REPLICATION-COMPETENT ADENOVIRUS TYPE 4 SARS-COV-2 VACCINES AND

THEIR USE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/138,221, filed January 15, 2021, which is herein incorporated by reference in its entirety.

FIELD

This disclosure concerns a recombinant replication-competent adenovirus type 4 (Ad4) expressing a SARS-CoV-2 spike protein and its use as an immunogenic composition for inhibiting SARS-CoV-2 infection and transmission.

BACKGROUND

Coronaviruses are a large family of viruses that typically cause mild to moderate upper respiratory tract disease; however, some members of this family can cause severe disease and death in humans. In the last 20 years, coronaviruses have caused three major outbreaks in humans resulting from severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, the latter of which first emerged in Wuhan, China in December 2019. As of January 2021, SARS-CoV-2 had infected more than 84 million people worldwide, leading to nearly 2 million deaths. Although several SARS-CoV-2 vaccines have been approved for use in the U.S. and other countries, a need remains for an effective SARS-CoV-2 vaccine that induces mucosal immunity and can be rapidly produced in large quantities.

SUMMARY

Disclosed herein are immunogenic compositions comprised of a replication-competent adenovirus type 4 (Ad4) expressing a SARS-CoV-2 spike (S) protein (“Ad4-Spike”), such as a wild- type or modified version of the S protein from the original Wuhan strain or from a SARS- CoV-2 variant, such as the beta (B.1.351) variant, the delta (B.1.617.2) variant, the gamma (P.l) variant, the delta plus variant, or the omicron (B.1.1.529) variant. In the disclosed Ad4 vector, the gene encoding the SARS-CoV-2 S protein is cloned into the E3 region of an Ad4 vaccine strain. To accommodate insertion of the S protein, at least a portion of the E3 region is deleted. The disclosed Ad4-Spike vaccines possess several important advantages over other proposed and licensed SARS-CoV-2 vaccine platforms. In particular, as a replicating vector, Ad4-Spike is capable of inducing a durable immune response, including mucosal immunity, which is an important factor for inhibiting both infection and transmission of the virus. Furthermore, Add- Spike vaccines can be rapidly produced to high titers at a relatively low cost.

Provided herein is a recombinant, replication-competent Ad4 expressing a SARS-CoV-2 S protein. The genome of the recombinant Ad4 includes a deletion in the adenovirus E3 region and an insertion of a coding sequence for the SARS-CoV-2 S protein. The SARS-CoV-2 S protein can be a native S protein or a modified S protein, such as a stabilized or truncated S protein. Additionally, the S protein can be from the Wuhan strain of SARS-CoV-2 or a variant thereof, such as a variant of concern (VOC).

Also provided is a recombinant, replication-competent Ad4 vector having a deletion in the adenovirus E3 region and an insertion of a coding sequence for the SARS-CoV-2 S protein. The SARS-CoV-2 S protein can be a native S protein or a modified S protein, such as a stabilized or truncated S protein, derived from either the Wuhan strain or a SARS-CoV-2 variant, such as a VOC.

Further provided are immunogenic compositions that include a recombinant Ad4 or a recombinant Ad4 vector disclosed herein, and a pharmaceutically acceptable carrier.

Also provided are methods of eliciting an immune response against SARS-CoV-2 in a subject and methods of immunizing a subject against SARS-CoV-2 infection by administering to the subject a therapeutically effective amount of a recombinant Ad4, a recombinant Ad4 vector, or an immunogenic composition disclosed herein. In some embodiments, the recombinant Ad4, recombinant Ad4 vector or immunogenic composition is administered to the upper respiratory tract, such as intranasally.

The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: SARS-CoV-2 spike expression of stabilized and truncated designs in transfected A549 Cells. A549 cells were transfected with a shuttle vector plasmid containing the gene for the SARS-CoV-2 spike protein from the Wuhan strain (nCoV). Four spike protein constructs were made: wild-type (WT), stabilized (PP), tail truncated (TT), and endocytosis motif truncated (noEndo). Controls included untransfected (unTF) cells and cells transfected with a plasmid expressing an HIV-1 envelope (Env) protein (FDE3). Expression of spike and Env was measured by flow cytometry using a SARS-CoV-2 spike protein-specific antibody and an HIV-1 Env-specific antibody (VRC01), respectively. SARS-CoV-2 spike protein expression in transfected A549 cells diminished with stabilizing mutations, truncation of the tail, and truncation of the endocytosis motif, relative to wild-type spike protein.

FIGS. 2A-2B: SARS-CoV-2 spike expression of stabilized and truncated designs in infected A549 Cells. Replicating adenovirus carrying a SARS-CoV-2 protein gene was used to infect A549 cells. Three spike protein designs based on the Wuhan strain were tested for expression on the surface of A549 cells: wild-type (nCoV-WT), PP-stabilized (nCoV-PP), and tail- truncated (nCoV-TT) spike protein. A replicating adenovirus expressing an HIV-1 Env protein (FDE3) was used as a positive control of infection and uninfected (unIF) cells were used a negative control. Expression of spike protein was measured by flow cytometry using a SARS-CoV-2 spike protein- specific antibody. Antibody VRC01 was used to detect expression of HIV Env. Expression of spike by nCoV-WT is shown in FIG. 2A; expression of spike by FDE3, nCoV-PP and nCoV-TT is shown in FIG. 2B. As shown in FIGS. 2A-2B, expression of spike protein was high from both the nCoV-WT and nCoV-PP constructs.

FIG. 3: Immunization with replicating Ad4 containing SARS-CoV-2 spike protein gene induces neutralization in rabbits. New Zealand white rabbits were immunized on day 0 and day 28 (indicated by the arrows) with 1.29 x 10 ⁹ infectious units (IFU) of purified replicating Ad4 nCoV- WT. Using a luciferase assay, serum neutralization against Wuhan SARS-CoV-2 pseudovirus was detected starting at 4 weeks post-immunization (prior to the second dose), and continued to increase up to 12 weeks post-immunization.

FIG. 4: Amino acid alignment of nCoV-PP, nCoV-WT, nCoV-Tail-Truncation, and nCoV- No-Endo spike proteins. Alignment displays locations of three mutations introduced to the SARS- Cov-2 wild-type (Wuhan) spike protein. nCoV-PP contains double proline stabilization substitutions at amino acid position 986 and 987; nCoV-Tail-Truncation includes a deletion of the terminal 24 amino acids at the cytoplasmic tail; and nCoV-No-Endo contains a deletion of the terminal endocytosis signaling motif (terminal five residues). Amino acid numbering is with reference to wild-type spike protein set forth herein as SEQ ID NO: 2.

FIGS. 5A-5B: Serum neutralization against Wuhan pseudovirus in a dose titration of intranasal Ad4-SARS-CoV-2w _u pp in hamsters. Syrian golden hamsters were intranasally administered 10 ² to 10 ⁷ infection forming units (IFU) of Ad4-SARS-CoV-2 Wuhan spike with PP stabilization (Ad4-SARS-CoV-2w _u pp). Serum neutralization against Wuhan pseudovirus was measured at week 4 (FIG. 5A) and week 8 (FIG. 5B). Strong neutralization was observed at both timepoints for the highest doses of Ad4-SARS-CoV-2w _u pp. FIGS. 6A-6E: Serum neutralization of intranasal Ad4-SARS-CoV-2 expressing the indicated VOC spike in hamsters. Syrian golden hamsters were immunized with intranasal Ad4 expressing stabilized spike proteins from either the Wuhan strain (Ad4-CoV2-Wuhan), the beta variant (Ad4-CoV2-SA), the delta variant (Ad4-CoV2-Indian) or the gamma variant (Ad4-CoV2- Brazil), or a stabilized chimeric spike protein having the beta variant RBD (Ad-CoV2-Wu/RBD- SA). An Ad4 expressing an influenza virus H5 hemagglutinin (Ad4-H5) and sham inoculation were included as negative controls. Serum neutralization against Wuhan pseudovirus (FIG. 6A) or delta pseudovirus (FIG. 6B) was determined 28 days following intranasal administration. In addition, serum neutralization against Wuhan pseudovirus (FIG. 6C), delta pseudovirus (FIG. 6D) and omicron pseudovirus (FIG. 6E) was determined 56 days following intranasal administration.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file, created on January 14, 2022, 199 KB, which is incorporated by reference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is the nucleotide sequence of the Ad4-SARS-CoV-2 spike vector.

TAAATTTAAATGAATTCCGTCAAGGGCGACACAAAAGGTATTCTAAATGCATAATAA ATACTGATAACATCTTATAGTTT GTATTATATTTTGTATTATCGTTGACATGTATAATTTTGATATCAAAAACTGATTTTCCC TTTATTATTTTCGAGATTTA TTTTCTTAATTCTCTTTAACAAACTAGAAATATTGTATATACAAAAAATCATAAATAATA GATGAATAGTTTAATTATAG GTGTTCATCAATCGAAAAAGCAACGTATCTTATTTAAAGTGCGTTGCTTTTTTCTCATTT ATAAGGTTAAATAATTCTCA TATATCAAGCAAAGTGACAGGCGCCCTTAAATATTCTGACAAATGCTCTTTCCCTAAACT CCCCCCATAAAAAAACCCGC CGAAGCGGGTTTTTACGTTATTTGCGGATTAACGATTACTCGTTATCAGAACCGCCCAGG ATGCCTGGCAGTTCCCTACT CTCGCCGCTGCGCTCGGTCGTTCGGCTGCGGGACCTCAGCGCTAGCGGAGTGTATACTGG CTTACTATGTTGGCACTGAT GAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGC AGAATATGTGATACAGGATA TATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAA ATGGCTTACGAACGGGGCGG AGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAG CCGTTTTTCCATAGGCTCCG CCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGG ACTATAAAGATACCAGGCGT TTCCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCA TTCCGCTGTTATGGCCGCGT TTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTG TATGCACGAACCCCCCGTTC AGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATG CAAAAGCACCACTGGCAGCA GCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAAC TGAAAGGACAAGTTTTGGTG ACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCG AAAAACCGCCCTGCAAGGCG _{GTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAG ATCATCTTATTAAGCTTAGAAAAAC} TCATCGAGCATCAAATGAAATTGCAATTTATTCATATCAGGATTATCAATACCATATTTT TGAAAAAGCCGTTTCTGTAA TGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGC GATTCCGACTCGTCCAACAT CAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCAT GAGTGACGACTGAATCCGGT GAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGC TCGTCATCAAAATCACTCGC ATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAATACGCGATCGCT GTTAAAAGGACAATTACAAA CAGGAATCGAGTGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTG AATCAGGATATTCTTCTAAT ACCTGGAACGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTA CGGATAAAATGCTTGATGGT CGGAAGTGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATT GGCAACGCTACCTTTGCCAT GTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAGCGATAGATTGTCGCACCTG ATTGCCCGACATTATCGCGA GCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGACGTT TCCCGTTGAATATGGCTCAT ATTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATA CATATTTGAATGTATTTAGA AAAATAAACAAATAGGGGTCAGTGTTACAACCAATTAACCAATTCTGAACATTATCGCGA GCCCATTTATACCTGAATAT

GGCTCATAACACCCCTTGTTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCC CATGCCGAACTCAGAAGTGAAAC

GCCGTAGCGCCGATGGTAGTGTGGGGACTCCCCATGCGAGAGTAGGGAACTGCCAGG CATCAAATAAAACGAAAGGCTCA

GTCGAAAGACTGGGCCTTTCGCCCGGGCTAATTAGGGGGTGTCGCCCTTATCGCTGA GGATCCATTTAAATTTAATTAAC

ATCATCAATAATATACCTTATTTTTTTTGTGTGAGTTAATATGCAAATAAGGCGTGA AAATTTGGGGATGGGGCGCGCTG

ATTGGCTGTGACAGCGGCGTTCGTTAGGGGCGGGGCAGGTGACGTTTTGATGACGCG ACTATGAGGAGGAGTTAGTTTGC

AAGTTCTGGTGGGGAAAAGTGACGTCAAACGAGGTGTGGTTTAAACACGGAAATACT CAATTTTCCCACGCTGTCTAACA

GGAAATGAGGTGTTTTTGGGCGGATGCAAGTGAAAACGGACCATTTTCGCGCGAAAA CTGAATGAGGAAGTGAAATCTGA

GTAATTTAGTGTTTATGACAGGGAGGAGTATTTGCCGAGGGCCGAGTAGACTTTGAC CGTTTACGTGGGGGTTTCGATTA

CCGTGTTTTTCACCTAAAGTTCCGCGTACGGTGTCAAAGTCCGGTGTTTTTACGTAG GTGTCAGCTGATCGTCAGGGTAT

TTAAACCTGCGCTCTGCAGTCAAGAGGCCACTCTTGAGTGCCAGCGAGAAGAGTTTT CTCCTCCGCGCCGCGAGTCAGAT

CTACACTTTGAAATATGAGGCACCTAAGAGACCTGCCCGATGAGGAAATTATCATCG CTTCCGGGAGCGAGATTCTGGAA

CTGGTGGTAAATGCTATGATGGGCGACGACCATCCGGAACCCCCCACCCCATTTGAG ACACCTTCGCTGCACGATTTGTA

TGATCTGGAGGTGGATGTGCCCGAGGACGACCCCAACGAGAAGGCGGTAAATGATTT ATTTAGCGATGCCGCGCTGCTAG

CTGCCGAGGAGGCTTCAAGCCCTAGCTCAGACAGCGACTCTTCACTGCATACCCCTA GACACGACAGAGGTGAGAAAGAG

ATCCCCGGGCTTAAATGGGAAAAGATGGACTTGCGTTGCTATGAGGAATGCCTGCCC CCAAGCGATGATGAGGACGAGCA

GGCGATTCAGAACGCAGCGAGCCATGGAGTGCAAGCCGTCAGCGAGAGCTTTGCACT GGACTGCCCGCCTTTGCCCGGAC

ACGGCTGTAAGTCTTGTGAATTTCATCGCATCAATACTGGAGATAAAGCTGTGTTAT GTGCACTTTGCTATATGAGAGCG

TACAACCATTGTGTTTACAGTAAGTGTGATTAAGTGAACTTTAAAGGGAGGCAAAGA GTAGGGTGACTGGGTGATGACTG

GTTTATTTATGTATATCTGTTTTTTATATAGGTCCCGTTTCTGACGCAGATGATGAG ACCCCCACTACAGAGTCCACTTT

GTCACCCCCTGAAATTGGCACGTCTCCATCTGACAATATTGTTAGACCAGTTCCTGT AAGAGCCACTGGGAGGAGAGCAG

CTGTAGAATGTTTGGATGATTTGCTTCAGGGTGGAGATGAACCTTTGGACTTGTGTA CCCGGAAACGCCCCAGGCATTAA

GTGCCACACATGTGTGTTTACTTGAGGTGATGTCAGTATTTATAGGGTGTGGAGTGC AATAAAATATGTGTTGACTTTAA

GTGCGTGGTTTATGACTCAGGGGAGGGGACTTTGGGTATATAAGCAGGTGCAGACCT GTGTGGTTAGCTCAGAGCGGTAT

GGAGATTTGGACGGTTTTGGAAGACTTTCACAAGACTAGGCAGCTGCTAGAGAACGC CTCGAACGGAGTCTCTTACCTGT

GGAGATTCTGCTTCGGCGGTGACCTAGCTAAGCTAGTCTATAGGGCCAAACAGGATT ATAGGGAACAATTTGAGGATATT

TTGAGAGAGTGTCCTGGTCTTTTTGACGCTCTTAACTTGGGCCATCAGTCTCACTTT AACCAGAGAATTTCAAGAGCCCT

TGACTTTACTACTCCTGGCAGAACCACTGCAGCAGTAGCCTTTTTTGCTTTTATTTT TGACAAATGGAGTCAAGAAACCC

ATTTCAGCAGGGATTACCAGCTGGATTTCTTAGCAGTAGCTTTGTGGAGAACATGGA AGTGCCAGCGCCTGAATGCAATC

TCCGGCTACTTGCCGGTACAGCCGCTAGACACTCTGAGGATCCTGAGTCTCCAGCAG CAGGAGGATCAAGAAGAGAATCC

GAGAGCCGGCCTGGACCCTCCGGCGGAGGAGTAGCTGACCTGTTTCCTGAACTGCAC CGGGTGCTGACTAGGTCTTCGAG

TGGTCGGGAGAGGGGTATTAAGCGGGAGAGGCATGATGAGACTAATCACAGAATTGA ACTGACTGTGGGTCTGATGAGCC

GCAAGCGTCCAGAAACAGTGTGGTGGTATGAGGTGCAGTCAACTGGCACAGATGAGG TGTCAGTCATGCATGAGAGATTT

TCCCTAGAACAAGTCAAGACTTGTTGGTTGGAGCCTGAGGATGATTGGGAGGTAGCC ATCAGGAATTATGCCAAGCTGGC

TCTGAGGCCAGATAGAAAGTACAAGATTACTAAGCTGATAAATATCAGAAATGCCTG CTACATCTCAGGGAATGGGGCTG

AAGTGGAGATCTGTCTCCAGGATAGAGTGGCTTTCAGATGCTGCATGATGAATATGT ACCCGGGAGTGGTGGACATGGAT

GGGGTCACCTTTATGAACATGAGGTTCAGGGGAGATGGGTATAATGGGACGGTCTTT ATGGCCAATACCAAGCTGACAGT

GCATGGATGCTCCTTCTTTGGGTTTAATAACACCTGCATCGAGGCTTGGGGTCAGGT CGGTGTTAAGGGGTGCAGTTTTT

CAGCCAACTGGATGGGGGTAGTGGGCAGGACCAAGAGTATGCTGTCTGTGAAGAAAT GCTTGTTTGAGAGGTGCCACCTG

GGGGTGATGAGCGAGGGCGAAGCCAGAATCCGCCACTGTGCCTCTACCGAGACGGGC TGTTTTGTGCTGTGCAAGGGCAA

TGCCAAGATCAAGCATAATATGATCTGTGGAGCCTCGGACGAGCGCGGCTACCAGAT GCTGACCTGCGCCGGTGGGAACA

GTCATATGCTGGCCGCCGTGCATGTGGCTTCCCATTCCCGCAAGCCCTGGCCTGAGT TCGAGCACAATGTCATGACCAGG

TGCAATATGCATCTGGGGGCTCGCCGAGGCATGTTTATGCCCTACCAGTGCAACCTG AATTATGTAAAGGTGCTCCTGGA

GCCCGATGTCATGTCCAGAGTGAGCCTGACGGGGGTGTTTGACATGAATGTGGAAGT GTGGAAGATTCTAAGATATGATG

AATACAAGACCAGGTGTCGAGCCTGCGAGTGCGGAGGGAAGCATGCCAGGTTCCAGC CCGTGTGTGTGGATGTGACGGAG

GACCTGCGACCCGATCATTTGGTGTTGTCCTGCACCGGGACGGAGTTCGGCTCCAGT GGGGAAGAATCTGACTAGAGTGA

GTAGTGTTTTGGGGAGGGAGAGGACCTGCATAAGGGGCAGAATGATTAAAATCTGTG CTTTTCTGTGTGTTGCAGCAGCA

TGAGCGGAAACGGCTCCTTTGAGGGAGGGGTATTCAGCCCTTATCTGACGGGGCGTC TCCCCTCCTGGGCGGGAGTGCGT

CAAAATGTGATGGGATCCACGGTGGACGGCCGGCCCGTACAGCCCGCGAACTCTTCA ACCCTGACCTATGCAACCCTGAG

CTCCTCGTCGGTGGACGCAGCTGCCGCCGCAGCTGCTGCTTCTGCCGCCAGCGCCGT GCGCGGAATGGCCATGGGCGCCG

GCTATTACGGCACTCTGGTGGCCAACTCGAGTTCCACTAATAATCCCGCCAGCCTGA ACGAGGAGAAGCTGCTGCTGTTG

ATGGCCCAGCTCGAGGCCTTGACCCAGCGCCTGGGCGAGCTGACCCAGCAGGTGGCT CAGCTGCAGGAGCAGACGCGGGC

CGCGGTTGCCACGGTGAAATCCAAATAAAAAATGAATCAATAAATAAACGGAGACGG TTGTTGATTTTAAAAATCAGAGT

CTGAATCTTTATTTGATTTTTCGCGCACGGTAGGCCCTGGACCACCGGCCTCGATCA TTGAGCACCCGGTGGATCTTTTC

CAAGACCCGGTAGAGGTGGGATTGGATATTGAGGTACATGGGCATGAGCCCGTCCCG GGGGTGAAGGTAGCTCCATTGCA

GGGCCTCGTGCTCGGGGGTGGTGTTGTAAATCACCCAGTCATAGCAGGGACGCAGGG CGTGGTGTTGCACAATATCTTTG

AGGAGGAGACTGATGGCCACGGGCAGCCCTTTGGTGTAGGTGTTTACAAACCTGTTG AGCTGGGAGGGATGCATGCGGGG

GGAGATGAGGTGCATCTTAGCCTGGATCTTCAGATTGGCGATGTTACCGCCCAGATC CCGCCTGGGATTCATGTTGTGCA

GGACCACCAGCACGGTGTATCCGGTGCACTTGGGGAATTTGTCATGCAACTTGGAAG GGAAGGCATGAAAGAATTTGGAG

ACGCCCTTGTGGCCGCCCAGGTTTTCCATGCACTCATCCATGATAATGGCTATGGGC CCGTGGGCGGCGGCTTGGGCAAA

GACGTTTCGGGGGTCGGACACATCGTAGTTGTGGTCCTGGGTGAGATCTTCATAGGC CATTTTAATGAATTTGGGGCGGA

GGGTGCCCGATTGGGGGACGAAGGTACCCTCAATCCCGGGGGCGTAGTTTCCCTCAC AGATCTGCATCTCCCAGGCCTTA AGCTCCGAGGGGGGGATCATGTCCACCTGCGGGGCGATAAAGAAAACGGTTTCCGGGGCG GGGGAGATGAGCTGGGCGGA AAGCAGGTTGCGGAGTAGCTGGGACTTACCGCAGCCGGTGGGGCCGTAGATAACCCCAAT GACCGGCTGCAGGTGGTAGT TGAGGGAGACACAGCTGCCGTCCTCCCTAAGAAGGGGGGCCACCTCGTTCATCATTTGGC GCACGTGCATGTTCTCGCGC ACCAGTTCCGCCAGGAGTCGCTCTCCGCCCAGCGAGAGGAGCTCCTGGAGCGAGGCGAAG TTTTTCAGCGGCTTGAGCCC GTCGGCCATGGGCATTTTGGAAAGGGTCTGTTGCAGGAGTTCCAAGCGGTCCCAGAGCTC GGTGATGTGCTCTACGGCAT CTCGATCCAGCAGACCTCCTCGTTTCGCGGGTTGGGGCGACTGCGGGAGTAGGGCGCCAG ACGATGGGCGTCCAGCGCGG CCAGGGTCCGGTCCTTCCAGGGTCGCAGCGTCCGCGTCAGGGTGGTCTCCGTCACGGTAA AGGGGTGCGCGCCGGGCTGG GCGCTTGCGAGGGTGCGCTTCAGGCTCATCCGGCTGGTCGAGAACCGCTCCCGATCGGCG CCCTGTGCGTCGGCCAGGTA GCAATTGACCATGAGTTCGTAGTTGAGCGCCTCGGCCGCGTGGCCTTTGGCGCGGAGCTT ACCTTTGGAAGTCTGCCCAC AGGCGGGACAGAGGAGGGACTTGAGGGCGTAGAGCTTGGGGGCGAGGAAGACGGACTCGG GGGCGTAGGCGTCCGCGCCG CAGTGGGCGCAGACGGTCTCGCACTCCACGAGCCAGGTGAGGTCGGGCTGATTGGGATCA AAAACCAGTTTTCCGCCGTT _{C TT TT TGATGCGT TTCT TACC TC TGGTCTCCATGAGC TCGTGTCCCCGC TGGGTGACAAAGAGGC TGTCCGTGTCCCCGT} AAACCGACTTTATGGGTCGGTCCTCGAGTGGGACGCCGCGGTCCTCGTCGTAGAGGAAAC CCGACCACTCTGAGACGAAG GCCCGGGTCCAAGCCAGCACGAAGGAGGCCACGTGGGAGGGATAGCGGTCGTTATCCACC AGCGGGTCCACCTTCTCCAG TGTATGCAAACACATGTCCCCCTCGTCCACATCCAGGAAGGTGATTGGCTTGTAAGTGTA GGCCACGTGACCGGGGGTCC CGGCCGGGGGGGTATAAAAGGGGGCGGGCCGCTGCTCGTCTTCACTGTCTTCCGGATCGC TGTCCAGGAGCGCCAGCTGT TGGGGTAGGTATTCCCTCTCAAAGGCGGGCATGACCTCCGCACTCAGGTTGTCAGTTTCT AGAAACGAGGAGGATTTGAT ATTGACGGTGCCGGCGGAGATGCCTTTCAAGAGCCCCTCGTCCATCTGGTCAGAAAAGAC AATCTTTTTGTTGTCGAGTT TGGTGGCGAAGGAGCCGTAGAGGGCGTTGGAGAGGAGCTTGGCGATGGAGCGCATGGTCT GGTTCTTTTCCTTGTCGGCG CGCTCCTTGGCGGCGATGTTGAGCTGCACGTACTCGCGCGCCACGCACTTCCATTCGGGG AAGACGGTGGTTAGCTCGTC TGGCACGATTCTGACCTGCCAGCCCCGGTTATGCAGGGTGATGAGGTCAACGCTGGTGGC CACCTCGCCGCGCAGGGGCT CGTTGGTCCAGCAGAGGCGGCCGCCCTTGCGCGAGCAGAAGGGGGGCAGGGGGTCCAGCA TAAGCTCGTCGGGGGGGTCA GCATCGATGGTGAAGATGCCTGGCAGGAGGTCGGGGTCGAAGTAGCTTATGCAGGTGCCC AGATCGTCCAGAGAAGCTTG CCATTCGCGCACGGCCAGCGCGCGCTCGTAGGGACTAAGGGGCGTGCCCCAGGGCATGGG GTGGGTGAGCGCGGAGGCGT ACATGCCGCAGATGTCGTAGACGTAGAGGGGCTCATCAAGGATGCCAATGTAGGTGGGGT AGCAGCGGCCCCCGCGGATG CTGGCGCGCACGTAGTCATACAACTCGTGCGAGGGGGCGAGGAGCCCGGCTCCGAGATTG GCGCGGCTGGGTTTTTCGGC GCGGTAGACGATCTGACGGAAGATGGCGTGGGAGTTGGAGGAGATGGTGGGTCTTTGGAA GATGTTGAAGTGGGCGTGGG GCAGGCCGACCGAGTCGCGGATGAAGTGGGCGTAGGAGTCTTGCAGCTTGGCGACAAGCT CGGCGGTGACGAGGACGTCC AGGGCGCAGTAGTCAAGGGTCTCTTGGATGATGTCATACTTGAGCTGGCCCTTTTGTTTC CACAGCTCGCGGTTGAGAAG GAACTCTTCGCGGTCCTTCCAGTACTCTTCAAGGGGGAACCCGTCCTGGTCGGCACGGTA AGAGCCTAGCATGTAGAACT GGTTAACGGCCTTGTAGGCGCAGCAGCCCTTCTCCACGGGGAGGGCATAGGCCTGGGCGG CCTTGCGCAGGGAGGTGTGC GTGAGGGCGAAGGTGTCCCTGACCATGACCTTTAGGAACTGGTGCTTGAAGTCGATATCG TCGCAGCCCCCCTGCTCCCA GAGCTGGAAGTCCGTGCGCTTCTTGTAGGCGGGGTTGGGCAAAGCGAAAGTAACATCGTT GAAGAGGATCTTGCCCGCGC GGGGCATAAAGTTGCGAGTGATGCGGAAAGGCTGGGGCACCTCGGCCCGGTTGTTGATGA CCTGGGCGGCGAGCACGATC TCGTCGAAGCCGTTAATGTTGTGGCCCACAATGTATAGTTCCACGAACCGCGGGCGGCCC TTGACGTGGGGCAGTTTCTT GAGCTCCTCGTAGGTGAGCTCGTCGGGGTCGCTGAGCCCGTGCTGCTCGAGGGCCCAGTC GGCGAGATGGGGGTTGGCGC GGAGGAAGGAAGTCCAGAGATCCACGGCCAGGGCGGTTTGCAGACGATCCCGGTACTGGC GGAACTGCTGACCCACGGCC _{ATT TT TTCGGGGGTGACGCAGTAGAAGGTGCGGGGGTCGCCGTGCCAACGGTCCCAT TT TAGC TGGAGGGCGAGATCAAG} GGCGAGCTCAACGAGCCGGTCGTCCCCGGAGAGTTTCATGACCAGCATGAAGGGGACGAG CTGCTTGCCGAAGGACCCCA TCCAGGTGTAGGTTTCCACATCGTAGGTGAGGAAGAGCCTTTCGGTGCGAGGATGCGAGC CGATGGGGAAGAACTGGATC TCCTGCCACCAGTTGGAGGAATGGCTGTTGATGTGATGGAAGTAGAAATGCCGACGGCGC GCCGAACATTCGTGCTTGTG TTTATACAAGCGGCCACAGTGCTCGCAACGCTGCACGGGATGCACGTGCTGCACGAGCTG TACCTGGGTTCCTTTGACGA GGAATTTCAGTGGGAAGTGGAGTCGTGGCGCCTGCATCTGGTGCTGTACTACGTCGTGGT GGTCGGCCTGGCCCTCTTCT GCCTCGATGGTGGTCATGCTGACGAGCCCGCGCGGGAGGCAGGTCCAGACCTCGGCGCGA ACGGGTCGGAGAGCGAGGAC GAGGGCGCGCAGGCCGGAGCTGTCCAGGGTCCTGAGACGCTGCGGAGTCAGGTCAGTGGG CAGCGGCGGCGCGCGGTTGA CTTGCAGGAGTTTTTCAAGGGCGCGCGGGAGGTCCAGATGGTACTTGATCTCCACCGCGC CGTTGGTGGCGACGTCGATG GCTTGCAGTGTCCCGTGCCCCTGGGGAGTGACCACCGTCCCCCGTTTCTTCTTGGCGGGC GGAAGCGGTTTGGCTTCCAT GGTTAAAAGCGGCGGCGAGGACGCGCGCCGGGCGGTAGGGGCGGCTCGGGACCCGGAGGC AGTGGTGGCAGGGGCACGTC GGCGCCGCGCGCGGGCAGGTTCTGGTACTGCGCCCGGAGAAGACTGGCGTGAGCGACGAC GCGACGGTTGACGTCCTGGA TCTGACGCCTCTGGGTGAAGGCCACGGGACCCGTGAGTTTGAACCTGAAAGACAGTTCGA CAGAATCAATCTCGGTATCA TTGACGGCGGCCTGCCGCAGAATCTCTTGCACGTCGCCCGAGTTGTCCTGGTAGGCAATC TCGGTCATGAACTGCTCGAT CTCCTCCTCCTGAAGGTCTCCGCGGCCGGCGCGCTCCACGGTGGCCGCGAGGTCGTTGGA GATGCGGCCCATGAGCTGCG AGAAGGCGTTCATGCCCGCCTCGTTCCAGACGCGGCTGTAAACCACGGCGCCCTCGGGAT CGCGGGCGCGCATGACCACC TGGGCGAGGTTGAGCTCCACGTGGCGCGCAAAAACCGCGTAGTTGCAGAGGCGCTGGTAG AGGTAGTTGAGCGTGGTGGC AATGTGCTCAGTGACAAAGAAGTACATAATCCAGCGGCGGAGCGGCATTTCGCTGACGTC GCCCAGGGCTTCCAAGCGCT CCATGGCCTCGTAAAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGTGCAGATACGG TCAAGTCCTCCTCCAGAAGA CGGATGAGCTCGGCGATGGTGGCGCGCACCTCGCGCTCGAAGGCTCCCGTGAGTTCCTCC ACTTCCTCCTCTTCATCCAC TAACATCTCTTCTACTTCCTCCTCAGGCGGTGGTGGCGGGGGAGGGGGCCTGCGTCGCCG GCGGCGCACGGGCAGACGGT CGATGAAACGCTCGATGGTCTCGCCGCGCCGGCGTCGCATGGTCTCGGTGACGGCGCGCC CGTCCTCGCGGGGTCGCAGC GTAAAGACGCCGCCGCGCATCTCCAGGTGGCCCGGGGGGTCCCCGTTGGGCAGGGAGAGT GCGCTGACGATGCATCTTAT CAATTGCCCCGTAGGGACTCCGCGCAAGGACCTAAGCGTCTCTAGATCCACGGGATCTGA AAACCGTTGAACGAAGGCTT CGAGCCAGTCGCAGTCGCAAGGTAGGCTGAGCACGGTTTCTTCTGGCGGCGGTGGGGTGT GGGCGGGGGCGATGCTGCTG GTGATGAAGTTGAAATAGGCGGTTCTGAGACGGCGGATGGTGGCGAGGAGCACCAGGTCT TTGGGCCCGGCTTGCTGGAT

