WO2021254473A1 - Immunogenic composition against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) - Google Patents
Immunogenic composition against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) Download PDFInfo
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- WO2021254473A1 WO2021254473A1 PCT/CN2021/100826 CN2021100826W WO2021254473A1 WO 2021254473 A1 WO2021254473 A1 WO 2021254473A1 CN 2021100826 W CN2021100826 W CN 2021100826W WO 2021254473 A1 WO2021254473 A1 WO 2021254473A1
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Definitions
- 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.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- COVID-19 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.
- ARDS acute respiratory distress syndrome
- COVID-19 vaccine To reduce the risk of SARS-CoV-2 infection without curtailing everyday activities, a COVID-19 vaccine is needed.
- a COVID-19 vaccine that is able to rapidly induce an immune response against SARS-CoV-2 is urgently needed.
- 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.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- 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.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- the present invention provides 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.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- the present invention provides a method for preventing a subject in need thereof from contracting COVID-19 disease, comprising administering to the subject an effective amount of the immunogenic composition of the present invention.
- the present invention provides use of the immunogenic 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.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- 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) .
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- the present invention provides use of the immunogenic composition of the present invention for preventing a subject in need thereof from contracting COVID-19 disease.
- 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. 2 shows the results from neutralization assays using sera from mice immunized with different formulations of the SARS-CoV-2 S-2P recombinant protein.
- 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.
- the antisera were subjected to neutralization assay with pseudovirus expressing SARS-CoV-2 spike protein to determine the ID 50 (left) and ID 90 (right) titers of neutralization antibodies. **p ⁇ 0.01, ***p ⁇ 0.001.
- FIG. 4 shows total anti-SIgG titers in mice immunized with S-2P with adjuvants.
- ELISA enzyme linked immunosorbent assay
- 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.
- 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 ⁇ g of S-2P with 10 ⁇ g CpG 1018 and 50 ⁇ g 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.
- FIG. 7 shows IFN- ⁇ /IL-4, IFN- ⁇ /IL-5, and IFN- ⁇ /IL-6 ratios.
- FIGS. 8A-8B show neutralizing antibody titers with pseudovirus assay in hamsters 2 weeks after second immunization.
- 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 ID 90 titers of neutralization antibodies (FIG. 8A) and total anti-SIgG antibody titers with ELISA (FIG.
- 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 ID 90 , 100 and 1, 638, 400 in IgG ELISA) . ***p ⁇ 0.001, ****p ⁇ 0.0001.
- 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 TCID 50 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) .
- FIG. 10 shows lung pathology scoring 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 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 comparisons test. ****p ⁇ 0.0001.
- 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.
- AEs Solicited adverse events
- FIGS. 12A-12C shows summary of humoral immune response in the Phase I clinical trial.
- Sera of participants vaccinated with 5, 15, or 25 ⁇ g of MVC-COV1901 were measured for anti-spike IgG by ELISA (FIG. 12A)
- 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. 13 shows summary of cellular immune response in the Phase I clinical trial.
- Cells were stimulated with a S1 peptides pool of peptides and incubated at 37°C for 24-48 hours.
- Cells stimulated with CD3-2 mAb served as a positive control.
- IFN- ⁇ (left 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. 14 shows neutralization of SARS-CoV-2 pseudovirus bearing wildtype or 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 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 ID 50 and ID 90 titers of neutralizing antibodies. Results are presented as bars representing geometric mean titers with symbols representing value of each sample.
- 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) .
- ID 50 neutralizing titers for low dose (LD; FIG. 15A) , mid-dose (MD; FIG. 15B) , high dose (HD; FIG. 15C) , ID 90 neutralizing titers for low dose (LD; FIG. 15D) , mid-dose (MD; FIG. 15E) , high dose (HD; FIG.
- the present invention relates to an immunogenic composition against SARS-CoV-2.
- the immunogenic composition comprises an antigenic recombinant protein and an adjuvant containing an aluminum-containing adjuvant and/or an unmethylated 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.
