ANTI-CORONAVIRUS VACCINES

Abstract

The invention provides stable coronavirus spike proteins. Immunogenic compositions comprising same and the methods of using these immunogenic compositions are also provided.

Claims

1. A composition comprising a coronavirus, a Spike protein of said coronavirus or an immunogenic fragment of said Spike protein, and an adjuvant comprising a saponin, a sterol, and a CpG-containing immunostimulatory oligonucleotide.

2. The composition according to claim 1, wherein said composition is essentially free of quaternary ammonium compounds and essentially free of a glycolipid according to Formula I: ##STR00003## wherein, R.sup.1 is hydrogen, or a saturated alkyl radical having up to 20 carbon atoms; X is —CH.sub.2—, —O— or —NH—; R.sup.2 is hydrogen, or a saturated or unsaturated alkyl radical having up to 20 carbon atoms; R.sup.3, R.sup.4, and R.sup.5 are independently hydrogen, —SO.sub.4.sup.2−, —PO.sub.4.sup.2−, —COC.sub.1-10 alkyl; R.sup.6 is L-alanyl, L-alpha-aminobutyl, L-arginyl, L-asparginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenyalany, L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl or their D-isomers.

3. The composition of claim 1, wherein the adjuvant consists of the saponin, the sterol, and the CpG-containing immunostimulatory oligonucleotide.

4. The composition according to claim 1, wherein the Saponin is Quil A and the sterol is selected from the group consisting of β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol.

5. The composition according to claim 4, wherein the saponin is present in the amount of about 20 μg per dose and the sterol is present in the amount of about 20 μg per dose.

6. A composition comprising a coronavirus, a Spike protein from said coronavirus or an immunogenic fragment of said Spike protein, and an adjuvant comprising a CpG containing immunostimulatory oligonucleotide and a glycolipid according to Formula I, ##STR00004## wherein, R.sup.1 is hydrogen, or a saturated alkyl radical having up to 20 carbon atoms; X is —CH.sub.2—, —O— or —NH—; R.sup.2 is hydrogen, or a saturated or unsaturated alkyl radical having up to 20 carbon atoms; R.sup.3, R.sup.4, and R.sup.5 are independently hydrogen, —SO.sub.4.sup.2−, —PO.sub.4.sup.2−, —COC.sub.1-10alkyl; R.sup.6 is L-alanyl, L-alpha-aminobutyl, L-arginyl, L-asparginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenyalany, L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl or their D-isomers.

7. The composition according to claim 6, wherein the glycolipid is N-(2-Deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamide or a salt thereof.

8. The composition according to claim 7 wherein the salt is an acetate.

9. The composition according to claim 8, wherein said composition is essentially saponin-free.

10. The composition according to claim 6, wherein the composition is essentially free of quaternary ammonium compounds.

11. The composition according to claim 6, wherein the adjuvant consists of the glycolipid and the CpG-containing immunostimulatory oligonucleotide.

12. The composition according to claim 6, wherein the glycolipid is present in the amount of about 250 μg per dose.

13. The composition according to claim 6, wherein the immunostimulatory oligonucleotide is a P-class immunostimulatory oligonucleotide characterized by the presence of one or more TLR-9 activating motif (s) and two palindromes or two complementarity areas.

14. The composition according to claim 13 wherein said P-class immunostimulatory oligonucleotide is 5′ modified.

15. The composition according to claim 14, wherein said P class immunostimulatory oligonucleotide comprises at least 22 contiguous nucleotides of SEQ ID NO: 8.

16. The composition according to claim 1, wherein the CpG containing immunostimulatory oligonucleotide is present in the amount of about 20 to about 50 μg per dose.

17. A composition comprising a coronavirus, a Spike protein from said coronavirus or an immunogenic fragment of said Spike protein, and an adjuvant comprising a saponin, a sterol, a quaternary ammonium compound, and a polyacrylic acid polymer.

18. The composition according to claim 17, wherein the Saponin is Quil A and the sterol is selected from the group consisting of β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol, and the quaternary ammonium compound is DDAB.

19. The composition according to claim 18, wherein said Quil A is present in the amount of about 20 μg per dose, the sterol is cholesterol and is present in the amount of about 20 μg per dose, the DDAB is present in the amount of about 10 μg per dose, and the polyacrylic acid polymer is present in the amount of about 0.05% v/v.

