT192759) was titrated was titrated to obtainto obtain

TCID50and TCID and Vero Vero E6 E6 cells cells(2.5 (2.5X X 10410cells/well) werewere cells/well) seeded in 96-well seeded plates plates in 96-well and incubated. The and incubated. The

sera underwent two-fold dilutions with the final dilution being 1:8192, and the diluted sera were

mixed with equal volume of viral solution containing 100 TCID50. The TCID. The serum-virus serum-virus mixture mixture was was

incubated and then added to the plates containing the Vero E6 cells, followed by further

incubation. The neutralizing titer was defined as the reciprocal of the highest dilution capable of

inhibiting 50% of cytopathic effect (CPE NT50), which NT), which was was calculated calculated inin using using the the Reed-Muench Reed-Muench

method. The National Institute for Biological Standards and Control (NIBSC; Potters Bar, UK)

reference serum sample 20/130, was analyzed using the same validated assays as a comparator.

Cellular

[0120] Cellular Immune Immune Response. Response. TheThe number number of of antigen-specific antigen-specific IFN-y IFN-y or or IL-4 IL-4 secreting secreting

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spot forming units (SFU) were determined by ELISpot assays. Cryopreserved peripheral blood

mononuclear cells (PBMC) were rapidly thawed and allowed to rest overnight. Cells were

dispensed at 1 X 105 cellsper 10 cells perwell wellfor forIFN- IFN-y ELISpot ELISpot assay assay (Human (Human IFN-y IFN-y ELISpot ELISpot Kit, Kit, Mabtech, Mabtech,

Stockholm, Sweden) or 2 X 105 cellsper 10 cells perwell wellfor forIL-4 IL-4ELISpot ELISpotassay assay(Human (HumanIFN-y IFN-yELISpot ELISpotKit, Kit,

Mabtech, Stockholm, Sweden). Cells were stimulated with a pool of peptides consisting mainly

of 15-mer sequences with 11 amino acids overlap, covering the N-terminal S1 domain of the S

protein of SARS-CoV-2 (PepTivator SARS-CoV-2 Prot_S1, Miltenyi Biotec) and incubated at

37 °C for 24-48 hours. Cells stimulated with CD3-2 mAb served as the positive control. IFN-y or IFN- or

IL-4 release were detected following the manuals and the spots were counted using the CTL

automatic ELISpot reader. The mean SFU counted in peptide pool stimulation triplicate was

calculated and normalized by subtracting the mean of the negative control replicates (control

media). Results were expressed as SFU per million PBMC.

[0121] Statistical Analysis. Safety analyses were performed on the total vaccinated group

(TVG) population who received at least 1 dose of vaccine. The immunogenicity endpoints

comprised the geometric mean titer (GMT) and seroconversion rate (SCR) of antigen specific

immunoglobulins and wild type virus and pseudovirus neutralizing antibody titers. SCR is

defined as the percentage of participants with 4-fold 4-foldincrease increasein intiters titersfrom fromthe thebaseline baselineor orfrom from

half of the lower limit of detection (LoD) if undetectable at baseline. The GMT and SCR are

presented with two-sided 95% CI. Antigen specific cellular immune responses are presented as

means determined by IFN-y ELISpot and IL-4 ELISpot.

[0122] Results

Safety.

[0123] Safety. No No SAESAE or or AESI AESI occurred occurred at at this this data data cut-off cut-off point. point. No No study study intervention intervention waswas

modified or interrupted. Occurrences of solicited AEs are summarized in Figure 11. The most

commonly reported local AEs were pain/tenderness (80.0%), while malaise/fatigue (28.9%) were

the most commonly reported systemic AEs among all treatment groups. All local and systemic

AEs were mild, except for one malaise/fatigue in the 25 ug µg dose group. No participant had fever.

Solicited AEs after the first and the second dose were similar. Evaluation of safety laboratory

values, ECG interpretation, and other unsolicited adverse events revealed no specific concern.

[0124] Humoral Immune Response. The humoral immunogenicity results are summarized in

FIGS. 12A to 12C. As shown in FIG. 12A, binding IgG titers to S protein increased rapidly after

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the second dose, with seroconversion in all participants by Day 43 and 57. The GMTs peaked at

Day 43 with a value of 7178.2 (95% CI: 4240.3 - 12151.7), 7746.1 (95% CI: 5530.2 - 10849.8),

11220.6 (95% CI: 8592.293 - 14652.84) in the 5 ug, µg, 15 ug, µg, and 25 ug µg dose groups, respectively.

The GMT levels in the 5 ug, µg, 15 ug, µg, and 25 ug µg dose groups on Day 43 ranged from 3.3 to 5.1

times the GMT of convalescent serum specimens. (2179.6, [95%CI: 1240.9 - 3828.4]) 3828.4]).