GCGCAGACGGTCGGCCATGCCCCAGGCGTGGTCCTGACACCTGGCCAGGTCCTTGTA GTAGTCCTGCATGAGCCGCTCCA

CGGGCACCTCCTCCTCGCCCGCGCGGCCGTGCATACGCGTGAGCCCAAACCCGCGCT GCGGCTGGACGAGCGCCAGGTCA

GCGACGACGCGCTCGGCGAGGATGGCCTGCTGGATCTGGGTGAGGGTGGTCTGGAAG TCGTCAAAGTCGACGAAGCGGTG

GTAGGCTCCGGTGTTAATGGTGTAGGAGCAGTTGGCCATGACGGACCAGTTGACAGT CTGGTGACCGGGCCGCGCGAGCT

CGTGGTACTTGAGGCGCGAGTAGGCGCGCGAGTCGAAGATGTAGTCGTTGCAGGTGC GCACCAGGTACTGGTAGCCGATG

AGGAAGTGCGGCGGCGGCTGGCGGTAGAGCGGCCATCGCTCGGTGGCGGGGGCGCCG GGCGCTAGGTCCTCGAGCATGGT

GCGGTGGTAGCCGTAGATGTACCTTGACATCCAGGTGATGCCGGCGGCGGTGGTGGA GGCGCGAGGGAACTCGCGGACGC

GGTTCCAGATGTTGCGCAGCGGCAGGAAGTAGTTCATGGTGGGCACGGTCTGGCCCG TGAGGCGCGCGCAGTCGTTGATG

CTCTAGACATACGGGCAAAAACGAAAGCGGTCAGCGGCTCGACTCCGTGGCCTGGAG GCTAAGCGAACGGGTTGGGCTGC

GCGTGTACCCCGGTTCGAATCTCGAATCAGGCTGGAGCCGCAGCTAACGTGGTACTG GCACTCCCGTCTCGACCCAGGCC

TGCACAAAACCTCCAGGATACGGAGGCGGGTCGTTTTGCAAATTTTTGGCGGTCGAA AAAAGCTAGTAAGCGCGGAAAGC

GGCCGACCGCAATGGCTCACTGCCGTAGATTGGAGAAGAATCGCCAGGGTTGCGTTG CGGTGTGCCCCGGTTCGAGACCG

CTCGGGTCGGCCGAATTCCGCGGCTAACGAGGGCGTGGCTGCCCCGTCGTTTCCAAG ACCCCATAAGCCAGCCGACTTCT

CCAGTTACGGAGCGAGCCCCTCTTTTGTTTTGTTTTTTGCCAGATGCATCCCGTACT GCGGCAGATGCGCCCCCACCCTC

CACCGCAACAGCAGCCCCCTCCTACGCAACAGCCGGCGCTTCTGCCTCCGCCCCAGC AGCAGCAACTTCCAGCCACTACC

GCCGCGGCCGCCGTGAGCGGGGCCGGGCAGAGTCAGTATGACCTGGCTTTGGAAGAG GGCGAGGGGCTGGCGCGCCTGGG

GGCGTCGTCGCCGGAGCGGCACCCGCGCGTGCAGATGAAAAGGGACGCTCGCGAGGC CTACGTGCCCAAGCAGAACCTGT

TCAGAGACAGGAGCGGCGAGGAGCCCGAGGAGATGCGCGCAGCCCGTTTCCACGCGG GGCGGGAGCTGCGGCGCGGCCTG

GACAGAAAGAGGGTGCTGAGGGACGAGGATTTCGAGGCGGACGAGCTGACGGGGATC AGCCCTGCGCGCGCGCACGTGGC

CGCGGCCAACCTGGTCACGGCGTACGAGCAGACCGTGAAGGAGGAGAGCAACTTCCA AAAATCCTTCAACAACCACGTGC

GCACCCTGATCGCGCGCGAGGAGGTGACCCTGGGCCTGATGCACCTGTGGGACCTGC TGGAGGCCATTGTGCAGAACCCC

ACCAGCAAACCGCTGACGGCGCAGCTGTTCCTGGTGGTGCAGCACAGTCGGGACAAC GAGACTTTTAGGGAGGCGCTGCT

GAATATCACCGAGCCCGAGGGCCGCTGGCTTCTGGACCTGGTGAATATTCTGCAGAG CATCGTGGTGCAGGAGCGCGGGC

TGCCGCTGTCCGAGAAGCTGGCGGCCATCAACTTTTCGGTGCTGAGTTTGGGCAAGT ACTACGCTAGGAAGATCTACAAG

ACCCCGTACGTGCCCATAGACAAGGAGGTGAAGATCGACGGGTTTTACATGCGCATG ACCCTGAAAGTGCTGACCCTGAG

CGACGATCTGGGGGTGTACCGCAACGACAGGATGCGCCGCGCGGTAAGCGCCAGCAG GCGGCGCGAGCTGAGCGATCAGG

AGCTGATGCACAGCCTGCAGCGGGCCCTGACCGGGGCCGGGACCGAGGGGGAGAGCT ACTTTGACATGGGCGCGGACCTG

CACTGGCAGCCCAGCCGCCGGGTCTTGGAAGCCGCGGCGGTCCCTTACGTAGAAGAG GTGGACGATGAGGATGAGGGCGA

GTACCTGGAAGACTGATGGCGCGACCGTATTTTTGCTAGATGCAGCAACAGCCACCT CCTGATCCCGCAATGCGGGCGGC

GCTGCAGAGCCAGCCGTCCGGCATTAACTCCTCGGACGATTGGACCCAGGCCATGCA ACGCATCATGGCGCTGACGACCC

GCAACCCCGAAGCCTTTAGACAGCAGCCCCAGGCCAACCGGCTCTCGGCCATCCTGG AGGCCGTGGTGCCCTCGCGCTCC

AACCCCACGCACGAGAAGGTGCTGGCCATCGTGAACGCGCTGGTTGAGAACAAGGCC ATTCGCGGCGACGAGGCCGGGCT

GGTGTACAACGCACTGCTGGAGCGCGTGGCCCGCTACAACAGCACCAACGTGCAGAC CAACCTGGACCGCATGGTGACCG

ACGTGCGCGAAGCCGTGGCCCAGCGCGAACGGTTCCACCGCGAGTCCAACCTGGGAT CCATGGTGGCACTGAACGCCTTC

CTCAGCACGCAGCCCGCCAACGTGCCCCGGGGCCAGGAGGACTACACCAACTTCATT AGCGCCCTGCGGCTAATGGTGAC

CGAGGTGCCCCAGAGCGAGGTGTACCAGTCGGGCCCGGACTACTTCTTCCAGACCAG TCGCCAGGGCTTGCAGACCGTGA

ACCTGAGTCAGGCTTTCAAGAACTTGCAGGGACTGTGGGGCGTGCAGGCTCCGGTCG GGGACCGCGCGACGGTGTCGAGC

CTGCTGACGCCGAACTCGCGCCTGCTGCTGCTGCTGGTGGCGCCCTTCACGGACAGC GGTAGTATCAACCGCAACTCGTA

CCTGGGCTACCTGATTAACCTGTACCGCGAGGCCATTGGCCAGGCGCACGTGGACGA GCAGACCTACCAGGAGATTACCC

ACGTGAGCCGCGCCCTTGGCCAGGACGACCCGGGCAATCTGGAAGCCACCCTGAACT TCTTGCTGACCAACCGGTCGCAG

AAGATCCCGCCCCAGTACGCGCTGAGCGCCGAGGAGGAGCGTATATTGAGATACGTG CAGCAAAGTGTGGGACTGTTCCT

GATGCAGGAGGGGGCCACCCCCAGCGCCGCGCTCGACATGACCGCGCGCAACATGGA GCCCAGCATGTACGCCAGTAATC

GCCCGTTTATTAATAAGCTGATGGACTACCTGCATCGGGCGGCCGCCATGAACTCTG ACTATTTCACCAACGCCATCCTG

AACCCCCACTGGCTCCCGCCGCCGGGGTTCTACACGGGCGAGTACGACATGCCCGAC CCCAATGACGGGTTTCTGTGGGA

CGACGTGGACAGCAGCGTGTTCTCCCCCCGACCGGGTGCTAACGAGCGCCCCTTGTG GAAGAAAGAGGGCAGCGACCGGC

GCCCGTCCTCGGCGCTGTCCGGCCGCACGGGTGCTGCCGCAGCGGTGCCCGAGGCCG CCAGTCCCTTTCCGAGCTTGTCA

CTGAACAGCGTCCGCAGTAGCGAGCTGGGCAGGATCACGCGCCCGCGCTTGCTGGGC GAGGAGGAGTACTTAAATAACTC

GCTGTTGAGGCCCGAGCGGGAGAAGAACTTCCCCAATAACGGGATAGAGAGTCTGGT GGATAAGATGAGCCGCTGGAAGA

CGTACGCGCATGAGCACAGGGACGATCCCCGGGCAACGCAGGGGGCCACCAGCCGGG GCAGTGCCGCCCGTAAACGCCGC

TGGCACGACAGGCAGCGGGGACTGATGTGGGACGATGAGGATTCCGCCGACGACAGC AGCGTGTTGGACTTGGGCGGGAG

TGGTGGTGGTAACCCGTTCGCTCACCTGCGCCCCCGCGTCGGGCGCCTGATGTAAAA AGAAACCAAAAATAAATGGTACT

CACCAAGGCCATGGCGACCAGCGTGCGTTCGTTTCTTCTCTGTTGTATCTAGTATGA TGAGGCGTGCGTACCCGGAGGGT

CCTCCTCCCTCGTACGAGAGCGTGATGCAGCAGGCAATGGCGGCGGCGGCGGCGATG CAGCCCCCGCTGGAGGCTCCTTA

CGTGCCACCGCGGTACCTGGCGCCTACGGAGGGGCGAAACAGCATTCGTTACTCGGA GCTGGCACCCTTGTACGATACCA

CCCGGTTGTACCTGGTGGACAACAAGTCGGCGGACATCGCCTCGCTGAACTACCAGA ACGACCACAGCAACTTTCTGACC

ACCGTGGTGCAGAACAACGATTTCACCCCCACGGAGGCCAGCACCCAGACCATCAAC TTTGACGAGCGCTCGCGGTGGGG

CGGTCAGCTGAAAACCATCATGCATACCAACATGCCCAACGTGAACGAGTTCATGTA CAGCAACAAGTTCAAGGCGCGGG

TCATGGTCTCCCGCAAGACCCCCAACGGGGTGACAGTAGGGGATGATTATGATGGTA GTCAGGATGAGCTGAAATACGAG

TGGGTGGAGTTTGAGCTGCCCGAAGGCAACTTCTCGGTGACCATGACCATTGACCTG ATGAACAACGCCATCATCGACAA

TTACTTGGCAGTGGGGCGGCAGAACGGGGTGCTGGAGAGCGACATCGGCGTGAAGTT CGACACCCGGAACTTCAGGCTGG

GTTGGGACCCCGTGACCGAGCTGGTCATGCCCGGGGTGTACACCAACGAGGCCTTCC ACCCCGACATCGTGCTGTTGCCC GGCTGCGGGGTGGACTTTACCGAGAGCCGCCTCAGTAATATGCTGGGCATCCGCAAGAGG CAGCCCTTCCAGGAGGGTTT CCAGATCATGTACGAGGACCTGGATGGAGGTAACATCCCCGCGCTCTTGGATGTCGAGGC CTATGAGAAAAGCAAGGAGG AGAGCGTCGCCGCGTCAACCGCAGCCGTAGCCACCGCCTCTACCGAGGTCCGGGGCGATA ATTTTGCTAGCGCCGCAGCA GTGGCGGCGGCCAAGGCTGATGAAACCGAAAGTAAGATAGTTATTCAGCCGGTGGAGAAG GATAGCAAGGATAGGAGCTA CAACGTGCTCTCGGACAAGAAAAACACCGCCTACCGCAGCTGGTACCTGGCCTACAACTA TGGCGACCACGAGAAGGGCG TGCGCTCCTGGACGCTGCTCACCACCTCGGACGTCACCTGCGGCGTGGAGCAAGTCTACT GGTCGCTGCCCGACATGATG CAAGACCCGGTCACCTTCCGCTCCACGCGTCAAGTTAGCAACTACCCGGTGGTGGGCGCC GAGCTCATGCCCGTCTACTC CAAGAGCTTCTTCAACGAGCAGGCCGTCTACTCGCAGCAGCTGCGCGCCTTCACCTCGCT CACGCACGTCTTCAACCGCT TCCCTGAGAACCAGATCCTCGTCCGCCCGCCCGCGCCCACCATTACCACCGTCAGTGAAA ACGTTCCTGCTCTCACAGAT CACGGGACCCTGCCGCTGCGCAGCAGTATCCGGGGAGTCCAGCGCGTGACCGTTACTGAC GCCAGACGCCGCACCTGCCC CTACGTCTACAAGGCCCTGGGCATAGTCGCGCCGCGCGTCCTCTCGAGCCGCACCTTCTA AAAAATGTCCATTCTCATCT CGCCCAGTAATAACACCGGTTGGGGTCTGCGCGCGCCCAGCAAGATGTACGGAGGCGCTC GCCAACGCTCCACGCAACAC CCCGTGCGCGTGCGCGGGCACTTCCGCGCTCCCTGGGGCGCCCTCAAGGGCCGCGTGCGG TCGCGCACCACCGTCGACGA CGTGATCGACCAGGTGGTGGCCGACGCTCGCAACTACACCCCCGCCGCCGCGCCCGTCTC CACCGTGGACGCCGTCATTG ACAGCGTGGTGTCCGACGCGCGCCGGTACGCCCGCGCCAAGAGCCGGCGGCGGCGCATCG CCCGGCGGCACCGTAGCACC ACCGCCATGCGTGCGGCGCGAGCCTTGCTGCGCAGGGCCAGGCGCACGGGACGCAGGGCC ATGCTCAGGGCGGCCAGACG CGCGGCTTCAGGCGCCAGCGCCGGCAGGACTCGGAGACGCGCGGCCACGGCGGCGGCAGC GGCCATAGCCAGCATGTCCC GCCCGCGGCGAGGGAACGTGTACTGGGTGCGCGACGCCGCCACCGGTGTGCGCGTGCCCG TGCGCACCCGCCCCCCTCGC ACTTGAAGATGTTCACTTCGCGATGTTGATGTGTCCCAGCGGCGAGGAGAAGGATGTCCA AGCGCAAATTCAAGGAAGAG ATGCTCCAGGTCATCGCGCCTGAGATCTACGGCCCCGCGGCGGCGGTGAAGGATGAAAGA AATCCCCGCAAAATCAAGCG GGTCAAAAAGGACAAAAAGGAAGAAGATGATGTGGACGATATGGTAGAGTTTGTGCGCGA GTTTGCCCCCCGGAGGCGCG TGCAGTGGCGCGGGCGGAAAGTGCGTCCGGTGCTGAGACCCGGCACCACGGTGGTTTTCG CGCCTGGCGAGCGGTCCGGC ACGACATCCAAGCGCTCCTACGATGAGGTGTACGGGGACGAGGATATTCTCGAGCAGGCG GCCGAGCGCCTGGGCGAGTT TGCTTACGGCAAGCGCAACCGCCTTGCGCCCCTGAAGGAAGAGGTGGTGTCCATCCCGCT GGACCACGGCAACCCCACGC CGAGTCTTAAGCCCGTGACCCTGCAGCAGGTGCTGCCGAGCGCGGCGCCGCGTCGGGGCT TGAAGCGCGAGGGCGAGGAT GTGTACCCCACCATGCAGCTGATGGTGCCCAAGCGCCAGAAGCTGGAAGACGTGCTGGAG ACCATGAAGGTGGACCCGGA CGTGCAGCCCGAGGTCAAGGTGAGGCCCATCAAGCAGGTGGCCCCGGGCCTTGGCGTGCA GACCGTGGACATCAAGATCC CCACGGAGCCCATGGAAACGCAGACCGAGGTCGTGAAGCCCATCACCAGCACCATGGAGG TGCAGACGGATCCTTGGATG CCGGCGGCGCCCCGAAAACCCCGGCGCAAGTACGGCGCGGCCAGCCTGCTGATGCCCAAC TACGCGCTGCATCCTTCCAT CATCCCCACGCCGGGCTACCGCGGCACGCGCTTCTACCACGGCTATACCGGCTCCCGCCG CCGCAAGACCACCACCCGCC GCCGTCGTCGCCGCACAGCTGCAACTCCCGCTGCCGCCCTGGTGCGGAGAGTGTACCGCC GCGGCCGCGCGCCTCTGACC CTGCCGCGGGCGCGCTACCACCCGAGCATTACCATTTAACTTTGCCGTCGCCTTTGCAGA TATGGCTCTCACATGCCGCA TTCGCGTCCCCATTACGGGCTACCGAGGAAGAAAACCGCGCCGTAGAAGGCTGGCGGGAA GCGGGATGCGCCGCCACCCC CACCGGCGGCGGCGCGCCATCAGCAAGCGGTTGGGGGGAGGCTTCCTGCCCGCGCTGATC CCCATCATCGCCGCGGCGAT CGGGGCGATCCCCGGCATTGCTTCCGTGGCGGTGCAGGCCTCTCAGCGCCACTGAGACAC ACACTTGGAAATTGTAATAA ACCCGAATGGACTCTGACGCTCCTGGTCCTGTGATGTGTTTTTGTAGACAGATGGAAGAC ATCAATTTTTCGTCCCTGGC TCCGCGACACGGCACGCGGCCGTTTATGGGCACCTGGAGCGACATCGGCACCAGCCAACT GAACGGGGGCGCCTTCAATT GGAGCAGTCTCTGGAGCGGGCTTAAGAATTTTGGGTCCACGCTTAAAACCTATGGCAGCA AGGCGTGGAACAGCACCACA GGGCAGGCGCTGAGAGATAAGCTGAAAGAGCAGAACTTCCAGCAGAAGGTAGTCGATGGC CTCGCCTCAGGCATCAACGG GGTGGTGGACCTGGCCAATCAGGCCGTGCAGCGGCAGATCAACAGCCGCCTGGACCCGGT TCCCCCCGCCGGCTCCGTGG AGATGCCGCAGGTGGAGGAGGAGCTGCCTCCCCTGGACAAGCGGGGCGACAAGCGTCCCC GTCCCGACGCGGAGGAGACG CTGCTGACGCACACGGACGAACCGCCCCCGTACGAGGAGGCGGTGAAACTGGGCCTGCCC ACCACGCGTCCCATTGCGCC TCTAGCTACCGGGGTGCTGAAACCCGAGAGTAGTAAGCCCGCGACCTTGGACTTGCCTCC TCCGCCCACTCCCCGCCCCT CCACAGTGGCTAAGCCCCTGCCGCCGGTGGCCGTGGCCCGCGCGCGACCGGGGGCTCGCC CTCAGGCGAACTGGCAGAGC ACTCTGAACAGCATCGTGGGTCTGGGAGTGCAGAGTGTGAAGCGCCGCCGCTGTTATTAA AAAACACTGTAGCGCTTAAC TTGCTTGTCTGTGTATATGTGTATGTCCGCCGCCGCTGCTGTCCAGAAGGAGGAGTGAAG AGAAAGGCGCGTCGTCGAGT TGCAAGATGGCCACCCCATCGATGCTGCCCCAGTGGGCGTACATGCACATCGCCGGACAG GACGCTTCGGAGTACCTGAG TCCGGGTCTGGTGCAGTTCGCCCGCGCCACAGACACCTACTTCAGTCTGGGGAACAAGTT TAGGAACCCCACGGTGGCGC CTACCCACGATGTGACCACCGACCGCAGCCAGCGGCTGACGCTGCGCTTTGTGCCCGTGG ACCGGGAGGACAACACCTAC TCGTACAAAGTGCGCTACACGCTGGCCGTGGGCGACAACCGCGTGCTGGACATGGCCAGC ACCTACTTTGACATCCGCGG CGTGCTGGATCGGGGCCCTAGCTTCAAACCCTACTCCGGCACTGCCTACAACAGCCTGGC TCCCAAGGGAGCGCCCAACA CCTGCCAGTGGAAGGATTCTGACAGCAAAATGCATACCTTTGGGGCAGCTGCCATGCCCG GTGTTACTGGGAAAAAGATA GAAGCTGATGGGCTGCCTATTAGAATAGATTCAACTTCTGGAACTGACACAGTAATTTAT GCTGATAAAACTTTCCAACC AGAACCACAAGTTGGAAATGACAGTTGGGTTGACACCAATGGTGCAGAGGAAAAATATGG AGGCAGAGCTCTAAAGGACA CTACAAAAATGAAACCCTGTTATGGTTCATTCGCCAAGCCTACCAACAAAGAAGGTGGTC AGGCTAACTTAAAAGATTCA GAACCCGCCGCCACCACTCCTAACTATGATATAGACCTGGCTTTCTTTGACAGCAAAACT ATTGTTGCTAACTACGATCC AGATATTGTAATGTACACAGAAAATGTTGACTTGCAGACTCCAGATACTCATATTGTATA CAAACCTGGAACAGAGGACA CCAGCTCTGAATCCAATTTGGGTCAGCAGGCCATGCCTAACAGACCCAACTACATTGGCT TCAGAGACAATTTTATCGGG CTCATGTACTACAACAGCACTGGCAATATGGGGGTGCTGGCCGGTCAGGCCTCTCAGCTG AATGCTGTGGTTGACTTGCA AGACAGAAACACTGAACTGTCCTACCAGCTCTTGCTTGACTCTCTGGGTGACAGAACCCG GTATTTCAGTATGTGGAATC AGGCGGTGGACAGCTATGATCCTGATGTGCGCATTATTGAAAACCATGGTGTGGAGGATG AATTGCCAAACTATTGCTTT CCGTTGAATGGTGTGGGATTGACAGACACTTACCAGGGTGTTAAAGTTAAAACAGATGCA GGTTCTGAAAAGTGGGACAA AGATGACACCACAGTTAGTAATGCTAATGAAATCCATGTAGGCAATCCTTTTGCCATGGA AATCAACATCCAAGCCAACC