- 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%to SEQ ID NO: 1.
- 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%, 94%, 95%, 96%, 97%, 98%, or 99%to SEQ ID NO: 2.
- 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.
- 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.
- a 0.5 ml dose of the immunogenic composition comprises from about 250 to about 500 ⁇ g Al 3+ , or about 375 ⁇ g Al 3+ .
- 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.
- ODN synthetic oligodeoxynucleotide
- a 0.5 ml dose of the immunogenic composition comprises from about 750 to about 3000 ⁇ g of the oligonucleotide, or wherein the immunogenic composition comprises about 750 ⁇ g, about 1500 ⁇ g, or about 3000 ⁇ g of the oligonucleotide.
- the present invention also relates to 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 the immunogenic composition of the present invention.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- 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.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- the present invention also relates to a method for preventing a subject in need thereof from contracting COVID-19 disease, comprising administering to the subject an effective amount of the immunogenic composition of the present invention.
- the present invention also relates to use of the immunogenic 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, the method comprising administering to the subject in need thereof an effective amount of the immunogenic composition.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- the immune response comprises production of neutralizing antibodies against SARS-CoV-2 and Th1-skewed immune response.
- 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.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- 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.
- the immunogenic composition is administered by intramuscular injection.
- an excipient includes one or more excipients.
- 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 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) or uracil (U) , or derivative thereof) base bonded to a sugar.
- the four nucleoside units (or bases) in DNA are called deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine.
- a nucleotide is a phosphate ester of a nucleoside.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- 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.
- the terms “immunogenic composition against severe acute 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 Th1-skewed immune response.
- the terms “aluminum-containing adjuvant” refers to an adjuvant including aluminum.
- 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.
- the aluminum-containing adjuvant is an aluminum-containing adjuvant approved for administration to humans by the FDA.
- the aluminum-containing adjuvant is an aluminum hydroxide adjuvant approved for administration to humans by the FDA.
- the aluminum-containing adjuvant is an aluminum phosphate adjuvant approved for administration to humans by the FDA.
- the terms “unmethylated cytosine-phosphate-guanosine (CpG) 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.
- the CpG-containing oligonucleotide contains palindromic hexamers following the general formula of: 5’-purine-purine-CG-pyrimidine-pyrimidine-3’.
- the unmethylated cytosine-phosphate-guanosine (CpG) motif has an oligonucleotide of SEQ ID NO: 8 (5’-TGACTGTGAACGTTCGAGATGA-3’) in which the Cs of the CGs are unmethylated.
- 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.
- the unmethylated cytosine-phosphate-guanosine (CpG) motif has an oligonucleotide of SEQ ID NO: 9 (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) , 5’-TCCATGGACGTTCCTGAGCGTT-3’ (SEQ ID NO: 11) , 5’-TCGTCGTTCGAACGACGTTGAT-3’ (SEQ ID NO: 12) , and 5’-TCGTCGACGATCGGC GCGCGCCG-3’ (SEQ ID NO: 13) .
- the CpG-containing 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.
- 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.
- 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.
- 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) .
- non-human primates e.g., monkeys
- farm animals e.g., farm animals
- rodents e.g., mice and rats
- pets e.g., dogs and cats
- dose refers to a measured portion of the immunogenic composition taken by (administered to or received by) a subject at any one time.
- isolated and purified refers to a material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment) .
- isolated, when used in reference to a recombinant protein, refers to a protein that has been removed from the culture medium of the host cell that produced the protein.
- “Stimulation” of a response or parameter includes eliciting and/or enhancing 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) .
- 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.
- the term “immunization” refers to a process that increases a mammalian subject’s reaction to antigen and therefore improves its ability to resist or overcome infection.
- vaccination refers to the introduction of vaccine into a body of a mammalian subject.
- 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.