[0125] As shown in FIG. 12B, no subject had detectable pseudovirus neutralizing titers (ID50) (ID)

at the lower limit of serum concentration tested (1:20 dilution) in the assay at baseline. At Day 43,

the pseudovirus neutralizing titers (ID50) showed (ID) showed peak peak GMTs GMTs ofof 538.5 538.5 (95% (95% CI: CI: 261.9 261.9 - - 1107.0), 1107.0),

993.1 (95% CI: 655.0 - 1505.7), and 1905.8 (95% CI: 1601.7 - 2267.8) in the 5 ug, µg, 15 ug, µg, and 25

ug µg dose groups, respectively. All participants (100%) seroconverted after the second dose. The

GMT levels in the 5 ug, µg, 15 ug, µg, and 25 ug µg dose groups on Day 43 ranged from 1.25 to 4.4 times

the GMT of convalescent serum specimens. (430.5, [95%CI: 274.9 - 674.0]).

[0126] TheThe results results of of wild-type wild-type SARS-CoV-2 SARS-CoV-2 neutralizing neutralizing antibody antibody titers titers areare summarized summarized in in

FIG. 12C. Before vaccination, no subject had detectable wild-type virus neutralizing titers (NT50) (NT)

at the lower limit of serum concentration tested (1:8 dilution) in the assay. After the second dose,

neutralizing responses were identified in serum samples from all participants in the 15 ug µg and 25

ug µg dose groups. At Day 43, the GMTs were 33.3 (95% CI: 18.5 - 59.9), 76.3 (95% CI: 53.7 -

108.3), and 167.4 (95% CI: 122.1 - 229.6) in the 5 ug, µg, 15 ug, µg, and 25 ug µg dose groups, respectively.

At Day 57, GMTs were similar in the 15 ug µg and 25 ug µg dose groups: 52.2 (95% CI: 37.9 - 71.8)

and 81.9 (95% CI: 55.8 - 120.2), respectively. The GMT levels in the 5 ug, µg, 15 ug, µg, and 25 ug µg dose

groups on Day 43 were 0.8, 1.8, and 3.9 times the GMT of convalescent serum specimens (42.7,

[95%CI: 26.4 - 69.0]; titers ranged from undetected to 631.0). All participants in 15 ug µg and 25 ug µg

dose groups seroconverted at Day 43 and Day 57; some were similar to the NIBSC reference

serum 20/130 (281.8).

Cellular

[0127] Cellular Immune Immune Response. Response. TheThe results results of of cellular cellular immune immune response response areare summarized summarized in in

FIG. 13. All participants had minimal IFN-y secreting TT cells IFN- secreting cells at at baseline. baseline. By By Day Day 57, 57, aa mean mean of of

161.3, 85.5 and 94. 9 IFN-y secreting T cells were observed per million cells in participants

vaccinated with 5 ug, µg, 15 ug, µg, and 25 ug, µg, respectively. Before vaccination, all participants had

minimal IL-4 secreting T cells. By Day 57, a mean of 24.1, 16.0 and 31.3 IL-4 secreting T cells

were observed per million cells in participants vaccinated with 5 ug, µg, 15 ug, µg, and 25 ug, µg,

respectively. The cellular immune response induced by MVC-COV1901 demonstrated

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substantially higher numbers of IFN-y- producingcells, IFN-- producing cells,suggesting suggestingaaTh1-skewed Thl-skewedimmune immune

response.

[0128] In conclusion, solicited adverse events were mostly mild and similar. No subject

experienced fever. After the second dose, of the three doses evaluated, both the 15 ug µg and 25 ug µg

dose elicited high neutralizing antibody responses with all participants seroconverting and a

Thl-skewed T cell immune response. Therefore, 15 µg Th1-skewed ug S-2P combined with CpG 1018 and

aluminum hydroxide was deemed adequate to elicit a profound humoral immune response. The

results also indicate that MVC-COV1901 vaccine was well tolerated and elicited robust immune

responses and is suitable for further development.