TGTGGAGGAACTTCCTCTATGCCAATGTTGCCCTCTATTTGCCTGATAAATACAAAT ACACACCGGCCAACATCACCCTG

CCCACCAACACCAACACCTACGAGTACATGAACGGCCGGGTGGTGGCGCCCTCGCTG GTGGACGCCTACATTAACATTGG

GGCGCGCTGGTCGCTGGACCCCATGGACAACGTAAATCCCTTCAACCACCACCGCAA TGCGGGCTTGCGCTACCGCTCCA

TGCTCCTGGGCAACGGGCGCTACGTGCCATTCCACATCCAGGTGCCCCAGAAATTTT TTGCCATTAAGAGCCTCCTGCTC

CTGCCCGGGTCCTACACCTACGAGTGGAACTTCCGCAAGGACGTCAACATGATCCTG CAGAGTTCCCTTGGCAACGACCT

GCGCACAGACGGGGCCTCCATCACCTTCACCAGCATTAACCTCTACGCCACCTTCTT CCCCATGGCGCACAACACCGCCT

CCACGCTTGAGGCCATGCTGCGCAACGACACCAATGACCAATCCTTCAACGACTACC TCTCGGCGGCCAACATGCTCTAT

CCCATCCCGGCCAACGCCACCAACGTGCCCATCTCCATCCCCTCGCGCAACTGGGCC GCCTTTCGCGGCTGGTCCTTCAC

GCGTCTCAAGACCAAAGAGACGCCCTCGCTGGGCTCCGGGTTCGACCCCTACTTCGT CTACTCGGGCTCCATCCCCTACC

TCGACGGCACCTTCTACCTCAACCACACCTTCAAGAAGGTCTCCATCACCTTCGACT CTTCCGTCAGCTGGCCCGGCAAC

GACCGGCTCCTGACGCCCAACGAGTTCGAAATCAAGCGCACCGTCGACGGCGAGGGA TACAACGTGGCCCAGTGCAACAT

GACCAAGGACTGGTTCCTGGTCCAGATGCTGGCCCACTACAACATCGGCTACCAGGG CTTCTACGTGCCCGAGGGCTACA

AGGACCGCATGTACTCCTTCTTCCGCAACTTCCAGCCCATGAGCCGCCAGGTGGTGG ACGAGGTTAACTACAAGGACTAC

CAGGCCGTCACCCTGGCCTACCAACACAACAACTCGGGCTTCGTTGGATACCTCGCG CCCACTATGCGCCAGGGCCAGCC

CTACCCCGCCAACTACCCCTACCCGCTCATCGGCAAGAGCGCCGTTACCAGCGTCAC CCAGAAAAAGTTCATCTGCGACA

GGGTCATGTGGCGCATCCCCTTCTCCAGCAACTTCATGTCCATGGGCGCGCTCACCG ACCTCGGCCAGAACATGCTCTAT

GCTAACTCCGCCCACGCGCTAGACATGAATTTCGAAGTCGACCCCATGGATGAGTCC ACCCTTCTCTATGTTGTCTTCGA

AGTCTTCGACGTCGTCCGAGTGCACCAGCCCCACCGCGGCGTCATTGAGGCCGTCTA CCTGCGCACCCCCTTCTCAGCCG

GTAACGCCACCACATAAATTCTTGCTTCTTGCAAGAAGCCATGGCCGCGGGCTCCGG CGAGCAGGAGCTCAGGGCCATCA

TCCGCGACCTGGGGTGCGGGCCCTACTTCCTGGGCACCTTCGATAAGCGATTCCCGG GATTCATGGCCCCGCACAAGGTG

GCCTGCGCCATCGTCAACACGGCCGGCCGCGAGACCGGGGGCGAGCATTGGCTGGCC TTCGCCTGGAACCCGCGCTCGAA

CACCTGCTACCTCTTCGACCCCTTCGGGTTCTCGGACCAGCGCCTCAAGCAAATCTA CCAGTTCGAGTACGAGGGACTGC

TGCGCCGCAGCGCCCTGGCCACCAAGGACCGCTGCGTTACCCTGGAAAAGTCCACCC AGACCGTGCAGGGTCCGCGTTCG

GCCGCCTGCGGGCTTTTCTGCTGCATGTTCCTACACGCCTTCGTGCACTGGCCCAAC CGCCCCATGGACAAAAATCCCAC

CATGAACTTGCTGACGGGGGTGCCCAACGGCATGCTCCAGTCGCCCCAGGTGGAACC TACCCTGCGCCGCAACCAGGAGG

CACTCTACCGCTTCCTCAACTCCCACTCTGCATACTTTCGCTCTCACCGCGCGCGCA TTGAGAAGGCCACCGCCTTCGAC

CGCATGAATCAAGACATGTAACAGTGTGTTTTAAAATATGTTTAATAAACAGCACTT TTTATGTGACACATGCATTTGAG

ATAATTTTATTCTTAAAAATCGAAGGGGTTCTGCCGGGAGGTTTCGGCATGGCCCGC GGGCAGGGACACGTTGCGGAACT

GGTACTTGGCCAGCCACTTGAACTCGGGGATCAGCAGTTTCGGCAGCAGGGTGTCGG GGAACGAGTCGGTCCACAGCTTC

CGCGTCAGTTGCAGGGCGCCCAGCAGGTCGGGCGCGGAGATCTTGAAATCGCAGTTG GGACCCGCGTTTTGCGCGCGAGA

GTTGCGGTACACAGGGTTGCAGCACTGGAACACCATCAGGGCCGGATGCTTCACGCT CGCCAGCACCGTAGCGTCGGTGA

TCCCGTCCACGTCGAGGTCTTCGGCGTTGGCCATCCCGAAGGGGGTCATCTTGCAGG TCTGCCGGCCCATGGTGGGCACG

CAGCCGGGCTTGTGGTTGCAATCGCAGTGCAGGGGGATCAGCATCATCTGGGCCTGG TCGGCGTTCATCCCCGGGTACAT

GGCCTTCATGAAAGCCTCCAGCTGCTTAAACGCCTGCTGGGCCTTGGCTCCCTCGGT GAAGAAGACCCCGCAGGACTTGC

TAGAAAACTGGTTGGTAGCGCACCCGGCGTCGTGCACGCAGCAGCGCGCGTCGTTGT TGGCCAGCTGCACCACGCTGCGC

CCCCAGCGGTTCTGGGTAATCTTGGCCCGGTCGGGGTTCTCCTTTAGCGCGCGTTGC CCGTTCTCGCTTGCCACATCCAT

CTCGATCATGTGCTCCTTCTGGATCATGGTGGTCCCGTGCAGGCACCGCAGCTTGCC CTCGACTTCGGTACAGCCGTGCA

GCCACAGCGCGCACCCCGTGCTCTCCCAGTTCTTGTGGGCGATCTGGGAATGCGCAT GCACGAACCCCTGCAGGAAGCGG

CCCATCATGGTCGTCAGGGTCTTGTTACTGGTAAAGGTCAGCGGAATGCCGCGGTGC TCCTCGTTGATGTACAGGTGGCA

GATGCGGCGATACACCTCGCCCTGCTCGGGCATCAGTTGGAAGTTGGATTTTAGGTC GCTTTCCACACGGTAGCGCTCCA

TCAGCATATTCATGATTTCCATGCCCTTCTCCCAGGCCGATACAATGGGCAGGCTCA GGGGGTTCGTCACCGCCATCTTA

GCGCTAGCAGCCTTCGTCAGCGGGTCGTTCTCATTGAGAGTCTCAAAGCTCCGCTTG CCGTCCTTCTCGGTGATCCGCAC

GGGGGGGTAGCTGAAGCCCACGGCCGCCAGCTCCTCCTCGGCCTCTCTTTCGTCCTC GCTGTCCTGGCTGACGTCCTGCA

GGGGCACATGCTTCGTTTTGCGGGGTTTCTTTTTGGGCGGCTGCTGCGGCGGCGGTG GTTGTTCCTGAGGCGAGGGGGAG

CGCGAGTTCTCGCTCACCACTACTATCTCTTCTTCTTGGTCCGAGGCCACGCGGCGG TAGGTATGTCTCTTCAGGGGCAG

AGGCGGAGGCGACGGGCTCTCGCGGCCCGGCGGGTGGCTGGCAGAGCCCCTTCCGCG ATCGGGGGTGCGCTCCCGGCGGC

GCTCTAACTGACTTCCTCCGCGGCCGGCCATTGTGTTCTCCTAGGGAACAACAACAA GCATGGAGACTCAGCCATCGTCG

CCAACCTCGCCATCTGCCCCCACCGCCGACAAGAAGCAGCAGCAGAATGAGAGCTTA ACCGCCCCGCCGCCCAGCCCCGC

CACCTTTGTCGCGGCCCCAGACATGCAAGAGATGGAGGAATCCATTCAGATTGACCT GGGCTATGTGACGCCCGCGGAGC

ACGAGGAGGAGCTTGCAGTGCGCTTTTCAACCCAGGAAGAGATACACCAAGAACAGC CAGAGCAGGAAGCAAAGAGCGAG

CATGACTACCTCCACCAGAGCGGGGGGGAGGACGCCCTCATCAAGCATCTGGCCCGG CAGGCCATCATCGTCAAGGACGC

GCTGCTTGACCGCACCGAGGTGCCCCTCAGCGTGGAGGAGCTCAGCCGCGCCTACGA GCTCAACCTCTTCTCGCCGCGCG

TGCCCCCCAAGCGCCAGCCCAACGGCACCTGCGAGCCCAACCCACGCCTCAACTTCT ACCCGGTCTTCGCGGTGCCCGAG

GCCCTGGCCACCTACCACATCTTTTTCAAGAACCAAAGGATCCCTGTCTCCTGTCGC GCCAACCGCACCCGCGCCGACTC

CCTTTTCAACCTGGGCCCCGGTGCCCGCCTACCTGATATCGCCTCCTTGGAAGAGGT TCCCAAGATCTTCGAGGGTCTGG

GCAGCGACGAGACTCGGGCCGCAAACGCTCTGCAAGGAGAAGGAGGAGATCATGAGC ACCACAGCGCCCTGGTGGAGTTG

GAAGGCGACAACGCGCGTCTGGCGGTGCTCAAGCGCACGATCGAGCTGACCCATTTC GCCTACCCGGCGCTTAACCTGCC

CCCCAAAGTCATGAGCACGGTTATGGATCAGGTGCTCATCAAGCGCGCGTCGCCCAT CTCCAAGGAGATGCAAGACCCCG

AGAGCTCCGAGGAGGGCAAGCCCGTGGTCAGCGACGAGCAGCTGGCGCGGTGGCTGG GACCCCAAGCTAGTCCCCAGAGC

TTGGAAGAGCGGCGCAAGCTCATAATGGCCGTGGTCCTGGTGACCGCGGAGCTGGAG TGTCTGCGCCGCTTCTTCGCCGA

CGCAGAAATTCTGCGCAAGGTCGAGGAGAACCTGCACTACATCTTCAGGCACGGGTT CGTACGCCAGGCCTGCAAGATCT CCAACGTGGAGCTGACCAACCTGGTCTCCTACATGGGCATCTTGCACGAGAACCGCCTGG GGCAGAACGTGCTGCACACC

ACCCTGCGCGGGGAGGCCCGCCGCGACTACATCCGCGACTGCGTTTACCTCTACCTC TGCCACACCTGGCAGACAGCCAT

GGGCGTGTGGCAGCAGTGTCTGGAGGAGCAGAACCTAAAAGAGCTCTGCAAGCTCCT GCAGAAGAACCTCAAGGCCCTGT

GGACCGGGTTCGACGAGCGCACCACCGCCTCGGACCTGGCAGACCTCATTTTCCCCG AGCGTCTCAGGCTGACGCTGCGC

AACGGTTTGCCCGACTTTATGAGTCAAAGCATGTTGCAAAACTTTCGCTCTTTCATC CTCGAACGCTCCGGGATCCTGCC

GGCCACCTGCTCCGCGCTGCCCTCGGACTTCGTGCCGCTGACCTTCCGCGAGTGCCC CCCGCCGCTGTGGAGCCACTGCT

ACCTGCTGCGCTTGGCCAACTACCTGGCCTACCACTCGGACGTGATCGAGGACGTCA GCAGCGAGGGCCTGCTCGAGTGC

CACTGCCGCTGCAACCTCTGCACGCCGCACCGCTCCCTGGCCTGCAACCCCCAGCTG CTGAGCGAGACCCAGATCATCGG

CACCTTCGAGTTGCAAGGGCCCGGCGATGAGGGTTCTGCCGCCAAGGGGGGTCTGAA ACTCACCCCGGGGCTGTGGACCT

CGGCCTACTTGCGCAAGTTCGTGCCCGAGGACTACCATCCCTTCGAGATCAGGTTCT ACGAGGACCAATCCCAGCCGCCC

AAGGCCGAGCTGTCGGCCTGCGTCATCACCCAGGGGGCGATCCTGGCCCAATTGCAA GCTATCCAGAAATCCCGCCAAGA

ATTCTTGCTGAAAAAGGGCCGCGGGGTCTACCTTGATCCCCAGACCGGTGAGGAGCT TAACCCCGGCTTCCCCCAGGATG

CCCCGAGGAAGCAGCAAGAAGCTGAAAGTGGAGCTGCCGCCCGTGGAGGATTTGGAG GAAGACTGGGAGAGCAGTCAGGC

AGAGGAGGAGGAGATGGAAGACTGGGACAGCACTCAGGCAGAGGACAGCCTGCAAGA CAGTCTGGAAGACGAGGAGGAGG

CAGAGGAGGTGGAAGAAGTAGCCGCCGCCGCCAGACCGTCGTCCTCGGCGGAGAAAG CAAGCAGCACGGATACCATCTCC

GCTCCGGGTCGGGGTCCCGCTCGACCCCACAGTAGATGGGACGAGACCGGGCGATTC CCGAACCCCACCACCCAGACCGG

TAAGAAGGAGCGGCAGGGATACAAGTCCTGGCGGGGGCACAAAAACGCCATCGTCTC CTGCTTGCAAGCTTGCGGGGGCA

ACATCTCATTCACCCGGCGCTACCTGCTCTTTCACCGCGGGGTGAACTTCCCCCGCA ACATCTTGCATTACTACCGTCAC

CTCCACAGCCCCTACTACTTCCAAGAAGAGGCAGAAAAAGACAAAACCAGCAGCTAG AAAATCCACAGCGGCGGCGGCGG

CAGGTGGACTGAGGATCGCGGCGAACGAGCCGGCGCAGACCCGGGAACTGAGGAACC GGATCTTTCCCACCCTCTATGCC

ATCTTCCAGCAGAGTCGGGGGCAGGAGCAGGAACTGAAAGTCAAGAACCGTTCTCTG CGCTCGCTCACCCGCAGTTGTCT

GTATCACAAGAGCGAAGACCAACTTCAGCGCACGCTTGAGGACGCCGAGGCTCTCTT CAACAAGTACTGCGCACTCACTC

TTAAAGAGTAGCCCGCGCCCGCCCACACACGGAAAAAGGCGGGAATTACGTCACCTG TGCACCCCCACCCAGCACCGCTA

TGAGCAAAGAAATTCCCACGCCTTACATGTGGAGCTACCAGCCCCAGATGGGCCTGG CCGCCGGCGCCGCCCAGGACTAC

TCCACCCGCATGAATTGGCTCAGCGCCGGGCCCGGGATGATCTCACGGGTGAATGAC ATCCGCGCCCACCGAAACCAGAT

ACTCCTAGAACAGTCAGCGCTCACCGCCACGCCCCGCAATCACCTCAATCCGCGTAA TTGGCCCGCCGCCCTAGTGTACC

AGGAAATTCCCCAGCCCACGACCGTACTACTTCCGCGAGACGCCCAGGCCGAAGTCC AGCTGACTAACTCAGGTGTCCAG

CTGGCGGGCGGCGCCACCCTGTGTCGTCACCACCCCGCTCAGGGTATAAAGCGGCTG GTGATCCGGGGCAGAGGCACACA

GCTCAACGACGAGGTGGTGAGCTCTTCACTGGGTTTGCGACCTGACGGAGTCTTCCA ACTCGCCGGATCGGGAAGATCTT

TTCGGGGCAACATCTCATTCACCCGGCGCTACCTGCTCTTTCACCGCGGGGTGAACT TCCCCCGCAACATCTTGCATTAC

TACCGTCACCTCCACAGCCCCTACTACTTCCAAGAAGAGGCAGAAAAAGACAAAACC AGCAGCTAGAAAATCCACAGCGG

CGGCGGCGGCAGGTGGACTGAGGATCGCGGCGAACGAGCCGGCGCAGACCCGGGAAC TGAGGAACCGGATCTTTCCCACC

CTCTATGCCATCTTCCAGCAGAGTCGGGGGCAGGAGCAGGAACTGAAAGTCAAGAAC CGTTCTCTGCGCTCGCTCACCCG

CAGTTGTCTGTATCACAAGAGCGAAGACCAACTTCAGCGCACGCTTGAGGACGCCGA GGCTCTCTTCAACAAGTACTGCG

CACTCACTCTTAAAGAGTAGCCCGCGCCCGCCCACACACGGAAAAAGGCGGGAATTA CGTCACCTGTGCACCCCCACCCA

GCACCGCTATGAGCAAAGAAATTCCCACGCCTTACATGTGGAGCTACCAGCCCCAGA TGGGCCTGGCCGCCGGCGCCGCC

CAGGACTACTCCACCCGCATGAATTGGCTCAGCGCCGGGCCCGGGATGATCTCACGG GTGAATGACATCCGCGCCCACCG

AAACCAGATACTCCTAGAACAGTCAGCGCTCACCGCCACGCCCCGCAATCACCTCAA TCCGCGTAATTGGCCCGCCGCCC

TAGTGTACCAGGAAATTCCCCAGCCCACGACCGTACTACTTCCGCGAGACGCCCAGG CCGAAGTCCAGCTGACTAACTCA

GGTGTCCAGCTGGCGGGCGGCGCCACCCTGTGTCGTCACCACCCCGCTCAGGGTATA AAGCGGCTGGTGATCCGGGGCAG

AGGCACACAGCTCAACGACGAGGTGGTGAGCTCTTCACTGGGTTTGCGACCTGACGG AGTCTTCCAACTCGCCGGATCGG

GAAGATCTTCCTTCACGCCTCGTCAGGCCGTGCTGACTTTGGAGAGTTCTTCCTCGC AACCTCGCTCGGGCGGCATCGGC

ACTCTCCAGTTTGTGGAGGAGTTCACTCCCTCGGTCTACTTCAACCCCTTCTCCGGC TCCCCCGGCCACTATCCGGACGA

GTTCATCCCGAACTTCGATGCCATCAGCGAATCGGTAGACGGCTACGATTGAATGTC CCATGGTGGCGCGGCTGACCTAG

CTCGGCTTCGACACCTGGACCACTGCCGCCGCTTTCGCTGCTTCGCTCGGGACCTCG CCGAGTTTACCTACTTTGAGCTG

TCCGAGGAGCACCCTCAGGGCCCGGCCCACGGAGTGCGGATCGTCGTCGAAGGGGGC CTAGACTCCCACCTGCTTCGTAT

CTTCAGCCAGCGCCCGATCCTGGTCCAGCGCCAACAGGGCAACACCCTCCTGACCCT TTACTGCATCTGCAACCACCCCG

GCCTGCACGAAAGTCTTTGTTGTCTGCTGTGTACTGAGTATAATAAAAGCTGAGATC AGCGACTACTCCGGACTCGATTG

TGTTCCAGCAGTCTGGCGATACCAAGGGTTGCATCCACTGCTCCTGCGACTCCCCCG AGTGCGTTCACACCCTCATCAAG

ACCCTATGCGGCCTCCGCGACCTCCTCCCCATGAACTAATCAACTAACCCCTTACCC CATTACCCATCCAGTAAAAAAAA

TAAAGATTAAAGAGACGATGATTTTGAATTACTAGTTATTAATAGTAATCAATTACG GGGTCATTAGTTCATAGCCCATA

TATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAA CGACCCCCGCCCATTGACGTCAA

TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGG TGGAGTATTTACGGTAAACTGCC

CACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAAT GACGGTAAATGGCCCGCCTGGCA

TTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATT AGTCATCGCTATTACCATGGTGA

TGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC CAAGTCTCCACCCCATTGACGTC

AATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAAC TCCGCCCCATTGACGCAAATGGG

CGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCACTGTCTTCCGGATCG CTGTCCAGGAGCGCCAGCTGTTG

GGCTCGCGGTTGAGAAGGAACTCTTCGCGGTCCTTCCAGTACTCTTCAAGGGGGAAC CCGTCCTGGTCGGCACGGGACTC

CGCGCAAGGACCTAAGCGTCTCCAGATCCACGGGATCTGAAAACCGTTGAACGAAGG CTTCGAGCCAGTCGCAGTCGCAA

GTCTAGAGCCACCATGTTCGTCTTCCTGGTCCTGCTGCCCCTGGTCTCATCTCAGTG CGTGAATCTGACTACAAGAACTC

AGCTGCCTCCCGCCTACACCAATTCCTTCACCCGGGGCGTGTACTATCCTGACAAGG TGTTTAGAAGCTCCGTGCTGCAC TCTACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGGTTCCACGCCATCCAC GTGAGCGGCACCAATGGCAC AAAGCGGTTCGACAATCCCGTGCTGCCTTTTAACGATGGCGTGTACTTCGCCTCTACCGA GAAGAGCAACATCATCAGAG GCTGGATCTTTGGCACCACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATG CCACCAACGTGGTCATCAAG GTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAGAACAAT AAGAGCTGGATGGAGTCCGA GTTTAGAGTGTATTCTAGCGCCAACAATTGCACATTTGAGTACGTGTCCCAGCCTTTCCT GATGGACCTGGAGGGCAAGC AGGGCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGATGGCTACTTCAAGA TCTACTCTAAGCACACCCCC ATCAACCTGGTGCGCGACCTGCCTCAGGGCTTCAGCGCCCTGGAGCCACTGGTGGATCTG CCTATCGGCATCAACATCAC CCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCCGGCGACTCCTCTAG CGGATGGACCGCAGGAGCAG CAGCCTACTATGTGGGCTATCTGCAGCCTAGGACCTTCCTGCTGAAGTACAACGAGAATG GCACCATCACAGACGCAGTG GATTGCGCCCTGGACCCCCTGAGCGAGACAAAGTGTACACTGAAGTCCTTTACCGTGGAG AAGGGCATCTATCAGACATC CAATTTCAGGGTGCAGCCAACCGAGTCTATCGTGCGCTTTCCTAATATCACAAACCTGTG CCCATTTGGCGAGGTGTTCA ACGCAACCAGGTTCGCAAGCGTGTACGCATGGAATAGGAAGCGCATCTCTAACTGCGTGG CCGACTATAGCGTGCTGTAC AACTCCGCCTCTTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCCACAAAGCTGAATGAC CTGTGCTTTACCAACGTGTA CGCCGATTCTTTCGTGATCAGGGGCGACGAGGTGCGCCAGATCGCACCTGGACAGACAGG CAAGATCGCCGACTACAATT ATAAGCTGCCAGACGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATT CCAAAGTGGGCGGCAACTAC AATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCTCT ACAGAGATCTACCAGGCCGG CAGCACCCCTTGCAATGGCGTGGAGGGCTTTAACTGTTATTTCCCACTGCAGTCCTACGG CTTCCAGCCCACAAACGGCG TGGGCTATCAGCCTTACCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCACCAGCAA CAGTGTGCGGACCCAAGAAG TCCACCAATCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGAACA GGCGTGCTGACCGAGTCCAA CAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGT GCGCGACCCACAGACCCTGG AGATCCTGGATATCACACCCTGCTCTTTCGGCGGCGTGAGCGTGATCACACCAGGAACCA ATACAAGCAACCAGGTGGCC GTGCTGTATCAGGACGTGAATTGTACCGAGGTGCCTGTGGCCATCCACGCCGATCAGCTG ACCCCAACATGGCGGGTGTA CAGCACCGGCTCCAACGTGTTCCAGACAAGAGCAGGATGCCTGATCGGAGCAGAGCACGT GAACAATTCCTATGAGTGCG ACATCCCAATCGGCGCCGGCATCTGTGCCTCTTACCAGACCCAGACAAACTCTCCAAGGA GAGCACGGAGCGTGGCATCC CAGTCTATCATCGCCTATACCATGTCCCTGGGCGCCGAGAATTCTGTGGCCTACTCTAAC AATAGCATCGCCATCCCAAC CAACTTCACAATCTCTGTGACCACAGAGATCCTGCCCGTGTCCATGACCAAGACATCTGT GGACTGCACAATGTATATCT GTGGCGATTCTACCGAGTGCAGCAACCTGCTGCTGCAGTACGGCAGCTTTTGTACCCAGC TGAATAGAGCCCTGACAGGC ATCGCCGTGGAGCAGGATAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAGATCTAC AAGACCCCCCCTATCAAGGA CTTTGGCGGCTTCAATTTTTCCCAGATCCTGCCTGATCCATCCAAGCCTTCTAAGCGGAG CTTTATCGAGGACCTGCTGT TCAACAAGGTGACCCTGGCCGATGCCGGCTTCATCAAGCAGTATGGCGATTGCCTGGGCG ACATCGCAGCACGGGACCTG ATCTGTGCCCAGAAGTTTAATGGCCTGACCGTGCTGCCACCCCTGCTGACAGATGAGATG ATCGCACAGTACACAAGCGC CCTGCTGGCAGGAACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGAT CCCCTTTGCCATGCAGATGG CCTATAGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGA TCGCCAATCAGTTTAACTCC GCCATCGGCAAGATCCAGGACAGCCTGTCCTCTACAGCCTCCGCCCTGGGCAAGCTGCAG GATGTGGTGAATCAGAACGC CCAGGCCCTGAATACCCTGGTGAAGCAGCTGAGCTCCAACTTCGGCGCCATCTCTAGCGT GCTGAATGATATCCTGAGCC GGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGATCACAGGCAGACTGCAGT CTCTGCAGACCTATGTGACA CAGCAGCTGATCAGGGCAGCAGAGATCAGGGCAAGCGCCAATCTGGCAGCAACCAAGATG TCCGAGTGCGTGCTGGGCCA GTCTAAGAGAGTGGACTTTTGTGGCAAGGGCTATCACCTGATGTCCTTCCCTCAGTCTGC CCCACACGGCGTGGTGTTTC TGCACGTGACCTACGTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCC ACGATGGCAAGGCCCACTTT CCAAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTC TACGAGCCCCAGATCATCAC CACAGACAATACCTTCGTGAGCGGCAACTGTGACGTGGTCATCGGCATCGTGAACAATAC CGTGTATGATCCACTGCAGC CCGAGCTGGACAGCTTTAAGGAGGAGCTGGATAAGTACTTCAAGAATCACACCTCCCCTG ACGTGGATCTGGGCGACATC AGCGGCATCAATGCCTCCGTGGTGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTG GCCAAGAATCTGAACGAGAG CCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCATGGTACAT CTGGCTGGGCTTCATCGCCG GCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGTATGACATCCTGCTGTTCTT GCCTGAAGGGCTGCTGTAGC TGTGGCTCCTGCTGTAAGTTTGATGAGGACGATTCCGAACCCGTGCTGAAGGGAGTGAAG CTGCATTACACCTGAGGATC CCTCGAGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCC TTGACCCTGGAAGGTGCCAC TCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCA TTCTATTCTGGGGGGTGGGG TGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGG TGGGCTCTATGGTGATCAAT AAAGAATCACTTACTTGAAATCTGAAACCAGGTCTCTGTCCATGTTTTCTGTCAGCAGCA CTTCGCTCCCCTCTTCCCAG CTCTGGTACTGCAGGCCCCGGCGGGCTGCAAACTTCCTCCACACTCTGAAGGGGATGTCA AATTCCTCCTGTCCCTCAAT CTTCATTTTTTATTTCTATTAGATGTCCAAAAAGCGCGCGCGGGTGGATGATGGCTTCGA CCCCGTGTATCCCTACGATG CAGACAACGCACCGACCGTGCCCTTCATCAACCCTCCCTTCGTCTCTTCAGATGGATTCC AAGAAAAGCCCCTGGGGGTG TTGTCCCTTAGGCTGGCCGACCCTGTCACCACCAAGAATGGGGAAATTACCCTCAAGCTG GGGGAGGGGGTGGACCTTGA CGACTCGGGAAAACTCATTGCAAACACAGTAAACAAGGCCATTGCCCCTCTCAGTTTTTC CAACAACACCATTTCCCTTA ACATGGATACCCCTTTATACACCAAAGATGGAAAACTATCCTTACAAGTTTCTCCACCAT TAAGTATATTAAAATCAACA ATTTTGAATACATTAGCTCTAGCTTTTGGCTCAGGTTTAGGACTCAGTGGCAGCGCCCTG GCAGTACAGTTAGCCTCTCC ACTTACATTTGATGATAAAGGGAATATAAAGATTACCCTAAACAGGGGATTGCATGTTAC AACAGGAGATGCAATTGAAA GCAACATCAGTTGGGCTAAAGGTATAAAATTTGAAGATGGTGCCATAGCTACAAACATTG GTAAGGGGCTAGAGTTCGGA ACCAGTAGTACAGAAACAGGAGTTAATAATGCTTATCCAATCCAAGTTAAACTTGGCTCT GGTCTCAGCTTTGACAGCAC AGGAGCCATAATGGCTGGCAATAAAGACTATGATAAATTAACTTTGTGGACAACGCCTGA CCCATCACCAAACTGTCAAA TACTTGCAGAAAATGATGCAAAACTAACACTTTGCTTAACTAAGTGTGACAGTCAAATAC TGGCCACTGTATCAGTTTTG GTTGTTAGAAGTGGAAACTTAAACCCAATTACTGGCACAGTAAGCAGTGCTCAAGTTTTT CTACGTTTTGATGCAAATGG

TGTTCTTTTAACAGAACACTCTACACTAAAAAAATACTGGGGCTACAAGCAAGGAGA TAGCATAGATGGCACTCCATACA

CCAATGCTGTTGGTTTTATGCCAAATTCAACAGCTTATCCAAAGACCCAAAGTTCTA CTACTAAAAATAATATAGTGGGT

CAAGTATACATGAATGGAGATGTTTCAAAACCCATGCTTCTTACTATAACTCTTAAT GGTACTGATGACACCACCAGTGC

ATACTCAATGTCATTTTCATACACCTGGACTAACGGAAGCTATATCGGAGCAACATT TGGAGCTAACTCATACACCTTCT

CCTACATAGCCCAACAATAATCCCACCCTGCATGCCAACCCACCTTTTCCCTCTATT TATAAATGGAAACTGAAACAAAA

ATAAAGTTCAAGTGTTTTATTGATTCAACAGTTTTTCACAGGATTCGAGTAGTTATT TTCCCTCCACCCTCCCATCTCAT

GGAATACACTATCCTCTCCCCACGCACAGCCTTAAACATCTGAATGCTATTGGTAAT GGACATGGTTTTGATCTCCACAT

TCCACACAGTTTCAGAGCGAGACAGTCTCGGGTCGGTCAAGGAGATGAAACCCTCCG GGCACTCCTGCATCTGCACCTCA

CAGTTCAACAGCTGAGGGCTGTCCTCGGTGATTGGAATCACAGTTATCTGGAATAAG AGCGATGAGAATCATAATCCGCA

AACGGGATCGGGCGGTTGTGGCGCATCAGGCCCCGCAGCAGTCGCTGTCTGCGCCGC TCCGTCAAGCTGCTACTCAAGGG

GTCCGGGTCCAGGGACTCCCTGCGCATGATGCCAATGGCCCTGAGCATCAGTCGCCT GGTACGGCGGGCGCAGCAGCGGA

TGCGGATCTCACTCAGGTCGGAGCAGTACGTGCAGCACAGCACCACCAAGTTGTTCA ACAGTCCATAGTTCAACGTGCTC

CAGCCAAAACTCATTTGTGGAACTATGCTGCCCACATGTCCATCGTACCAGATCCTG ATGTAAATCAGGTGGCGTCCCCT

CCAGAACACACTGCCCATGTACATGATCTCCTTGGGCATGTGCAGGTTCACCACCTC CCGGTACCACATCACCCGCTGGT

TGAACATGCAGCCCTGGATAATTCTGCGGAACCAGATGGCAAGTACCGTCCCGCCCG CCATGCAGCGCAGGGACCCCGGG

TTCTGGCAATGGCAGTGGATCACCCACCGCTCGCGACCGTGGATCAACTGGGAACTA AACAAGTCTATGTTGGCACAGCA

CAGGCACACGCTCATGCATGTCTTCAGCACTCTCAATTCCTCGGGGGTCAGGACCAT ATCCCAGGGCACAGGGAACTCTT

GCAGGACAGTGAACCCGGCCGAACAGGGCAATCCTCGCACGGAACTTACATTGTGCA TGGACAGGGTATCGCAATCAGGC

AGCACCGGATGATCCTCCACCAGAGAAGCGCGGCTCTCGGTCTCCTCACAGCGAGGT AAGGTGGCCGGCGGTTGGTACGG

ATGATGGCGAGATAACGCTAATCGTGTTCTGGATCGTGTCATGATGGAGCTGTTTCC GGACATTTTCGTATTTCACAAAG

CAGAACCTGGTCCGGGCACTGCACACCGCTCGTCGGCGACGGTCTCGGCGCTTCGAG CGCTCAATGTTGAAGTTATAGAA

CAGCCACTCCCTCAGAACGTGCAGTATCTCCTGAGCCTCTTGGGTGATGAAAATCCC ATCCGCCCTGATGGCTCTGATTA

CATCAACCACGGTGGAATGGGCCAAACCCAGCCAGATGATGCAATTTTGTTGGGTTT CGGTGACGGCGGGGGAGGGAAGA

ACAGGAAGAACCATGATTAACTTTATTCCAAACGGTCTCGGAACACTTCAAAATGCA GGTCCCGGAGGTGGCACCTCTCG

CCCCCACTGTGTTGGTGGAAAATAACAGCCAGGTCAAAGGTAACACGGTTCTCGAGA TGTTCCACGGTGGCTTCCAGCAA

AGCCTCCACGCGCACATCCAGAAACAAGAGGACAGCGAAAGCGGGAGCGTTTTCTAA TTCCTCAATCATCATATTACACT

CCTGCACCATGCCTAGATAATTTTCATTTTTCCAGCCTTGAATGATTCGTATTAGTT CCTGAGGTAAATCCAAGCCAGCC

ATGATAAAAAGCTCGCGCAGAGCGCCCTCCACCGGCATTCTTAAGCACACCCTCATA ATTCCAACAGATTCTGCTCCTGG

TTCACCTGTAGTAGATTAACAAGTGGAATATCAATTGCTCTGCCGCAATCCCTAAGC TCCTCCCTTAGCAGTAACTGTAT

GTACTCATTCATATCTTCTCCGAAATTTTTAGCCATAGGACCACCAGGAACAAGAGA AGGGCAAGCCACATTACAGATAA

AGCGAAGTCCTCCCCAGTGAGCATTGCCAAATGTAAGATTGAAATAAGCATGCTGGC TAGACCCGGTGATATCTTCCAGA

TAACTGGACAGAAAATCAGGCAAGCAATTTTTAAGAAAATTAACAAAAGAAAAGTCG TCTAGGTGCACGTTTAGAGCCTC

AGGAACAACGATGGAATAAGTGCAAGGAGTACGTTCCAGCATGGTTAGTGTTTTTGG TGATCTGTAGAACAAAAAATAAA

CATGCAATATTAAACCATGCTAGCCTGGCGAACAGGTGGATAAATCACTCTTTCCAA CACCAGGCAGGCTACAGGGTCTC

CGGCGCGACCATTGTAGAAGCTGACATTATGATTAAAAAGCATCACCGACAGACCTT CCCGGTGGCCGGCATGGATGATT

CGAGAAGAAGCATACACTCCGGGAACATTGGCGTCCGTGAGTGAAAAAAAGCGACCT ATAAAGCCTTGAGGCACTACAAT

GCTTAATCTTAATTCCAGCAAAGCGACCCCATGCGGATGAAGCACAAAATTGGCAGG TGCGTAAAAAATGTAATTACTCC

CCTTCTGCACAGGCAGCAAAGCCCCCGCTCCCTCCAGAAACACATACAAAACCTGAG CGTCCATAGCTTACCGAGCACGG

CAGGCGCAAGAGTCAGAGAAAAAGCTGAGCTCTAACCTAACTGCCCGCTTCTGTACT CAATATATAGCCCTAACCTCACT

GACGTAAAGGCCAAGGTCTAAAAATACCCGCCAACACGCCCAGAAACCGGTGACACA CTAAAAAAATACGTGCACTTCCT

CAAACGCCCAAACTGGCGTCATTTCCGGTTTCCCACGCTACGTCACCTCTCAACGAC TTTCAAATTCCGTCGACCGTTAA

ACACATCAGTTACCCCGCCCCTAACGAACGCCGCTGTCACAGCCAATCAGCGCGCCC CATCCCCAAATTTTCACGCCTTA

TTTGCATATTAACTCACACAAAAAAAATAAGGTATATTATTGATGATGAAGCTTTTA AT

SEQ ID NO: 2 is the amino acid sequence of a wild-type SARS-CoV-2 (Wuhan strain) spike protein deposited under GenBank Accession No. YP_009724390.1.