20. The composition according to claim 1, wherein the coronavirus is SARS-2 coronavirus and the antigen is the Spike protein or the immunogenic fragment thereof.

21. The composition according to claim 20, wherein the Spike protein is at least 90% identical to SEQ ID NO: 13, with a proviso that said protein is in a pre-fusion state.

22. The composition according to claim 21, wherein amino acids at positions 973 and 974 are substituted with proline residues.

23. The composition according to claim 20, comprising a mutation in SEQ ID NO: 15.

24. The composition according to claim 23, wherein SEQ ID NO: 15 is replaced with SEQ ID NO: 16.

25. The composition according to claim 20, wherein said Spike protein or the immunogenic fragment thereof further comprises SEQ ID NO: 12.

26. The composition according to claim 20, wherein the Spike protein comprises SEQ ID NO: 17 or a sequence at least 99% identical thereto).

27. The composition according to claim 1, wherein said a Spike protein of said coronavirus or an immunogenic fragment of said Spike protein is present in the amount of about 20 μg per dose.

28. A method of inducing an immune response in a subject in need thereof, the method comprising administering to said subject the composition according to claim 1.

29. The method according to claim 28 wherein said subject is a canine.

30. The method according to claim 28 wherein said immunogenic composition is administered to said subject in a prime administration and in a boost administration, wherein the boost administration is between about 14 and about 42 days after the prime administration.

31. The method according to claim 28 wherein said immune response is a protective immune response.

32. The method according to claim 31 wherein said protective immune response is retained for at least six months after the boost administration.

33. The method according to claim 32 wherein said protective immune response is retained for at least 12 months after the boost administration.

Description

EXAMPLES

Example 1

[0097] The objective of the study is to evaluate the efficacy of a recombinant SARS-CoV-2 trimer spike protein vaccine in dogs via the generation of antibodies with the ability to neutralize SARS-CoV-2 in vitro. The vaccine protein is recognized as a target of antibody mediated binding. The protein is similar to that utilized for human SARS and MERS vaccines.

[0098] Six- to eleven-month-old male (castrated) and female beagle dogs were used in this study. Nine of these dogs had previous exposure to Canine Parvovirus (orally administered MLV vaccine and CPV2c challenge), Canine Parainfluenza Virus (orally administered MLV vaccine and CPIV challenge strain D008), and canine distemper virus (orally administered MLV vaccine). Six of these dogs had previous exposure to canine distemper virus (orally administered MLV vaccine). The dogs were healthy and negative to SARS-CoV-2 via PCR by oropharyngeal swabs prior to Day 0.

[0099] Animals were maintained in an appropriate housing environment to meet USDA Animal Welfare Regulations (9 Code of Federal Regulations, Chapter 1, Subchapter A—Animal Welfare), AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) and Institutional Animal Care and Use Committee (IACUC) guidelines. The dogs were fed dry food suitable for the age and nutritional requirements of the animals, moistened if necessary, and provided ad libitum at least once daily through the course of the study. Canned food or non-medicated nutritional supplements were given as needed. Water was available ad libitum at all times.

[0100] The dogs were randomly assigned to one of three groups, as provided in Table 1.

TABLE-US-00002 TABLE 1 Trt No. of Vaccination Blood End of Group Animals Details Day Dose Route Collection Study T01 5 REHYDRAGEL ® ONLY (Control) 0, 21 1.0 mL SubQ 0, 21, 42 42 20 μg recombinant Trimer Spike T02 5 protein (SEQ ID NO: 17) with 1% v/v REHYDRAGEL ® adjuvant, Q.S. with 0.063% PBS (LP), pH = 7.4 T03 5 20 μg recombinant Trimer Spike protein (SEQ ID NO: 17) with adjuvant containing Quil A - 20 μg; Cholesterol - 20 μg; SEQ ID NO: 8 - 20 μg per dose), Q.S. with 0.063% PBS (LP), pH = 7.4

[0101] Blood for pre-screening (approximately 3.0-6.0 mL) was collected prior to Day 0 for titer screening. Blood was collected in SST tubes from all animals.