- a plasmid having a polynucleotide encoding the residues 1-1208 of SARS-CoV-2 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 site (SEQ ID NO: 3) , an 8x His Tag, and a Twin-Strep Tag (SEQ ID NO: 4) was transfected into ExpiCHO-Scells (Thermo Fisher Scientific, Waltham, MA, 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 (Al (OH) 3 ) or aluminum phosphate (AlPO 4 ) as the immunogenic compositions against SARS-CoV-2.
- an unmethylated CpG motif CpG 1018, SEQ ID NO: 8
- aluminum-containing adjuvant such as aluminum hydroxide (Al (OH) 3 ) or aluminum phosphate (AlPO 4 ) as the immunogenic compositions against SARS-CoV-2.
- This example provides a description of preclinical studies to assess the immunogenicity of the immunogenic compositions against SARS-CoV-2 obtained from Example 1 in mice.
- 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%CO 2 .
- CMV/R-SARS-CoV-2 spike plasmid 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.
- 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%CO 2 . Pseudovirus infectivity was assessed in 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) .
- RLU relative luciferase units
- 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.
- SARS-CoV-2 S-2P recombinant protein (0.1 ⁇ g/mouse and 1 ⁇ g/mouse) formulated with aluminum phosphate elicited greater neutralization than the recombinant protein (0.1 ⁇ g/mouse and 1 ⁇ g/mouse) alone.
- 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) .
- SARS-CoV-2 S-2P recombinant protein (afinal concentration of 10 ⁇ g or 50 ⁇ g/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 ⁇ L intramuscularly (50 ⁇ L into each hind leg) . Two weeks after the final immunization, sera were collected for measurement of antibody responses.
- 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 ⁇ g/mouse) and a higher dose (5 ⁇ g/mouse) .
- SARS-CoV-2 S-2P recombinant protein (1 ⁇ g/mouse) formulated with aluminum hydroxide alone elicited greater neutralization than the recombinant protein (1 ⁇ g/mouse and 5 ⁇ g/mouse) formulated with CpG alone.
- the recombinant protein formulated with CpG alone elicited neutralizing activity in a dose-dependent manner.
- SARS-CoV-2 spike protein SEQ ID NO: 7
- SEQ ID NO: 7 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 pCMV ⁇ 8.91 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 unit (TU) of SARS-CoV-2 pseudotyped lentivirus was estimated by using cell viability assay in response to the limited dilution of lentivirus.
- 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.
- HEK293-hAce2 cells (2 ⁇ 10 4 cells/well) were seeded in 96-well white isoplates and incubated for overnight. Sera were heated at 56 °Cfor 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.
- pseudovirus 1000 TU
- the culture medium was replaced with 50 ⁇ L of fresh medium. On the following day, the culture medium was replaced with 100 ⁇ L of fresh medium.
- Cells were lysed at 72 h post infections and relative luciferase units (RLU) were measured. The luciferase activity was detected by Tecan i-control (Infinite 500) .
- the 50%and 90%inhibition dilution titers (ID 50 and ID 90 ) were calculated considering uninfected cells as 100%neutralization and cells transduced with only virus as 0%neutralization. Reciprocal ID 50 and ID 90 geometric mean titers (GMT) were both determined as ID 90 titers are useful when ID 50 titer levels are consistently saturating at the upper limit of detection.
- the diluted sera were mixed with an equal volume of SARS-CoV-2 virus at 100 TCID 50 /50 ⁇ L (hCoV-19/Taiwan/CGMH-CGU-01/2020, GenBank accession MT192759) and incubated at 37 °Cfor 2 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 ID 50 and log 90%end point for ID 90 titers.
- mice Female BALB/c and C57BL/6 mice were obtained from the Labotory Animal Center, Academia Sinica, Taiwan and BioLASCO Taiwan Co. Ltd.
- 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 were euthanized and splenocytes were isolated and stimulated with S-2P protein (2 ⁇ g/well) as previously described (Lu et al. Immunology, 130 (2) : 254–261, 2010) .
- S-2P protein 2 ⁇ g/well
- the culture supernatant from the 96-well microplates was harvested to analyze the levels of cytokines by ELISA using 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) .