[0129] Example5 5Evaluation

[0129] Example Evaluationofofthe theNeutralizing NeutralizingAbility Abilityofofa aCpG-adjuvanted CpG-adjuvantedS-2P S-2P

Subunit Vaccine "MVC-COV1901" Against SARS-CoV-2 Variants of Concern (VoCs)

[0130] Since the beginning of the COVID-19 pandemic, mutants have been detected

periodically. A number of them, termed Variants of Concern (VoCs), were found to carry

mutations in the crucial receptor-binding domain (RBD), a prime target for antibody recognition

and neutralization. The most representative of these VoC, all bearing an N501 N501YY mutation mutation in in the the

spike RBD, are B.1.1.1.7 (Alpha variant), B.1.351 (Bata variant), and P1 (Gamma variant). The

VoCs with these mutations were found to decrease neutralization capabilities of monoclonal

antibodies and vaccine-induced antibodies, and this could potentially render current therapeutics

and vaccines ineffective (Garcia-Beltran et al., Cell, 184(9):2372-2383.e9, 2021). This Example

provides a study involving investigation of neutralizing ability of MVC-COV1901 vaccine

against SARS-CoV-2 VoCs using sera from two sources: rat sera from animal toxicology studies

and human sera from phase 1 clinical trial.

[0131] A. A. Neutralizing Neutralizing ability ability of of MVC-COV1901 MVC-COV1901 vaccine vaccine against against SARS-CoV-2 SARS-CoV-2 VoCs VoCs in in rats. rats.

[0132] Materials and

[0132] Materials and Methods Methods

[0133] Animal studies. Crl:CD Sprague Dawley (SD) rats were obtained from BioLASCO

Taiwan Co. Ltd. (Taipei, Taiwan), and studies were conducted in the Testing Facility for

Biological Safety, TFBS Bioscience Inc. (New Taipei City, Taiwan). Immunization of SD rats

were carried out as described in Example 2, section C. Briefly, rats were immunized three times

at two weeks apart with 5, 25, or 50 ug µg of S-2P protein adjuvanted with 1,500 ug µg of CpG 1018

and 750 ug µg of aluminum hydroxide. The sera were harvested two weeks after the second

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PCT/CN2021/100826

immunization (Day 29) or two weeks after the third immunization (Day 43) and subjected to

neutralization assay with pseudovirus expressing SARS-CoV-2 Wuhan wildtype (WT) or B.1.351

variant (Beta variant) spike proteins.

[0134] Pseudovirus neutralization assay. Lentivirus expressing the SARS-CoV-2 spike

proteins of the Wuhan-Hu-1 wildtype strain (WT) was constructed, and the neutralization assay

performed as described in Example 2, section C. Lentiviruses expressing B.1.351 variant (Beta

variant) spike proteins were constructed in the same manner but with the wild-type spike protein

sequence replaced with the variant sequence (GenBank Accession No. MZ314998.1).

[0135] Statistical analysis. Prism 6.01 (GraphPad Software Inc., San Diego, CA, USA) was

used for statistical analysis. Two-way ANOVA with Tukey's multiple comparison test and

Kruskal-Wallis with corrected Dunn's multiple comparisons test were used to calculate

significance asas significance noted in respective noted figure in respective descriptions. figure * p < 0.05, descriptions. * p ** p < 0.01, < 0.05, p <*** p < 0.001, 0.01, p < 0.001,

*****p<0.0001. p < 0.0001.

[0136] Results

[0137] MVC-COV1901-induced antibodies in rats effectively neutralized variants comparable

to the wildtype. As shown in FIG. 14, at Day 29 and 43, the antibodies retained effectiveness

against the B.1.351 (Beta variant), although the titers were reduced. Notably, sera sampled two

weeks after the third immunization (Day 43) had higher ID50 and ID and IDID90 geometric geometric meanmean titers titers

(GMT) than sera sampled two weeks after the second immunization (Day 29), suggesting a trend

towards improved neutralization activity against this VoC with a third immunization. The effect is

especially pronounced in the low (5 ug) µg) dose. By Day 43, all dose groups achieved similar levels

of GMTs against B.1.351 at ID50 (FIG. ID (FIG. 14, 14, left left panel) panel) and and ID90 ID9 (FIG. (FIG. 14,14, right right panel). panel).

[0138] To To sumsum up,up, thethe ratrat study study shows shows that that by by using using a three-dose a three-dose regimen, regimen, we we were were able able to to

induce similar levels of neutralizing titer across three-dose groups. Given that the three-dose

regimen resulted in high immunogenicity against the variant, these results could be extrapolated

to humans in that an extra immunization could be a strategy to increase immunity against the

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VoCs.

[0139]

[0139] B.B.Neutralizing Neutralizingability abilityofofMVC-COV1901 MVC-COV1901vaccine vaccineagainst againstSARS-CoV-2 SARS-CoV-2VoCs VoCsinin

human.

[0140] Materials and Methods

Clinicaltrial.

[0141] Clinical trial. Forty-five Forty-five (45) (45)human humansubjects fromfrom subjects the age the of 20 of age to 20 49 were to 49enrolled in were enrolled in

a prospective, open-labeled, single-center dose-escalation phase 1 study with three separate

sub-phases for participants from 20 to less 50 years of age. Each sub-phase had 15 participants.