[0102] Blood samples (approximately 6.0-12.0 mL or as appropriate for individual dog weight and blood collection guidelines) for serology were collected in SST tubes from all animals on Days 0 and 21, either a day before vaccination (i.e., day −1 and day 20) or the day of the vaccination but before the vaccination itself. On Day 42 (the end of the study) the maximum blood volume was calculated for each animal based on individual animal weight and IACUC guidelines. Blood was collected in SST tubes from all animals.

[0103] All animals were observed once on Day -1, twice on Day 0 (prior to and 3-6 hours post-vaccination), once daily on Days 1-5, twice on Day 21 (prior to and 3-6 hours post-vaccination), once daily on Days 22-26. Clinical observations were for approximately 30 minutes per session.

[0104] Injection site observations were recorded on Study Days 0 (prior to vaccination and 3-6 hours after), 1 through 5 for the first injection site (left shoulder). Injection site observations were recorded on Study Days 21 (prior to vaccination and 3-6 hours after), 22 through 26 (right shoulder).

[0105] The vaccines were well-tolerated by the dogs. No injection site pain or swelling observed during the study. Mild elevations of tympanic temperatures were observed in all study groups post-both vaccinations. No abnormal clinical signs were observed in any animals.

[0106] For the measurement of SN titer, a known quantity of the virus was combined with different dilutions of inactivated sera from the test animals. SN titer was measured by assessing viability of Vero E6 cells after the cells were incubated with the mixture of the virus and different dilution of the sera. See Tan et al., Nat Biotech 38:1073-78 (September 2020) and Wang et al., J Immunol. Methods 301:21-30 (2005).

[0107] For the determination of ELISA titer, plates were coated with 100 μl/well of 250 ng/ml protein (SEQ ID NO: 13) in coating buffer.

[0108] Peroxidase conjugated rabbit anti-dog IgG (H+L), polyclonal antibody (Jackson ImmunoResearch # 304-035-003, lot 135618) was used as a secondary antibody and TMB Microwell Peroxidase Substrate DAKO True Blue #1601 was used as the substrate. Sera were initially diluted 1:300 followed by 1:3 serial dilutions. Secondary antibody was diluted 1:30,000 in PBST (PBS+0.05% (w/v) TWEEN®-20). 100 μl/well of sample serum dilution was added to the plates, and the plates were incubated and incubated at room temperature for 60 minutes. Secondary antibody was diluted 1:30,000 in dilution buffer, and 100 μl/well of this solution was added to the wells, and the reaction proceeded for 30 minutes. The plates were washed (4× with PBST) after sample incubation and after incubation with the secondary antibody.

[0109] Lateral flow test is a semi-quantitative test. Generally, it is a binary test to determine whether the animal has or lacks antibodies to SARS-CoV2 by the presence or the absence of the visible band indicating the presence of the antibodies. However, but lateral flow device may also measure the intensity of the band thus providing a semi-quantitative measure of the amount of the antibodies. For convenience, this semi-quantitative measure is referred to as LF titer, or “titer measured by LF” or the like. It should be understood, however, that as applied to the Lateral Flow measurements, the term “titer” is not a titer, in the strict sense.

[0110] Lateral Flow titer was measured by loading the sample and a chase buffer containing a blocker protein such as BSA, a buffer to maintain pH, Tween 20, sodium azide, and polyethylene glycol (PEG) 8000 to the sample well of a lateral flow device in which they are absorbed by a pad. The sample and buffer are wicked via capillary action through a deposit of colloidal gold conjugated with SEQ ID NO: 13.

[0111] The recombinant Spike protein-colloidal gold conjugate was prepared by adding a saturating quantity of protein to the gold and incubated for 10 minutes, followed by the addition of a BSA blocker and a stabilizer buffer including BSA and sucrose.

[0112] The antibody-gold complex continues to migrate down the test strip until it crosses a line of deposited reagent (Protein A or G) to immobilize antibodies. The cross-linking of the antibody-gold complex to the reagent on this line results in an accumulation of colloidal gold on the line, and a visible red line is formed.

[0113] SN titers as well as lateral flow measurements and ELISA for individual animals are summarized in tables 2, 3, and 4, respectively.