- the OD450 values were read by Multiskan GO (Thermo Fisher Scientific) .
- 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) and Day 15 (for repeat-dose study) .
- the observation period was 14 days (for single-dose 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.
- alum 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 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 ⁇ g of S-2P with CpG 1018 and alum.
- S-2P was able to inhibit SARS-CoV-2 at a concentration of 1 ⁇ g, although at lower potency than that of pseudovirus (FIG. 3, FIG. 5) .
- the reciprocal ID 50 GMT of 1 ⁇ g 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-SIgG titers were all found to be highly correlated with Spearman’s rank correlation coefficients greater than 0.8.
- CpG 1018 induced Th1 immunity.
- 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.
- S-2P adjuvanted with alum induced limited amounts of IFN- ⁇ and IL-2, the representative cytokines of Th1 response.
- significant increases in IFN- ⁇ and IL-2 were detected most strongly in high antigen dose plus CpG 1018 and alum.
- S-2P did not result in systemic adverse effects in rats.
- 5 ⁇ g, 25 ⁇ g or 50 ⁇ g of S-2P adjuvanted with 1500 ⁇ g CpG 1018 or 750 ⁇ g CpG 1018 combined with 375 ⁇ g 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.
- 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.
- S-2P prefusion spike protein
- CpG 1018 and alum elicited Th1-dominant immune responses with high neutralizing antibody levels in mice and showed no major adverse effects in rats.
- the inventors have demonstrated in this Example that the S-2P combined with adjuvant CpG 1018 in combination with alum induced potent 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.
- Lentivirus expressing the Wuhan-Hu-1 strain SARS-CoV-2 spike protein was constructed and the neutralization assay 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 (ID 50 and ID 90 ) were calculated referencing uninfected cells as 100%neutralization and cells transduced with only virus as 0%neutralization.
- Total serum anti-SIgG titers were detected with direct ELISA using custom 96-well plates coated with S-2P antigen.
- 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 1 x 10 4 PFU of SARS-CoV-2 TCDC#4 (hCoV-19/Taiwan/4/2020, GISAID Accession ID: EPI_ISL_411927) intranasally in a volume of 100 ⁇ L 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 TCID 50 assay) .
- the left lung was fixed in 4%paraformaldehyde for histopathological examination.
- TCID 50 cell culture infectious assay
- TCID 50 tissue culture infectious dose
- RNA sample Five (5) ⁇ L was added into a total 25 ⁇ L 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 ⁇ L 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.
- dNTP deoxyribonucleoside triphosphate
- 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) .
- 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 3 , 10 4 , or 10 5 PFU of SARS-CoV-2 and euthanized on Day 3 or 6 after infection for tissue sampling. Following infection of 10 3 to 10 5 PFU of SARS-CoV-2, the hamsters exhibited dose-dependent weight loss. Hamsters infected with 10 3 PFU gained weight while 10 4 and 10 5 PFU-infected hamsters experienced progressively severe weight loss at 6 days post-infection (dpi) .
- Adjuvanted S-2P protected hamsters from clinical signs and viral load after SARS-CoV-2 challenge.
- hamsters were challenged with 10 4 PFU of SARS-CoV-2 virus and body weights were tracked up to 3 or 6 days post 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 50 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) .
- viral load, especially viral titer measured by TCID 50 dropped noticeably at 6 dpi in 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) .
- 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.
- SARS-CoV-2 subunit vaccine i.e., the immunogenic composition of the present invention.
- 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.
- MVC-COV1901 is formulated in with three different dosages of SARS-CoV-2 Spike (S) protein with CpG 1018 and aluminum hydroxide as adjuvants.
- S SARS-CoV-2 Spike
- Each MVC-COV1901 vaccine contains 5, 15, or 25 ⁇ g of S-2P adjuvanted with 750 ⁇ g of CpG 1018 and 375 ⁇ g (Al equivalent to weight) of aluminum hydroxide, administered as a single 0.5 mL intramuscular (IM) injection.