The three different dose levels employed in this clinical trial are low dose (LD; 5 ug), µg), mid-dose

(MD; 15 ug) µg) and high dose (HD; 25 ug) µg) of S-2P protein adjuvanted with 750 ug µg of CpG 1018

and 375 ug µg of aluminum hydroxide for phase 1a, la, 1b, and 1c, respectively. The vaccination

schedule consisted of two doses, administered by intramuscular (IM) injection of 0.5 mL in the

deltoid region of the non-dominant arm, preferably 28 days apart, on Day 1 and Day 29. On Day

57 (4 weeks after the second administration), serum samples were taken for pseudovirus

neutralization assays. The clinical trial is described in greater detail in Example 4.

[0142] Pseudovirus neutralization assay. Lentivirus expressing the SARS-CoV-2 spike

proteins of the Wuhan-Hu-1 wildtype strain (WT) was constructed, and the neutralization assay

performed as described in Example 2, section C. Lentiviruses expressing D614G, B.1.1.7 (Alpha

variant; GenBank Accession No. MZ314997.1), B.1.351 (Beta variant; GenBank Accession No.

MZ314998.1), P1 (Gamma variant; GenBank Accession No. LR963075), and B.1.429 (Epsilon

variant; GenBank Accession No. MW591579) spike proteins were constructed in the same

manner but with the wild-type spike protein sequence replaced with the respective variant

sequences.

Statisticalanalysis.

[0143] Statistical analysis. Methods Methodsofofstatistical analysis statistical were were analysis performed as described performed in the as described in the

previous section.

[0144] Results

[0145] Human antisera from vaccination with MVC-COV1901 neutralized D614G, B.1.1.7

(Alpha), P1 (Gamma) variants, but neutralization was diminished with B.1.351 (Beta) and

B.1.429 (Epsilon) variants. FIGS. 15A-C present the data from pseudovirus neutralization assays

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Claims (13)

What is claimed is: 01 Oct 2025

1. An immunogenic composition against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising an antigenic recombinant protein and an adjuvant 5 consisting of aluminum hydroxide and an unmethylated cytosine-phosphate-guanosine (CpG) motif consisting of a synthetic oligodeoxynucleotide (ODN) of SEQ ID NO: 8, wherein the antigenic recombinant protein substantially consists of residues 14-1208 of 2021290765

SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization 10 domain.

2. The immunogenic composition of claim 1, wherein the residues 14-1208 of SARS-CoV- 2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 – 685 comprise an amino acid sequence of SEQ ID NO: 1 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% to SEQ ID NO: 1.

15 3. The immunogenic composition of claim 1 or 2, wherein the C-terminal T4 fibritin trimerization motif comprises an amino acid sequence of SEQ ID NO: 2 or the amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% to SEQ ID NO: 2.

4. The immunogenic composition of any one of claims 1-3, wherein the antigenic recombinant protein comprises an amino acid sequence of SEQ ID NO: 5 or 6 or the 20 amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% to SEQ ID NO: 5 or 6.

5. The immunogenic composition of any one of claims 1-4, wherein a 0.5 ml dose of the immunogenic composition comprises from about 250 to about 500 µg Al3+, or about 375 µg Al3+.

6. The immunogenic composition of any one of claims 1-5, wherein a 0.5 ml dose of the 25 immunogenic composition comprises from about 750 to about 3000 μg of the oligonucleotide, or wherein the immunogenic composition comprises about 750 μg, about 1500 μg, or about 3000 μg of the oligonucleotide.

7. A method for eliciting an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a subject in need thereof, comprising administering to 30 the subject an effective amount of an immunogenic composition of any one of claims 1- 6.

8. The method of claim 7, wherein the immune response comprises production of 01 Oct 2025

neutralizing antibodies against SARS-CoV-2 and Th1-skewed immune response.

9. Use of the immunogenic composition of any one of claims 1-6 for eliciting an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a 5 subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the immunogenic composition.

10. The use of claims 9, wherein the immune response comprises production of neutralizing 2021290765

antibodies against SARS-CoV-2 and Th1-skewed immune response.

11. Use of the immunogenic composition of any one of claims 1-6 for protecting a subject 10 in need thereof from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the method comprising administering to the subject in need thereof an effective amount of the immunogenic composition.

12. Use of the The immunogenic composition of any one of claims 1-6 for preventing a subject in need thereof from contracting COVID-19 disease, the method comprising 15 administering to the subject in need thereof an effective amount of the immunogenic composition.

13. The use of any one of claims 9-12, wherein the immunogenic composition is administered by intramuscular injection.