TABLE-US-00003 TABLE 2 Animal Treatment SN Day SN Day SN Day ID Group 0 21 42 6591558 T01 <32 <32 <32 6591183 T01 <32 <32 <32 6586457 T01 <32 <32 <32 6586384 T01 <32 <32 <32 6586279 T01 <32 <32 <32 6591094 T02 <32 <64 >2048 6590969 T02 <32 272 >2048 6586490 T02 <32 55 563 6586341 T02 <32 536 >2048 6586287 T02 <32 <32 1026 6591540 T03 <32 489 >2048 6591442 T03 <32 441 >2048 6586422 T03 <32 258 >2048 6586350 T03 <32 538 >2048 6586295 T03 <32 160 >2048

TABLE-US-00004 TABLE 3 Day 0 Day 21 Day 42 Animal Group Visual Titer Visual Titer Visual Titer 6586279 T01 Neg 4469 Neg 3411 Neg 5571 6586384 T01 Neg 7147 Neg 11932 Neg 4243 6586457 T01 Neg 3598 Neg 6952 Neg 5706 6591183 T01 Neg 5632 Neg 10458 Neg 6727 6591558 T01 Neg 3873 Neg 4010 Neg 7843 6586287 T02 Neg 2420 Pos 20082 Pos 850922 6586341 T02 Neg 7025 Pos 365099 Pos 908487 6586490 T02 Neg 5456 Pos 295387 Pos 749547 6590969 T02 Neg 5731 Pos 650084 Pos 964848 6591094 T02 Neg 3604 Pos 132687 Pos 775755 6586295 T03 Neg 3230 Pos 558603 Pos 757122 6586350 T03 Neg 2552 Pos 695001 Pos 935679 6586422 T03 Neg 3661 Pos 556414 Pos 985040 6591442 T03 Neg 8359 Pos 541499 Pos 727335 6591540 T03 Neg 3632 Pos 755504 Pos 793830 Titer Titer Titer Animal Group Day 127 Day 155 Day 187 6586279 T01 2,763 4,684 6,038 6586384 T01 8,636 8,043 2,793 6586457 T01 9,804 5,889 6,162 6591183 T01 4,145 10,615 7,083 6591558 T01 5,717 7,436 3,506 6586287 T02 115,359 95,210 82,828 6586341 T02 353,214 264,908 257,688 6586490 T02 134,871 81,540 23,998 6590969 T02 395,128 187,301 144,476 6591094 T02 254,027 142,923 144,150 6586295 T03 467,165 475,987 474,329 6586350 T03 361,288 419,237 548,015 6586422 T03 363,699 424,946 649,238 6591442 T03 351,571 405,438 446,510 6591540 T03 463,024 437,149 495,005

[0114] All animals from group T01 were negative and all animals from groups T02 and T03 were positive on days 127, 155, and 187 by visual observation.

TABLE-US-00005 TABLE 4 ELISA ELISA ELISA ELISA ELISA ELISA ELISA Animal Group Day 0 Day 21 Day 42 Day 99 Day 127 Day 155 Day 187 6586279 T01 300 100 <1000 <300 <300 300 300 6586384 T01 100 100 <1000 300 300 300 300 6586457 T01 100 100 <1000 <300 <300 <300 300 6591183 T01 100 100 <1000 300 300 300 300 6591558 T01 100 100 <1000 <300 <300 300 300 6586287 T02 300 8100 81000 8,100 2,700 900 900 6586341 T02 100 8100 27000 24,300 8,100 2,700 2,700 6586490 T02 100 8100 27000 24,300 2,700 900 900 6590969 T02 100 2700 81000 24,300 8,100 900 900 6591094 T02 100 8100 27000 8,100 2,700 900 900 6586295 T03 100 8100 243000 24,300 24,300 8,100 8,100 6586350 T03 100 8100 81000 24,300 24,300 8,100 8,100 6586422 T03 100 8100 243000 24,300 8,100 8,100 8,100 6591442 T03 100 8100 243000 72,900 8,100 8,100 8,100 6591540 T03 100 24300 243000 24,300 8,100 8,100 8,100

[0115] No control animals seroconverted, as expected. In contrast each of the animals in group T02 and T03 seroconverted. All animals in groups T02 and T03 had protective titer on day 42.

Example 2

[0116] The objective of the study is to evaluate the efficacy of a recombinant SARS-CoV-2 trimer spike protein vaccine in cats via the generation of antibodies with the ability to neutralize SARS-CoV-2 in vitro.