- IM intramuscular
- 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 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.
- 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 ⁇ g 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.
- Cohort 1a Four sentinel participants were to be recruited to receive vaccine with 5 ⁇ g of S-2P to evaluate the preliminary safety data of the vaccine. If no ⁇ Grade 3 adverse 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 1a and Phase 1b would proceed.
- AE Grade 3 adverse event
- SAE serious adverse event
- Cohort 1b Another 4 sentinel participants were to be enrolled to receive vaccine with 15 ⁇ g of S-2P. If no ⁇ Grade 3 AE or SAE occurred within 7 days after the first dose in the 4 sentinel participants, dosing of the remaining participants in Phase 1b and Phase 1c would proceed.
- Cohort 1c Another 4 sentinel participants would be enrolled to receive vaccine with 25 ⁇ g of S-2P. If no ⁇ Grade 3 AE or SAE occurred within 7 days after the first dose in the 4 sentinel participants, dosing of the remaining participants in Phase 1c would proceed.
- ECG electrocardiogram
- 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 and immunology evaluation.
- the immunogenicity endpoints were to evaluate neutralizing antibody titers and 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- ⁇ ELISpot and IL-4 ELISpot.
- 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.
- ELISA enzyme-linked immunosorbent assay
- 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 10 4 cells/well) .
- the amount of pseudovirus entering the cells was calculated by lysing and measuring the relative luciferase units (RLU) .
- ID 50 Fifty percent inhibition dilution (concentration) titers (ID 50 ) were calculated considering uninfected cells as 100%neutralization and cells transduced with virus as 0%neutralization and reciprocal ID 50 geometric mean titers (GMT) were both determined.
- SARS-CoV-2 virus (hCoV-19/Taiwan/CGMH-CGU-01/2020, GenBank accession MT192759) was titrated to obtain TCID 50 and Vero E6 cells (2.5 x 10 4 cells/well) were seeded in 96-well plates 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 TCID 50 . The serum-virus mixture 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 NT 50 ) , which was calculated in using the Reed-Muench method.
- CPE NT 50 50%of cytopathic effect
- the number of antigen-specific IFN- ⁇ or IL-4 secreting 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 10 5 cells per well for IFN- ⁇ ELISpot assay (Human IFN- ⁇ ELISpot Kit, Mabtech, Sweden) or 2 x 10 5 cells per well for IL-4 ELISpot assay (Human IFN- ⁇ ELISpot Kit, Mabtech, Sweden) .
- IFN- ⁇ ELISpot assay Human IFN- ⁇ ELISpot Kit, Mabtech, Sweden
- IL-4 ELISpot assay Human IFN- ⁇ ELISpot Kit, Mabtech, 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- ⁇ 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.
- 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.
- GCT geometric mean titer
- SCR seroconversion rate
- the GMT and SCR are presented with two-sided 95%CI.
- Antigen specific cellular immune responses are presented as means determined by IFN- ⁇ ELISpot and IL-4 ELISpot.
- FIGS. 12A to 12C The humoral immunogenicity results are summarized in FIGS. 12A to 12C.
- binding IgG titers to S protein increased rapidly after 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 ⁇ g, 15 ⁇ g, and 25 ⁇ g dose groups, respectively.
- the GMT levels in the 5 ⁇ g, 15 ⁇ g, and 25 ⁇ g 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] ) .
- the GMT levels in the 5 ⁇ g, 15 ⁇ g, and 25 ⁇ g 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] ) .
- GMTs were similar in the 15 ⁇ g and 25 ⁇ g 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 ⁇ g, 15 ⁇ g, and 25 ⁇ g 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 ⁇ g and 25 ⁇ g dose groups seroconverted at Day 43 and Day 57; some were similar to the NIBSC reference serum 20/130 (281.8) .
- VoCs Variants of Concern
- RBD crucial receptor-binding domain
- 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 ⁇ g of S-2P protein adjuvanted with 1, 500 ⁇ g of CpG 1018 and 750 ⁇ g of aluminum hydroxide.