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRS SVLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFD NPVLPFNDGVYFASTEKSNI IRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY S SANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNI DGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINI TRFQT LLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTI TDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRV QPTES IVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSF VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSN LKPFERDI STE IYQAGSTPC NGVEGFNCYFPLQSYGFQPTNGVGYQPYRWVL SFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFL PFQQFGRDIADTTDAVRDPQTLE ILDI TPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQS I IAYTMSLGAENSVAYSNNS IAIPTNFTI SVTTE ILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQV KQIYKTPPIKDFGGF NFSQI LPDPSKPSKRSF IEDLLFNKVTLADAGF IKQYGDCLGDIAARDL ICAQKFNGLTVLPPLLTDEMIAQYTSALLAG TITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLS STASALGKLQDVVNQNAQALN TLVKQLS SNFGAI SSVLNDIL SRLDKVEAEVQI DRLI TGRLQSLQTYVTQQLIRAAE IRASANLAATKMSECVLGQSKRV DFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTH WFVTQRNFYEPQIITTDNT

FVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLIDL

QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFD EDDSEPVLKGVKLHYT

SEQ ID NO: 3 is the amino acid sequence of a stabilized SARS-CoV-2 spike protein with a double proline substitution (nCoV-PP).

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFSNVTWFHAIHVSGTNGTKRFD NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFL GVYYHKNNKSWMESEFRVY SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGF SALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRV QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCY GVSPTKLNDLCFTNVYADSF VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSN LKPFERDISTEIYQAGSTPC NGVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTNLVKNKCVNF NFNGLTGTGVLTESNKKFL PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEV PVAIHADQLTPTWRVYSTGS NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLG AENSVAYSNNSIAIPTNFTI SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE VFAQVKQIYKTPPIKDFGGF NFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTV LPPLLTDEMIAQYTSALLAG TITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSS TASALGKLQDVVNQNAQALN TLVKQLSSNFGAI SSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECV LGQSKRV DFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTH WFVTQRNFYEPQIITTDNT FVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLIDL

QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFD EDDSEPVLKGVKLHYT

SEQ ID NO: 4 is the amino acid sequence of a tail-truncated SARS-CoV-2 spike protein (nCoV-TT).

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFSNVTWFHAIHVSGTNGTKRFD NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFL GVYYHKNNKSWMESEFRVY SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGF SALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRV QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCY GVSPTKLNDLCFTNVYADSF VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSN LKPFERDISTEIYQAGSTPC NGVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTNLVKNKCVNF NFNGLTGTGVLTESNKKFL PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEV PVAIHADQLTPTWRVYSTGS NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLG AENSVAYSNNSIAIPTNFTI SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE VFAQVKQIYKTPPIKDFGGF NFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTV LPPLLTDEMIAQYTSALLAG TITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSS TASALGKLQDVVNQNAQALN TLVKQLSSNFGAI SSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECV LGQSKRV DFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTH WFVTQRNFYEPQIITTDNT FVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLIDL

QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCS

SEQ ID NO: 5 is the amino acid sequence of a SARS-CoV-2 spike protein lacking the C- terminal endocytosis motif (nCoV-noEndo).

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFSNVTWFHAIHVSGTNGTKRFD NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFL GVYYHKNNKSWMESEFRVY SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGF SALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRV QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCY GVSPTKLNDLCFTNVYADSF VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSN LKPFERDISTEIYQAGSTPC NGVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTNLVKNKCVNF NFNGLTGTGVLTESNKKFL PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEV PVAIHADQLTPTWRVYSTGS NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLG AENSVAYSNNSIAIPTNFTI SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE VFAQVKQIYKTPPIKDFGGF NFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTV LPPLLTDEMIAQYTSALLAG TITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSS TASALGKLQDVVNQNAQALN TLVKQLSSNFGAI SSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECV LGQSKRV DFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTH WFVTQRNFYEPQIITTDNT FVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDD SEPVLKGV

SEQ ID NO: 6 is a nucleic acid sequence encoding a SARS-CoV-2 spike protein.

ATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACA ACCAGAACTCAATTACCCCCTGC ATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGT TTTACATTCAACTCAGGACT TGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCA ATGGTACTAAGAGGTTTGAT AACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATA ATAAGAGGCTGGATTTTTGG TACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGT TATTAAAGTCTGTGAATTTC AATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGG AAAGTGAGTTCAGAGTTTAT TCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAA GGAAAACAGGGTAATTTCAA AAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCA CACGCCTATTAATTTAGTGC GTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTA ACATCACTAGGTTTCAAACT TTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCT GGTGCTGCAGCTTATTATGT GGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGA TGCTGTAGACTGTGCACTTG ACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATC AAACTTCTAACTTTAGAGTC CAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAA GTTTTTAACGCCACCAGATT TGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGT CCTATATAATTCCGCATCAT TTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTA ATGTCTATGCAGATTCATTT GTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGAT TATAATTATAAATTACCAGA TGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGG TAATTATAATTACCTGTATA GATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATC AGGCCGGTAGCACACCTTGT AATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACT AATGGTGTTGGTTACCAACC ATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACC TAAAAAGTCTACTAATTTGG TTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTG AGTCTAACAAAAAGTTTCTG CCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAG ACACTTGAGATTCTTGACAT TACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCA GGTTGCTGTTCTTTATCAGG ATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGC GTGTTTATTCTACAGGTTCT AATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATAT GAGTGTGACATACCCATTGG TGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGT AGCTAGTCAATCCATCATTG CCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCA TACCCACAAATTTTACTATT AGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATG TACATTTGTGGTGATTCAAC TGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGTGCTTT AACTGGAATAGCTGTTGAAC AAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAA TTAAAGATTTTGGTGGTTTT AATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGAT CTACTTTTCAACAAAGTGAC ACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAG AGACCTCATTTGTGCACAAA

AGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAAT ACACTTCTGCACTGTTAGCGGGT ACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATG CAAATGGCTTATAGGTTTAA TGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATT TAATAGTGCTATTGGCAAAA TTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACC AAAATGCACAAGCTTTAAAC ACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATC CTTTCACGTCTTGACAAAGT TGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATA TGTGACTCAACAATTAATTA GAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTAC TTGGACAATCAAAAAGAGTT GATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTA GTCTTCTTGCATGTGACTTA TGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGC ACACTTTCCTCGTGAAGGTG TCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAA TCATTACTACAGACAACACA TTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCT TTGCAACCTGAATTAGACTC ATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGG TGACATCTCTGGCATTAATG CTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAA ATGAATCTCTCATCGATCTC CAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTT ATAGCTGGCTTGATTGCCAT AGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGCTG TTGTTCTTGTGGATCCTGCT GCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCAAATTACATTACACAT AA

SEQ ID NO: 7 is the amino acid sequence of a stabilized SARS-CoV-2 beta variant spike protein with a double proline substitution.

MFVFLVLLPLVSSQCVNFTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFSNVTWFHAIHVSGTNGTKRFA NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFL GVYYHKNNKSWMESEFRVY SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRGLPQGF SALEPLVDLPIGINITRFQT LHI SYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSF TVEKGIYQTSNFRVQPT ESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIR GDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKP FERDI STEI YQAGSTPCNGV KGFNCYFPLQSYGFQPTYGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVNFNFN GLTGTGVLTESNKKFLPFQ QFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVA IHADQLTPTWRVYSTGSNVF QTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSI IAYTMSLGVENSVAYSNNSIAIPTNFTI SVT TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFA QVKQIYKTPPIKDFGGFNFS QILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPP LLTDEMIAQYTSALLAGTIT SGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTAS ALGKLQDWNQNAQALNTLV KQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN LAATKMSECVLGQSKRVDFC GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWF VTQRNFYEPQI ITTDNTFVS GNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEI DRLNEVAKNLNESLIDLQEL GKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEP VLKGVKLHYT

SEQ ID NO: 8 is the amino acid sequence of a stabilized, double proline-substituted, chimeric SARS-CoV-2 spike protein comprising the RBD of the beta variant and remaining sequence from the Wuhan strain.

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFSNVTWFHAIHVSGTNGTKRFD NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFL GVYYHKNNKSWMESEFRVY SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGF SALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRV QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCY GVSPTKLNDLCFTNVYADSF VIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSN LKPFERDISTEIYQAGSTPC NGVKGFNCYFPLQSYGFQPTYGVGYQPYRWVLSFELLHAPATVCGPKKSTNLVKNKCVNF NFNGLTGTGVLTESNKKFL PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEV PVAIHADQLTPTWRVYSTGS NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLG AENSVAYSNNSIAIPTNFTI SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE VFAQVKQIYKTPPIKDFGGF NFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTV LPPLLTDEMIAQYTSALLAG TITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSS TASALGKLQDVVNQNAQALN TLVKQLSSNFGAI SSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECV LGQSKRV DFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTH WFVTQRNFYEPQIITTDNT FVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDD SEPVLKGVKLHYT

SEQ ID NO: 9 is the amino acid sequence of a stabilized SARS-CoV-2 delta variant spike protein with a double proline substitution.

MFVFLVLLPLVSSQCVNLTTTTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFSNVTWFHAIHVSGTNGTKRFD NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFL DVYYHKNNKSWMKSEFRVY SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPHGF SALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRV QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCY GVSPTKLNDLCFTNVYADSF VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSN LKPFERDISTEIYQAGSTPC NGVQGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTNLVKNKCVNF NFNGLTGTGVLTESNKKFL PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEV PVAIHADQLTPTWRVYSTGS NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSRRRARSVASQSIIAYTMSLG AENSVAYSNNSIAIPTNFTI SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE VFAQVKQIYKTPPIKDFGGF NFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTV LPPLLTDEMIAQYTSALLAG TITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSS TASALGKLQDVVNQNAQALN TLVKQLSSNFGAI SSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECV LGQSKRV DFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTD WFVTQRNFYEPQIITTDNT FVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDD SEPVLKGVKLHYT

SEQ ID NO: 10 is the amino acid sequence of a stabilized SARS-CoV-2 gamma variant spike protein with a double proline substitution. MFVFLVLLPLVSSQCVNFTNRTQLPSAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFD

NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNY PFLGVYYHKNNKSWMESEFRVY

SSANNCTFEYVSQPFLMDLEGKQGNFKNLSEFVFKNIDGYFKIYSKHTPINLVRDLP QGFSALEPLVDLPIGINITRFQT

LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLS ETKCTLKSFTVEKGIYQTSNFRV

QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTF KCYGVSPTKLNDLCFTNVYADSF

VIRGDEVRQIAPGQTGTIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFR KSNLKPFERDISTEIYQAGSTPC

NGVKGFNCYFPLQSYGFQPTYGVGYQPYRWVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFL

PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNC TEVPVAIHADQLTPTWRVYSTGS

NVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTM SLGAENSVAYSNNSIAIPTNFTI

SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKN TQEVFAQVKQIYKTPPIKDFGGF

NFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNG LTVLPPLLTDEMIAQYTSALLAG

TITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDS LSSTASALGKLQDVVNQNAQALN

TLVKQLSSNFGAI SSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAAIKMSECV LGQSKRV

DFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSN GTHWFVTQRNFYEPQIITTDNT

FVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASFVNIQKEIDRLNEVAKNLNESLIDL

QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFD EDDSEPVLKGVKLHYT

SEQ ID NO: 11 is the amino acid sequence of a stabilized SARS-CoV-2 delta plus variant spike protein with a double proline substitution.

MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFSNVTWFHAIHVSGTNGTKRFD NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFL DVYYHKNNKSWMESGVYSS ANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSA LEPLVDLPIGINITRFQTLL ALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCT LKSFTVEKGIYQTSNFRVQP TESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGV SPTKLNDLCFTNVYADSFVI RGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLK PFERDISTEIYQAGSKPCNG VEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTNLVKNKCVNFNF NGLTGTGVLTESNKKFLPF QQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPV AIHADQLTPTWRVYSTGSNV FQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSRRRARSVASQSIIAYTMSLGAE NSVAYSNNSIAIPTNFTISV TTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVF AQVKQIYKTPPIKDFGGFNF SQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLP PLLTDEMIAQYTSALLAGTI TSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTA SALGKLQNVVNQNAQALNTL VKQLSSNFGAI SSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECV LGQSKRVDF CGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWF VTQRNFYEPQIITTDNTFV SGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLIDLQE LGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSE PVLKGVKLHYT

SEQ ID NO: 12 is the amino acid sequence of a stabilized SARS-CoV-2 omicron variant spike protein with a double proline substitution.

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFSNVTWFHVISGTNGTKRFDNP VLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLDH KNNKSWMESEFRVYSSANN CTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPI IVEPERDLPQGFSALEPLVDLPIGINITRFQTLLA LHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL KSFTVEKGIYQTSNFRVQPT ESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRI SNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIR GDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKP FERDI STEI YQAGNKPCNGV AGFNCYFPLRSYSFRPTYGVGHQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVNFNFN GLKGTGVLTESNKKFLPFQ QFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVA IHADQLTPTWRVYSTGSNVF QTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSI IAYTMSLGAENSVAYSNNSIAIPTNFTI SVT TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFA QVKQIYKTPPIKYFGGFNFS QILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFKGLTVLPP LLTDEMIAQYTSALLAGTIT SGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTAS ALGKLQDWNHNAQALNTLV KQLSSKFGAISSVLNDIFSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN LAATKMSECVLGQSKRVDFC GKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWF VTQRNFYEPQI ITTDNTFVS GNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEI DRLNEVAKNLNESLIDLQEL

GKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDD SEPVLKGVKLHYT SEQ ID NO: 13 is a codon-optimized nucleic acid sequence encoding a stabilized SARS- CoV-2 beta variant spike protein with a double proline substitution.

ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGAGCTCCCAGTGCGTGAACTTCACC ACAAGAACCCAGCTGCCCCCTGC CTACACCAATTCCTTCACAAGGGGCGTGTACTATCCCGACAAGGTGTTTCGCTCTAGCGT GCTGCACTCCACACAGGATC TGTTTCTGCCTTTCTTTTCTAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCA ATGGCACAAAGCGGTTCGCC AATCCAGTGCTGCCCTTTAACGACGGCGTGTACTTCGCCTCCACCGAGAAGTCTAACATC ATCAGAGGCTGGATCTTTGG CACCACACTGGATAGCAAGACACAGTCCCTGCTGATCGTGAACAATGCCACCAACGTGGT CATCAAGGTGTGCGAGTTCC AGTTTTGTAATGACCCATTCCTGGGCGTGTACTATCACAAGAACAATAAGTCTTGGATGG AGAGCGAGTTTAGGGTGTAC TCCTCTGCCAACAATTGCACATTTGAGTACGTGAGCCAGCCCTTCCTGATGGACCTGGAG GGCAAGCAGGGCAATTTCAA GAACCTGCGCGAGTTCGTGTTTAAGAATATCGATGGCTACTTCAAGATCTACTCCAAGCA CACCCCAATCAACCTGGTGA GGGGACTGCCACAGGGCTTCTCTGCCCTGGAGCCACTGGTGGACCTGCCCATCGGCATCA ACATCACCCGCTTTCAGACA CTGCACATCAGCTACCTGACACCAGGCGATAGCTCCTCTGGATGGACCGCAGGAGCAGCA GCCTACTATGTGGGCTACCT GCAGCCCAGGACCTTCCTGCTGAAGTATAACGAGAATGGCACCATCACAGACGCAGTGGA TTGCGCCCTGGACCCCCTGT CTGAGACCAAGTGTACACTGAAGAGCTTTACCGTGGAGAAGGGCATCTACCAGACAAGCA ATTTCCGGGTGCAGCCTACC GAGTCCATCGTGAGATTTCCCAATATCACAAACCTGTGCCCTTTTGGCGAGGTGTTCAAC GCCACCCGCTTCGCCAGCGT GTATGCCTGGAATAGGAAGCGCATCTCCAACTGCGTGGCCGACTATTCTGTGCTGTACAA CAGCGCCTCCTTCTCTACCT TTAAGTGCTACGGCGTGAGCCCCACAAAGCTGAATGACCTGTGCTTTACCAACGTGTATG CCGATTCCTTCGTGATCAGG GGCGACGAGGTGCGCCAGATCGCACCAGGCCAGACAGGCAATATCGCCGACTACAACTAT AAGCTGCCTGACGATTTCAC CGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATAGCAAAGTGGGCGGCAACTACAA TTATCTGTACCGGCTGTTTA GAAAGTCTAACCTGAAGCCATTCGAGAGGGACATCTCCACAGAGATCTACCAGGCCGGCT CTACCCCCTGCAATGGCGTG AAGGGCTTTAACTGTTATTTCCCTCTGCAGAGCTACGGCTTCCAGCCAACCTACGGCGTG GGCTATCAGCCCTACCGCGT GGTGGTGCTGTCTTTTGAGCTGCTGCACGCACCTGCAACAGTGTGCGGCCCAAAGAAGAG CACCAATCTGGTGAAGAACA AGTGCGTGAACTTCAACTTCAACGGACTGACCGGCACAGGCGTGCTGACCGAGTCCAACA AGAAGTTCCTGCCTTTTCAG CAGTTCGGCCGGGACATCGCCGATACCACAGACGCCGTGAGAGACCCTCAGACCCTGGAG ATCCTGGATATCACACCATG CTCCTTCGGCGGCGTGTCTGTGATCACACCAGGCACCAATACAAGCAACCAGGTGGCCGT GCTGTACCAGGGCGTGAATT GTACCGAGGTGCCCGTGGCAATCCACGCAGACCAGCTGACCCCTACATGGAGGGTGTATT CTACCGGCAGCAACGTGTTC CAGACACGCGCCGGATGCCTGATCGGAGCAGAGCACGTGAACAATAGCTACGAGTGCGAT ATCCCTATCGGCGCCGGCAT CTGTGCCTCCTATCAGACCCAGACAAACTCCCCACGGAGAGCCCGGTCTGTGGCAAGCCA GTCCATCATCGCCTACACCA TGAGCCTGGGCGTGGAGAACAGCGTGGCCTATTCCAACAATTCTATCGCCATCCCTACCA ACTTCACAATCTCCGTGACC ACAGAGATCCTGCCAGTGAGCATGACCAAGACATCCGTGGACTGCACAATGTACATCTGT GGCGATTCCACCGAGTGCTC TAACCTGCTGCTGCAGTATGGCTCTTTTTGTACCCAGCTGAATAGAGCCCTGACAGGCAT CGCCGTGGAGCAGGACAAGA ACACACAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCACCCATCAAGGACT TTGGCGGCTTCAACTTCAGC CAGATCCTGCCCGATCCTAGCAAGCCATCCAAGCGGTCTTTTATCGAGGACCTGCTGTTC AACAAGGTGACCCTGGCCGA TGCCGGCTTCATCAAGCAGTACGGCGATTGCCTGGGCGACATCGCAGCCAGAGACCTGAT CTGTGCCCAGAAGTTTAATG GCCTGACCGTGCTGCCTCCACTGCTGACAGATGAGATGATCGCCCAGTATACATCTGCCC TGCTGGCAGGAACCATCACA AGCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATCCCCTTTGCCATGCAGATGGCC TACAGGTTCAACGGCATCGG CGTGACCCAGAATGTGCTGTATGAGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGC CATCGGCAAGATCCAGGACT CTCTGAGCTCCACAGCAAGCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAGAACGCCC AGGCCCTGAATACCCTGGTG AAGCAGCTGTCTAGCAACTTCGGCGCCATCTCCTCTGTGCTGAATGATATCCTGAGCCGG CTGGACCCTCCTGAGGCAGA GGTGCAGATCGACCGGCTGATCACAGGCAGACTGCAGTCCCTGCAGACCTACGTGACACA GCAGCTGATCAGGGCAGCAG AGATCAGGGCATCTGCCAATCTGGCCGCCACCAAGATGAGCGAGTGCGTGCTGGGCCAGT CCAAGAGAGTGGACTTTTGT GGCAAGGGCTACCACCTGATGAGCTTCCCACAGTCCGCCCCTCACGGCGTGGTGTTTCTG CACGTGACCTATGTGCCAGC CCAGGAGAAGAACTTCACCACAGCACCAGCCATCTGCCACGATGGCAAGGCACACTTTCC TCGGGAGGGCGTGTTCGTGA GCAACGGCACCCACTGGTTTGTGACACAGAGAAATTTCTACGAGCCACAGATCATCACCA CAGACAATACCTTCGTGAGC GGCAACTGTGACGTGGTCATCGGAATCGTGAACAATACCGTGTACGATCCTCTGCAGCCA GAGCTGGACTCTTTTAAGGA GGAGCTGGATAAGTATTTCAAGAATCACACCAGCCCCGACGTGGATCTGGGCGACATCTC TGGCATCAATGCCAGCGTGG TGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTGGCCAAGAATCTGAACGAGTCCC TGATCGATCTGCAGGAGCTG GGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGC CTGATCGCCATCGTGATGGT GACCATCATGCTGTGCTGTATGACAAGCTGCTGTTCCTGCCTGAAGGGCTGCTGTTCTTG TGGCAGCTGCTGTAAGTTTG ATGAGGACGATAGCGAGCCTGTGCTGAAGGGCGTGAAGCTGCACTATACCTGA