[0117] Healthy domestic short hair cats of approximately ten months of age were used in the study. The cats were acclimatized for at least 14 days prior to use in the study.

[0118] All animals in the study were healthy prior to Day 0. All animals in the study were negative to SARS-CoV-2 by PCR via nasal swab and serology prior to Day 0. The animals were maintained in an appropriate housing environment to meet USDA Animal Welfare Regulations. Environmental conditions and floor space were consistent with the standard practices of the testing facility. The animas were fed with a diet according to their age requirement and provided with water ad libitum.

[0119] The randomization was produced using a SAS (SAS release 9.4 or higher, SAS Institute, Cary, N.C.) program developed specifically for the study with the ranuni function used to generate random numbers.

[0120] The animals were treated as summarized in Table 5 below.

TABLE-US-00006 TABLE 5 Trt No. of Vaccination Blood End of Group Animals Details Day Dose Route Collection Study T01 5 Control: Adjuvant only, as in T02 0, 21 1.0 mL SubQ 0, 21, 42 42 T02 5 Per dose: 20 μg recombinant Trimer Spike protein (SEQ ID NO: 17) adjuvant containing 20 μg Quil A, 20 μg Cholesterol, 10 μg DDA, 0.05% v/v CARBOPOL ®, Q.S. with DMEM PBS, pH = 7.5 T03 5 Per dose: 20 μg recombinant Trimer Spike protein (SEQ ID NO: 17) with adjuvant containing 250 μg BAY1005 ® acetate, 50 μg SEQ ID NO: 8, Q.S. with DMEM PBS, pH = 6.8)

[0121] The animals were observed, and tympanic temperature was measured twice daily on day 0 (prior to and 3-6 hours post-vaccination), once daily on days -1, 1-5, twice on day 21 (prior to and 3-6 hours after the vaccination, once daily on days 22-26. Clinical observations were for approximately 30 minutes per session.

[0122] Injection site reactions were observed daily for 5 days after each vaccination (days 1-5 and 22-26) or until the reactions were no longer visible for call cats.

[0123] The vaccines were well-tolerated. No injection site pain or swelling were observed while on study. No abnormal clinical observations or elevated temperatures were observed while on study.

[0124] Antibody responses to the vaccination were measure by the measurement of serum neutralizing titer, by lateral flow assay and by ELISA.

[0125] SN titers and LF titers were measured as described in Example 1.

[0126] ELISA Titers were measured as described in Example 1 except in this study, the starting dilution was 1:100 (rather than 1:300) and the secondary antibody was diluted 1:40,000 rather than 1:30,000. The results for the individual animals are provided in tables 6 (SN titer), 7 (Lateral Flow), and 8 (ELISA).

TABLE-US-00007 TABLE 6 Animal Group SN, Day 0 SN Day 21 SN Day 42 M191610 T01 <32 <32 <32 M191687 T01 <32 <32 <32 M191814 T01 <32 <32 <32 M191962 T01 <32 <32 <64 M192021 T01 <32 <32 <32 M191628 T02 <32 <32 >2048 M191644 T02 <32 <32 >2048 M191733 T02 <32 >2048 >2048 M191776 T02 <32 >2048 >2048 M191989 T02 <32 1371 >2048 M191725 T03 <32 558 >2048 M191857 T03 <32 >2048 >2048 M191920 T03 <32 361 >2048 M192004 T03 <32 550.5 >2048 M191602 T03 <32 >2048 >2048