- the sera were harvested two weeks after the second 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.
- 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) .
- MVC-COV1901-induced antibodies in rats effectively neutralized variants comparable to the wildtype.
- the antibodies retained effectiveness against the B. 1.351 (Beta variant) , although the titers were reduced.
- sera sampled two weeks after the third immunization (Day 43) had higher ID 50 and ID 90 geometric mean 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 ⁇ g) dose.
- all dose groups achieved similar levels of GMTs against B. 1.351 at ID 50 (FIG. 14, left panel) and ID 90 (FIG. 14, right panel) .
- the rat study shows that by using a three-dose regimen, we were able 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 VoCs.
- 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.
- IM intramuscular
- 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.
- FIGS. 15A-C present the data from pseudovirus neutralization assays of human sera with the panel of WT, D614G, B. 1.1.7 (Alpha) , B. 1.351 (Beta) , P1 (Gamma) , and B. 1.429 (Epsilon) variants.
- FIGS. 15A-C present the data from pseudovirus neutralization assays of human sera with the panel of WT, D614G, B. 1.1.7 (Alpha) , B. 1.351 (Beta) , P1 (Gamma) , and B. 1.429 (Epsilon) variants.
- the titers of neutralizing antibodies of all groups (LD, MD, and HD groups) against D614G and B.
- vaccinated phase 1 human subjects showed more reduced but still appreciable neutralization abilities against the B. 1.351 (Beta) , P1 (Gamma) , and B. 1.429 (Epsilon) variants at ID 90 , especially at higher doses.
- the results indicate that two doses of MVC-COV1901 were able to elicit neutralizing antibodies against SARS-CoV-2 variants in a dose-dependent manner.
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Abstract
Description
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 (afinal concentration of 1 μg or 10 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 μL intramuscularly (50 μL into each hind leg) . Two weeks after the final immunization, sera were collected for measurement of antibody responses.
Mouse immunizations. BALB/c mice aged 6-8 weeks (Laboratory Animal Center, Taiwan) (N = 6/group) were vaccinated with the SARS-CoV-2 S-2P recombinant protein at 0 and 3rd week. SARS-CoV-2 S-2P recombinant protein (afinal concentration of 10 μg or 50 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 μL intramuscularly (50 μL into each hind leg) . Two weeks after the final immunization, sera were collected for measurement of antibody responses.
Immunization of mice. Female BALB/c and C57BL/6 mice were obtained from the Labotory 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 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-SIgG 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.
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 μg of S-2P protein adjuvanted with 150 μg CpG 1018 and 75 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 1 x 10 4 PFU of SARS-CoV-2 TCDC#4 (hCoV-19/Taiwan/4/2020, GISAID Accession ID: EPI_ISL_411927) intranasally in a volume of 100 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
Claims (15)
- 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.
- 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.
- 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.
- 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 amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99%to SEQ ID NO: 5 or 6.
- The immunogenic composition of any one of claims 1-4, wherein 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.
- The immunogenic composition of any one of claims 1-5, wherein a 0.5 ml dose of the immunogenic composition comprises from about 250 to about 500 μg Al 3+, or about 375 μg Al 3+.
- The immunogenic composition of any one of claims 1-6, wherein 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.
- The immunogenic composition of any one of claims 1-7, wherein a 0.5 ml dose of the 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.
- 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 any one of claims 1-8.
- The method of claim 9, wherein the immune response comprises production of neutralizing antibodies against SARS-CoV-2 and Th1-skewed immune response.
- Use of the immunogenic composition of any one of claims 1-8 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.
- The use of claims 11, wherein the immune response comprises production of neutralizing antibodies against SARS-CoV-2 and Th1-skewed immune response.
- Use of the immunogenic composition of any one of claims 1-8 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.
- Use of the immunogenic composition of any one of claims 1-8 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.
- The use of any one of claims 11-14, wherein the immunogenic composition is administered by intramuscular injection.
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