TABLE-US-00008 TABLE 7 Day 0 Day 21 Day 42 Day 181 Day 265 Animal Group Visual Titer Visual Titer Visual Titer Titer Titer M191610 T01 Neg 8973 Neg 9288 Neg 10309 18580 N/A M191687 T01 Neg 9545 Neg 10443 Neg 11800 16804 N/A M191814 T01 Neg 9169 Neg 5292 Neg 7804 13916 N/A M191962 T01 Neg 11614 Neg 7924 Neg 17164 9359 N/A M192021 T01 Neg 5862 Neg 14558 Neg 12855 19127 N/A M191628 T02 Neg 7526 Pos 188166 Pos 521160 171951 128861 M191644 T02 Neg 10431 Pos 293915 Pos 342056 245437 171481 M191733 T02 Neg 10775 Pos 221439 Pos 401849 186440 179934 M191776 T02 Neg 14205 Pos 247635 Pos 462343 314616 288543 M191989 T02 Neg 5853 Pos 226459 Pos 411664 267826 208967 M191725 T03 Neg 9878 Pos 142507 Pos 375131 534069 626340 M191857 T03 Neg 14056 Pos 243948 Pos 252345 287385 304955 M191920 T03 Neg 6730 Pos 164120 Pos 438883 412096 440451 M192004 T03 Neg 11400 Pos 157025 Pos 540735 437773 494968 M191602 T03 Neg 7066 Pos 20709 Pos 363478 398709 623569 Day 419 Animal Group Visual Titer M191610 T01 N/A N/A M191687 T01 N/A N/A M191814 T01 N/A N/A M191962 T01 N/A N/A M192021 T01 N/A N/A M191628 T02 Pos 72,433 M191644 T02 Pos 73,167 M191733 T02 Pos 138,427 M191776 T02 Pos 149,226 M191989 T02 Pos 159,924 M191725 T03 Pos 263,788 M191857 T03 Pos 374,334 M191920 T03 Pos 153,351 M192004 T03 Pos 188,604 M191602 T03 Pos 383,614 N/A - Sample not analyzed

[0127] All animals from group T01 were negative and all animals from groups T02 and T03 were positive on days 181 and 265 by visual observation.

TABLE-US-00009 TABLE 8 ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA Animal Group Day 0 Day 21 Day 42 Day 86 Day 115 Day 148 Day 181 Day 265 M191610 T01 900 300 300 300 900 900 900 N/A M191687 T01 100 100 100 <300 <300 <300 <300 N/A M191814 T01 900 300 300 300 900 300 300 N/A M191962 T01 300 300 300 300 300 <300 300 N/A M192021 T01 100 100 100 <300 300 <300 <300 N/A M191628 T02 300 24300 72900 72900 72900 72900 72900 24300 M191644 T02 300 24300 72900 72900 24300 24300 24300 24300 M191733 T02 900 72900 72900 72900 24300 24300 24300 24300 M191776 T02 100 24300 72900 72900 24300 24300 24300 24300 M191989 T02 300 24300 72900 72900 72900 72900 24300 24300 M191725 T03 900 8100 72900 72900 72900 72900 72900 72900 M191857 T03 300 24300 >218700 218700 72900 72900 72900 72900 M191920 T03 300 24300 >218700 218700 72900 72900 72900 72900 M192004 T03 300 8100 >218700 72900 72900 72900 72900 72900 M191602 T03 300 8100 >218700 218700 72900 218700 72900 72900 ELISA ELISA ELISA ELISA ELISA ELISA Animal Group D289 D300 D328 D356 D384 D419 M191610 T01 N/A N/A N/A N/A N/A N/A M191687 T01 N/A N/A N/A N/A N/A N/A M191814 T01 N/A N/A N/A N/A N/A N/A M191962 T01 N/A N/A N/A N/A N/A N/A M192021 T01 N/A N/A N/A N/A N/A N/A M191628 T02 24,300 24,300 24,300 24,300 24,300 24,300 M191644 T02 24,300 24,300 24,300 8,100 8,100 8,100 M191733 T02 24,300 24,300 24,300 24,300 24,300 24,300 M191776 T02 24,300 24,300 8,100 8,100 8,100 24,300 M191989 T02 24,300 24,300 24,300 24,300 8,100 24,300 M191725 T03 72,900 72,900 72,900 72,900 72,900 72,900 M191857 T03 72,900 24,300 24,300 24,300 24,300 72,900 M191920 T03 72,900 72,900 72,900 72,900 72,900 72,900 M192004 T03 72,900 72,900 72,900 72,900 24,300 24,300 M191602 T03 72,900 72,900 72,900 72,900 72,900 72,900 N/A - Sample not analyzed

[0128] These data demonstrate that the vaccines according to the invention cause robust immune response against COVID-19 spike protein and that the immune response persists for at least 265 days or more, e.g., twelve months or more, or thirteen months or more, or 419 days.

[0129] All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein fully incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.