SEQ ID NO: 14 is a codon-optimized nucleic acid sequence encoding a stabilized, double proline-substituted, chimeric SARS-CoV-2 spike protein comprising the RBD of the beta variant and remaining sequence from the Wuhan strain.

ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGAGCTCCCAGTGCGTGAACCTGACC ACAAGGACCCAGCTGCCCCCTGC CTACACCAATTCCTTCACACGGGGCGTGTACTATCCCGACAAGGTGTTTAGATCTAGCGT GCTGCACTCCACACAGGATC TGTTTCTGCCTTTCTTTTCTAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCA ATGGCACAAAGCGGTTCGAC AATCCAGTGCTGCCCTTTAACGATGGCGTGTACTTCGCCTCCACCGAGAAGTCTAACATC ATCAGAGGCTGGATCTTTGG

CACCACACTGGACAGCAAGACACAGTCCCTGCTGATCGTGAACAATGCCACCAACGT GGTCATCAAGGTGTGCGAGTTCC

AGTTTTGTAATGATCCATTCCTGGGCGTGTACTATCACAAGAACAATAAGTCTTGGA TGGAGAGCGAGTTTCGCGTGTAC

TCCTCTGCCAACAATTGCACATTTGAGTACGTGAGCCAGCCCTTCCTGATGGACCTG GAGGGCAAGCAGGGCAATTTCAA

GAACCTGAGGGAGTTCGTGTTTAAGAATATCGATGGCTACTTCAAGATCTACTCCAA GCACACCCCAATCAACCTGGTGC

GCGACCTGCCACAGGGCTTCTCTGCCCTGGAGCCACTGGTGGATCTGCCCATCGGCA TCAACATCACCCGGTTTCAGACA

CTGCTGGCCCTGCACAGAAGCTACCTGACACCAGGCGACAGCTCCTCTGGATGGACC GCAGGAGCAGCAGCCTACTATGT

GGGCTACCTGCAGCCCAGGACCTTCCTGCTGAAGTATAACGAGAATGGCACCATCAC AGACGCAGTGGATTGCGCCCTGG

ACCCCCTGTCTGAGACCAAGTGTACACTGAAGAGCTTTACCGTGGAGAAGGGCATCT ACCAGACAAGCAATTTCAGGGTG

CAGCCTACCGAGTCCATCGTGCGCTTTCCCAATATCACAAACCTGTGCCCTTTTGGC GAGGTGTTCAACGCCACCCGCTT

CGCCAGCGTGTATGCCTGGAATAGGAAGCGCATCTCCAACTGCGTGGCCGACTATTC TGTGCTGTACAACAGCGCCTCCT

TCTCTACCTTTAAGTGCTACGGCGTGAGCCCCACAAAGCTGAATGACCTGTGCTTTA CCAACGTGTATGCCGATTCCTTC

GTGATCAGGGGCGACGAGGTGCGCCAGATCGCACCAGGCCAGACAGGCAATATCGCC GACTACAACTATAAGCTGCCTGA

CGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATAGCAAAGTGGG CGGCAACTACAATTATCTGTACC

GGCTGTTTAGAAAGTCTAACCTGAAGCCATTCGAGAGGGACATCTCCACAGAGATCT ACCAGGCCGGCTCTACCCCCTGC

AATGGCGTGAAGGGCTTTAACTGTTATTTCCCTCTGCAGAGCTACGGCTTCCAGCCA ACCTACGGCGTGGGCTATCAGCC

CTACCGCGTGGTGGTGCTGTCTTTTGAGCTGCTGCACGCACCTGCAACAGTGTGCGG CCCAAAGAAGAGCACCAATCTGG

TGAAGAACAAGTGCGTGAACTTCAACTTCAACGGACTGACCGGCACAGGCGTGCTGA CCGAGTCCAACAAGAAGTTCCTG

CCTTTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTGCGCGACCCT CAGACCCTGGAGATCCTGGATAT

CACACCATGCTCCTTCGGCGGCGTGTCTGTGATCACACCAGGCACCAATACAAGCAA CCAGGTGGCCGTGCTGTACCAGG

GCGTGAATTGTACCGAGGTGCCCGTGGCAATCCACGCAGACCAGCTGACCCCTACAT GGCGGGTGTATTCTACCGGCAGC

AACGTGTTCCAGACAAGAGCCGGATGCCTGATCGGAGCAGAGCACGTGAACAATAGC TACGAGTGCGATATCCCTATCGG

CGCCGGCATCTGTGCCTCCTATCAGACCCAGACAAACTCCCCACGGAGAGCCCGGTC TGTGGCAAGCCAGTCCATCATCG

CCTACACCATGAGCCTGGGCGCCGAGAACAGCGTGGCCTATTCCAACAATTCTATCG CCATCCCTACCAACTTCACAATC

TCCGTGACCACAGAGATCCTGCCAGTGAGCATGACCAAGACATCCGTGGACTGCACA ATGTACATCTGTGGCGATTCCAC

CGAGTGCTCTAACCTGCTGCTGCAGTATGGCTCTTTTTGTACCCAGCTGAATAGAGC CCTGACAGGCATCGCCGTGGAGC

AGGACAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCAC CCATCAAGGACTTTGGCGGCTTC

AACTTCAGCCAGATCCTGCCCGATCCTAGCAAGCCATCCAAGCGGTCTTTTATCGAG GACCTGCTGTTCAACAAGGTGAC

CCTGGCCGATGCCGGCTTCATCAAGCAGTACGGCGATTGCCTGGGCGACATCGCAGC CAGAGACCTGATCTGTGCCCAGA

AGTTTAATGGCCTGACCGTGCTGCCTCCACTGCTGACAGATGAGATGATCGCCCAGT ATACATCTGCCCTGCTGGCAGGA

ACCATCACAAGCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATCCCCTTTGCC ATGCAGATGGCCTACAGATTCAA

CGGCATCGGCGTGACCCAGAATGTGCTGTATGAGAACCAGAAGCTGATCGCCAATCA GTTTAACTCCGCCATCGGCAAGA

TCCAGGACTCTCTGAGCTCCACAGCAAGCGCCCTGGGCAAGCTGCAGGATGTGGTGA ATCAGAACGCCCAGGCCCTGAAT

ACCCTGGTGAAGCAGCTGTCTAGCAACTTCGGCGCCATCTCCTCTGTGCTGAATGAT ATCCTGAGCCGGCTGGACCCACC

AGAGGCAGAGGTGCAGATCGACCGGCTGATCACAGGCAGACTGCAGTCCCTGCAGAC CTACGTGACACAGCAGCTGATCA

GGGCAGCAGAGATCAGGGCATCTGCCAATCTGGCCGCCACCAAGATGAGCGAGTGCG TGCTGGGCCAGTCCAAGAGAGTG

GACTTTTGTGGCAAGGGCTACCACCTGATGAGCTTCCCACAGTCCGCCCCTCACGGC GTGGTGTTTCTGCACGTGACCTA

TGTGCCAGCCCAGGAGAAGAACTTCACCACAGCACCAGCCATCTGCCACGATGGCAA GGCACACTTTCCCCGGGAGGGCG

TGTTCGTGAGCAACGGAACCCACTGGTTTGTGACACAGCGCAATTTCTACGAGCCAC AGATCATCACCACAGACAATACA

TTCGTGTCCGGCAACTGTGACGTGGTCATCGGAATCGTGAACAATACCGTGTACGAT CCTCTGCAGCCAGAGCTGGACTC

TTTTAAGGAGGAGCTGGATAAGTATTTCAAGAATCACACCAGCCCCGACGTGGATCT GGGCGACATCTCTGGCATCAATG

CCAGCGTGGTGAACATCCAGAAGGAGATCGACAGGCTGAACGAGGTGGCCAAGAATC TGAACGAGTCCCTGATCGATCTG

CAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGC TTCATCGCCGGCCTGATCGCCAT

CGTGATGGTGACCATCATGCTGTGCTGTATGACAAGCTGCTGTTCCTGCCTGAAGGG CTGCTGTTCTTGTGGCAGCTGCT

GTAAGTTTGATGAGGACGATAGCGAGCCTGTGCTGAAGGGCGTGAAGCTGCACTATA CCTGA

SEQ ID NO: 15 is a codon-optimized nucleic acid sequence encoding a stabilized SARS-

CoV-2 delta variant spike protein with a double proline substitution.

ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGAGCTCCCAGTGCGTGAACCTGACC ACAACCACACAGCTGCCCCCTGC CTATACCAATTCCTTCACACGCGGCGTGTACTATCCTGACAAGGTGTTTCGGTCTAGCGT GCTGCACTCCACACAGGATC TGTTTCTGCCATTCTTTTCTAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCA ATGGCACAAAGCGGTTCGAC AATCCAGTGCTGCCCTTTAACGATGGCGTGTACTTCGCCTCCACCGAGAAGTCTAACATC ATCCGGGGCTGGATCTTTGG CACCACACTGGACAGCAAGACACAGTCCCTGCTGATCGTGAACAATGCCACCAACGTGGT CATCAAGGTGTGCGAGTTCC AGTTTTGTAATGATCCCTTCCTGGACGTGTACTATCACAAGAACAATAAGTCTTGGATGA AGAGCGAGTTTAGAGTGTAT TCCTCTGCCAACAATTGCACATTTGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAG GGCAAGCAGGGCAATTTCAA GAACCTGAGAGAGTTCGTGTTTAAGAATATCGATGGCTACTTCAAGATCTACTCCAAGCA CACCCCAATCAACCTGGTGA GGGACCTGCCACACGGCTTCTCTGCCCTGGAGCCACTGGTGGATCTGCCCATCGGCATCA ACATCACCAGATTTCAGACA CTGCTGGCCCTGCACAGGAGCTACCTGACACCCGGCGACAGCTCCTCTGGATGGACCGCC GGCGCTGCCGCCTACTATGT GGGCTATCTGCAGCCTCGCACCTTCCTGCTGAAGTACAACGAGAATGGCACCATCACAGA CGCAGTGGATTGCGCCCTGG ACCCCCTGTCTGAGACCAAGTGTACACTGAAGAGCTTTACCGTGGAGAAGGGCATCTATC AGACAAGCAATTTCCGCGTG CAGCCAACCGAGTCCATCGTGCGGTTTCCCAATATCACAAACCTGTGCCCTTTTGGCGAG GTGTTCAACGCAACCAGGTT

CGCAAGCGTGTACGCATGGAATCGCAAGCGGATCTCCAACTGCGTGGCCGACTATTC TGTGCTGTACAACAGCGCCTCCT

TCTCTACCTTTAAGTGCTATGGCGTGAGCCCAACAAAGCTGAATGACCTGTGCTTTA CCAACGTGTACGCCGATTCCTTC

GTGATCCGGGGCGACGAGGTGCGGCAGATCGCACCAGGACAGACAGGCAAGATCGCA GACTACAATTATAAGCTGCCTGA

CGATTTCACCGGCTGCGTGATCGCCTGGAACTCTAACAATCTGGATAGCAAAGTGGG CGGCAACTACAATTATAGATACA

GGCTGTTTAGAAAGTCTAATCTGAAGCCATTCGAGAGGGACATCTCCACAGAGATCT ACCAGGCCGGCTCTACCCCCTGC

AATGGCGTGCAGGGCTTTAACTGTTATTTCCCTCTGCAGAGCTACGGCTTCCAGCCA ACCAACGGCGTGGGCTATCAGCC

CTACCGGGTGGTGGTGCTGTCTTTTGAGCTGCTGCACGCACCTGCAACAGTGTGCGG ACCAAAGAAGAGCACCAATCTGG

TGAAGAACAAGTGCGTGAACTTCAACTTCAACGGACTGACCGGAACAGGCGTGCTGA CCGAGTCCAACAAGAAGTTCCTG

CCATTTCAGCAGTTCGGCAGAGACATCGCCGATACCACAGACGCCGTGAGGGACCCT CAGACCCTGGAGATCCTGGATAT

CACACCATGCTCCTTCGGCGGCGTGTCTGTGATCACACCCGGCACCAATACAAGCAA CCAGGTGGCCGTGCTGTATCAGG

GCGTGAATTGTACCGAGGTGCCAGTGGCAATCCACGCAGACCAGCTGACCCCTACAT GGCGCGTGTACTCTACCGGCAGC

AACGTGTTCCAGACAAGGGCAGGATGCCTGATCGGAGCAGAGCACGTGAACAATAGC TATGAGTGCGATATCCCCATCGG

CGCCGGCATCTGTGCCTCCTACCAGACCCAGACAAACTCCCGGAGAAGGGCCAGATC TGTGGCCAGCCAGTCCATCATCG

CCTATACCATGAGCCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAATTCTATCG CCATCCCTACCAACTTCACAATC

TCCGTGACCACAGAGATCCTGCCAGTGAGCATGACCAAGACATCCGTGGACTGCACA ATGTATATCTGTGGCGATTCCAC

CGAGTGCTCTAACCTGCTGCTGCAGTACGGCTCTTTTTGTACCCAGCTGAATAGGGC CCTGACAGGAATCGCAGTGGAGC

AGGACAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCAC CCATCAAGGACTTTGGCGGCTTC

AACTTCAGCCAGATCCTGCCCGATCCTAGCAAGCCCTCCAAGCGGAGCTTCATCGAG GACCTGCTGTTCAACAAGGTGAC

CCTGGCCGATGCCGGCTTCATCAAGCAGTATGGCGATTGCCTGGGCGACATCGCAGC AAGGGACCTGATCTGTGCCCAGA

AGTTTAATGGCCTGACCGTGCTGCCTCCACTGCTGACAGATGAGATGATCGCCCAGT ACACATCTGCCCTGCTGGCAGGA

ACCATCACAAGCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATCCCTTTTGCC ATGCAGATGGCCTATCGCTTCAA

CGGCATCGGCGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAATCA GTTTAACTCCGCCATCGGCAAGA

TCCAGGACTCTCTGAGCTCCACAGCAAGCGCCCTGGGCAAGCTGCAGGATGTGGTGA ATCAGAACGCCCAGGCCCTGAAT

ACCCTGGTGAAGCAGCTGTCTAGCAACTTCGGCGCCATCTCCTCTGTGCTGAATGAT ATCCTGAGCAGACTGGACCCCCC

CGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGGCTGCAGTCCCTGCAGAC CTACGTGACACAGCAGCTGATCA

GGGCCGCCGAGATCAGGGCCTCTGCCAATCTGGCCGCCACCAAGATGAGCGAGTGCG TGCTGGGCCAGTCCAAGAGGGTG

GATTTTTGTGGCAAGGGCTATCACCTGATGAGCTTCCCACAGTCCGCCCCTCACGGA GTGGTGTTTCTGCACGTGACCTA

CGTGCCAGCCCAGGAGAAGAACTTCACCACAGCACCAGCAATCTGCCACGACGGCAA GGCACACTTTCCAAGAGAGGGCG

TGTTCGTGAGCAACGGCACCGATTGGTTTGTGACACAGAGGAATTTCTACGAGCCCC AGATCATCACCACAGACAATACA

TTCGTGTCCGGCAACTGTGACGTGGTCATCGGCATCGTGAACAATACCGTGTATGAT CCTCTGCAGCCAGAGCTGGACTC

TTTTAAGGAGGAGCTGGATAAGTACTTCAAGAATCACACCAGCCCCGACGTGGATCT GGGCGACATCTCTGGCATCAATG

CCAGCGTGGTGAACATCCAGAAGGAGATCGACCGGCTGAACGAGGTGGCCAAGAATC TGAACGAGTCCCTGATCGATCTG

CAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCTTGGTATATCTGGCTGGGC TTCATCGCCGGCCTGATCGCCAT

CGTGATGGTGACCATCATGCTGTGCTGTATGACAAGCTGCTGTTCCTGCCTGAAGGG CTGCTGTTCTTGTGGCAGCTGCT

GTAAGTTTGATGAGGACGATAGCGAGCCAGTGCTGAAGGGCGTGAAGCTGCACTACA CCTGA

SEQ ID NO: 16 is a codon-optimized nucleic acid sequence encoding a stabilized SARS-

CoV-2 gamma variant spike protein with a double proline substitution.

ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGAGCTCCCAGTGCGTGAATTTCACC AACAGAACACAGCTGCCTTCTGC CTACACCAATAGCTTCACACGGGGCGTGTACTATCCAGACAAGGTGTTTAGATCTAGCGT GCTGCACAGCACACAGGATC TGTTTCTGCCATTCTTTTCCAACGTGACCTGGTTCCACGCCATCCACGTGTCCGGCACCA ATGGCACAAAGCGGTTCGAC AATCCCGTGCTGCCTTTTAACGATGGCGTGTACTTCGCCTCCACCGAGAAGTCTAACATC ATCAGAGGCTGGATCTTTGG CACCACACTGGACAGCAAGACACAGTCCCTGCTGATCGTGAACAATGCCACCAACGTGGT CATCAAGGTGTGCGAGTTCC AGTTTTGTAATTATCCCTTCCTGGGCGTGTACTATCACAAGAACAATAAGTCTTGGATGG AGAGCGAGTTTAGGGTGTAC TCCTCTGCCAACAATTGCACATTTGAGTATGTGAGCCAGCCTTTCCTGATGGACCTGGAG GGCAAGCAGGGCAATTTCAA GAACCTGAGCGAGTTCGTGTTTAAGAATATCGATGGCTACTTCAAGATCTACTCCAAGCA CACCCCCATCAACCTGGTGC GCGACCTGCCTCAGGGCTTCTCTGCCCTGGAGCCCCTGGTGGATCTGCCTATCGGCATCA ACATCACCCGGTTTCAGACA CTGCTGGCCCTGCACAGAAGCTACCTGACACCCGGCGACAGCTCCTCTGGATGGACCGCC GGCGCTGCCGCCTACTATGT GGGCTACCTGCAGCCTAGGACCTTCCTGCTGAAGTATAACGAGAATGGCACCATCACAGA CGCAGTGGATTGCGCCCTGG ACCCCCTGTCCGAGACCAAGTGTACACTGAAGTCTTTTACCGTGGAGAAGGGCATCTACC AGACATCTAATTTCAGGGTG CAGCCAACCGAGAGCATCGTGCGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAG GTGTTCAACGCCACCCGCTT CGCCAGCGTGTATGCCTGGAATAGGAAGCGCATCAGCAACTGCGTGGCCGACTATTCCGT GCTGTACAACAGCGCCTCCT TCTCTACCTTTAAGTGTTACGGCGTGTCTCCTACAAAGCTGAATGACCTGTGCTTTACCA ACGTGTATGCCGATAGCTTC GTGATCAGGGGCGACGAGGTGCGCCAGATCGCACCAGGACAGACCGGAACAATCGCAGAC TACAATTATAAGCTGCCTGA CGATTTCACCGGCTGCGTGATCGCCTGGAACTCCAACAATCTGGATTCTAAAGTGGGCGG CAACTACAATTATCTGTACC GGCTGTTTAGAAAGTCCAACCTGAAGCCATTCGAGCGGGACATCAGCACAGAGATCTACC AGGCAGGCTCCACCCCATGC AATGGAGTGAAGGGCTTTAACTGTTATTTCCCACTGCAGAGCTACGGCTTCCAGCCCACA TATGGCGTGGGCTATCAGCC TTACAGAGTGGTGGTGCTGTCCTTTGAGCTGCTGCACGCACCAGCAACAGTGTGCGGACC CAAGAAGTCTACCAATCTGG TGAAGAACAAGTGCGTGAACTTCAACTTCAACGGACTGACCGGAACAGGCGTGCTGACCG AGTCCAACAAGAAGTTCCTG CCATTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTGCGCGACCCACAG ACCCTGGAGATCCTGGATAT

CACACCCTGCAGCTTCGGCGGCGTGTCCGTGATCACACCAGGAACCAATACAAGCAA CCAGGTGGCCGTGCTGTACCAGG

GCGTGAATTGTACCGAGGTGCCTGTGGCAATCCACGCAGACCAGCTGACCCCAACAT GGCGGGTGTATTCTACCGGCAGC

AACGTGTTCCAGACAAGAGCCGGCTGCCTGATCGGCGCCGAGTATGTGAACAATTCT TACGAGTGCGATATCCCTATCGG

CGCCGGCATCTGTGCCAGCTACCAGACCCAGACAAACAGCCCACGGAGAGCACGGTC CGTGGCAAGCCAGTCCATCATCG

CCTACACCATGTCTCTGGGCGCCGAGAATAGCGTGGCCTATTCCAACAATTCTATCG CCATCCCAACCAACTTCACAATC

TCCGTGACCACAGAGATCCTGCCCGTGTCTATGACCAAGACAAGCGTGGACTGCACA ATGTACATCTGTGGCGATTCCAC

CGAGTGCTCTAACCTGCTGCTGCAGTATGGCAGCTTTTGTACCCAGCTGAATAGAGC CCTGACAGGCATCGCCGTGGAGC

AGGACAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCCC CTATCAAGGACTTTGGCGGCTTC

AACTTCAGCCAGATCCTGCCTGATCCAAGCAAGCCATCCAAGAGGTCTTTTATCGAG GACCTGCTGTTCAACAAGGTGAC

CCTGGCCGATGCCGGCTTCATCAAGCAGTACGGCGATTGCCTGGGCGACATCGCAGC AAGGGACCTGATCTGTGCCCAGA

AGTTTAATGGCCTGACCGTGCTGCCACCCCTGCTGACAGATGAGATGATCGCCCAGT ATACATCCGCCCTGCTGGCCGGC

ACCATCACATCTGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATCCCCTTTGCC ATGCAGATGGCCTACAGGTTCAA

CGGCATCGGCGTGACCCAGAATGTGCTGTATGAGAACCAGAAGCTGATCGCCAATCA GTTTAACTCCGCCATCGGCAAGA

TCCAGGACTCCCTGAGCTCCACAGCCTCTGCCCTGGGCAAGCTGCAGGATGTGGTGA ATCAGAACGCCCAGGCCCTGAAT

ACCCTGGTGAAGCAGCTGTCTAGCAACTTCGGCGCCATCTCCTCTGTGCTGAATGAT ATCCTGAGCCGGCTGGACCCCCC

CGAGGCAGAGGTGCAGATCGACCGGCTGATCACCGGCAGACTGCAGAGCCTGCAGAC CTACGTGACACAGCAGCTGATCA

GGGCCGCCGAGATCAGGGCATCCGCCAATCTGGCCGCCATCAAGATGTCTGAGTGCG TGCTGGGCCAGAGCAAGAGAGTG

GACTTTTGTGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCCGCCCCACACGGA GTGGTGTTTCTGCACGTGACCTA

TGTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCCACGATGGCAA GGCCCACTTTCCAAGGGAGGGCG

TGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCTACGAGCCCC AGATCATCACCACAGACAATACC

TTCGTGAGCGGCAACTGTGACGTGGTCATCGGCATCGTGAACAATACCGTGTACGAT CCACTGCAGCCCGAGCTGGACTC

CTTTAAGGAGGAGCTGGATAAGTATTTCAAGAATCACACCTCTCCCGACGTGGATCT GGGCGACATCTCCGGCATCAATG

CCTCTTTCGTGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTGGCCAAGAATC TGAACGAGTCCCTGATCGATCTG

CAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGC TTCATCGCCGGCCTGATCGCCAT

CGTGATGGTGACCATCATGCTGTGCTGTATGACAAGCTGCTGTTCCTGCCTGAAGGG CTGCTGTTCTTGTGGCAGCTGCT

GTAAGTTTGATGAGGACGATAGCGAGCCTGTGCTGAAGGGCGTGAAGCTGCACTATA CCTGA

SEQ ID NO: 17 is a codon-optimized nucleic acid sequence encoding a stabilized SARS-

CoV-2 delta plus variant spike protein with a double proline substitution.

ATGTTTGTGTTTCTGGTGCTGCTGCCACTGGTGAGTAGCCAGTGTGTGAACCTGAGA ACCCGAACACAGCTGCCTCCTGC CTATACCAACAGCTTCACCAGAGGCGTGTACTACCCTGACAAGGTGTTCCGATCTAGCGT GCTCCATAGCACCCAGGACC TGTTCTTGCCTTTTTTCTCTAACGTGACATGGTTCCACGCCATTCACGTGTCTGGCACCA ACGGAACAAAAAGATTCGAC AACCCTGTGCTGCCCTTCAACGACGGTGTCTATTTTGCCAGCACCGAGAAGAGCAACATC ATCAGAGGCTGGATCTTCGG AACCACCCTGGACAGCAAGACCCAGAGCCTGCTGATCGTCAATAACGCAACAAATGTGGT GATCAAGGTGTGCGAGTTCC AATTTTGCAACGATCCTTTCCTGGATGTGTACTACCACAAGAACAACAAAAGCTGGATGG AAAGTGGAGTTTATAGCAGC GCCAACAACTGCACCTTCGAGTACGTGAGCCAACCTTTCCTGATGGACCTCGAAGGGAAA CAGGGCAACTTCAAGAACCT TAGAGAGTTCGTCTTTAAGAACATCGACGGCTACTTTAAAATCTACTCCAAGCACACCCC CATCAACCTGGTGCGGGACC TGCCTCAGGGCTTTAGCGCGCTGGAACCCTTGGTTGACCTGCCCATCGGCATCAACATCA CTAGATTCCAGACCCTTCTG GCCCTCCACCGGTCTTACCTGACACCTGGCGACAGTAGTTCTGGCTGGACAGCCGGCGCC GCTGCCTACTACGTGGGCTA TCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCTGT GGATTGCGCCCTGGACCCTC TGTCCGAAACCAAGTGCACACTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCT CTAACTTCCGGGTGCAGCCT ACTGAAAGCATCGTGCGGTTCCCAAACATTACAAACCTGTGCCCTTTCGGAGAAGTTTTC AACGCCACTCGCTTCGCCTC TGTCTATGCCTGGAACAGAAAGCGGATCAGCAATTGTGTGGCCGATTACAGCGTGCTGTA CAACAGCGCCAGCTTTTCTA CATTCAAGTGCTACGGCGTGTCTCCCACCAAGCTGAATGATCTGTGCTTCACCAACGTGT ACGCCGACTCGTTTGTGATC CGGGGAGACGAAGTGCGCCAGATCGCCCCTGGGCAGACAGGAAACATCGCCGATTACAAT TACAAACTGCCTGACGATTT TACAGGATGTGTGATAGCTTGGAACTCCAACAACCTCGACAGCAAAGTGGGCGGCAACTA CAATTACCGGTACAGACTGT TTAGAAAGAGCAACCTAAAACCCTTCGAGAGAGATATCTCTACCGAGATCTACCAGGCCG GCAGCAAGCCTTGTAATGGC GTTGAGGGCTTCAACTGTTACTTCCCTCTGCAGAGCTACGGCTTCCAGCCCACCAACGGC GTCGGGTACCAGCCTTACAG AGTTGTGGTTCTGAGCTTCGAGCTGCTCCACGCTCCTGCCACCGTGTGTGGTCCTAAGAA AAGCACCAACCTGGTGAAGA ACAAGTGCGTGAATTTCAATTTCAACGGCCTGACAGGCACAGGCGTGCTGACCGAGAGCA ACAAAAAGTTCCTGCCCTTC CAGCAGTTCGGCAGAGATATTGCCGATACCACAGACGCCGTGCGGGACCCTCAAACCCTG GAAATCTTGGACATCACACC TTGCAGCTTCGGCGGAGTGTCTGTGATCACTCCCGGGACCAACACCAGCAACCAGGTTGC CGTGCTGTACCAGGGCGTCA ACTGCACCGAAGTGCCAGTGGCTATACACGCCGACCAGCTGACCCCTACATGGCGGGTGT ACAGCACCGGCAGCAACGTG TTCCAGACCAGAGCCGGCTGCCTGATCGGCGCAGAGCACGTGAACAACTCTTATGAATGC GACATCCCCATCGGAGCCGG CATTTGCGCCAGCTACCAGACACAGACCAATAGCAGAAGACGGGCTAGAAGCGTGGCCTC GCAGAGCATAATCGCATACA CAATGAGCCTGGGAGCCGAGAACAGCGTGGCCTACAGCAACAATAGTATCGCCATCCCCA CAAATTTTACCATCAGCGTG ACAACCGAAATCCTGCCAGTGAGCATGACAAAGACCAGCGTCGACTGCACAATGTACATA TGTGGCGATAGCACGGAGTG CAGCAATCTGCTGCTCCAATACGGCAGCTTCTGCACCCAGCTGAATCGGGCACTGACCGG CATCGCCGTGGAACAGGATA AAAATACCCAGGAGGTGTTTGCCCAGGTGAAGCAGATATATAAGACCCCTCCGATCAAGG ACTTCGGAGGCTTCAATTTC AGCCAGATCCTGCCCGATCCAAGCAAGCCTAGCAAGCGGTCCTTCATCGAGGATCTGCTG TTCAATAAGGTGACCCTGGC

CGACGCCGGATTCATCAAACAGTACGGCGACTGCCTGGGCGACATCGCCGCCAGAGA TCTGATCTGTGCTCAAAAGTTCA

ACGGACTGACAGTCCTGCCACCTCTGTTGACAGATGAAATGATCGCTCAGTACACCT CCGCCCTCCTGGCCGGGACGATC

ACCTCTGGATGGACCTTCGGCGCCGGCGCTGCACTGCAGATCCCTTTCGCCATGCAG ATGGCCTACAGATTCAACGGCAT

CGGAGTGACCCAAAACGTCCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAA CTCTGCTATCGGCAAGATCCAGG

ACAGCCTCAGCAGCACCGCCAGCGCCCTGGGCAAACTCCAGAACGTGGTGAACCAGA ACGCACAGGCCCTGAATACCCTG

GTGAAGCAGCTGAGCAGCAACTTCGGCGCTATCAGCTCTGTGCTGAACGACATCCTG AGCAGACTGGACCCTCCCGAGGC

CGAGGTGCAGATTGACAGGCTGATCACAGGCAGACTGCAGTCGCTGCAAACTTACGT GACCCAGCAACTGATCCGGGCCG

CCGAAATCAGGGCCAGCGCCAACCTGGCTGCTACAAAGATGTCCGAATGCGTGTTGG GCCAGTCCAAGAGAGTGGACTTC

TGCGGCAAGGGATACCACCTGATGAGCTTCCCTCAGTCCGCTCCCCACGGCGTCGTG TTCCTGCATGTGACATACGTGCC

CGCCCAGGAGAAGAATTTCACCACCGCCCCTGCCATCTGCCACGACGGCAAGGCCCA CTTCCCCAGAGAGGGCGTGTTCG

TGTCCAACGGCACCCACTGGTTCGTGACCCAGCGGAACTTCTACGAGCCTCAGATCA TCACCACCGATAACACATTCGTG

TCCGGCAACTGCGACGTGGTTATCGGCATCGTGAACAATACCGTGTACGACCCTCTG CAGCCAGAACTGGATTCTTTTAA

GGAAGAGCTGGACAAATACTTTAAGAACCACACATCTCCTGATGTGGACCTGGGCGA CATCAGCGGCATCAACGCCTCCG

TGGTCAACATCCAAAAGGAGATCGATAGACTGAACGAGGTGGCCAAGAACCTCAACG AGTCTCTGATTGACCTGCAGGAG

CTGGGCAAGTACGAGCAGTACATCAAGTGGCCTTGGTACATCTGGCTGGGCTTCATC GCCGGCCTGATCGCTATCGTCAT

GGTGACCATCATGCTGTGCTGTATGACCTCCTGCTGCAGCTGTCTGAAAGGCTGCTG TTCTTGCGGCAGCTGTTGCAAGT

TTGACGAGGACGACTCCGAGCCCGTGCTGAAGGGGGTGAAGCTGCACTACACGTGA

SEQ ID NO: 18 is a codon-optimized nucleic acid sequence encoding a stabilized SARS- CoV-2 omicron variant spike protein with a double proline substitution.

ATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGTCTAGCCAATGTGTGAACCTGACA ACAAGGACCCAGCTTCCCCCAGC TTACACCAATTCATTTACAAGAGGCGTGTATTACCCCGATAAGGTGTTCCGAAGCAGCGT GCTGCACAGCACCCAGGATC TCTTCCTGCCTTTTTTCAGCAATGTGACTTGGTTCCACGTGATCAGCGGAACCAACGGCA CCAAGCGGTTTGACAATCCT GTGCTGCCCTTCAACGACGGCGTGTACTTCGCCAGCATCGAGAAGAGCAACATTATCCGG GGCTGGATCTTCGGCACCAC CCTCGATAGCAAGACCCAGAGCTTACTGATCGTAAACAACGCCACCAATGTCGTAATCAA GGTCTGTGAATTTCAGTTCT GCAACGACCCCTTTCTGGACCACAAGAACAACAAGTCGTGGATGGAAAGCGAGTTCAGAG TGTACAGCTCCGCTAACAAT TGTACATTCGAGTACGTGTCTCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGAAAC TTCAAGAATCTGAGGGAGTT CGTGTTCAAAAACATCGACGGCTACTTCAAGATCTACAGCAAGCATACCCCCATCATCGT TGAACCTGAGAGAGACCTGC CACAGGGTTTCAGCGCTCTGGAGCCTCTGGTTGACCTGCCCATCGGCATCAACATCACCC GGTTTCAGACACTGTTAGCC CTGCATAGATCTTACCTGACCCCAGGCGATTCTTCCTCTGGCTGGACCGCCGGAGCCGCA GCCTACTACGTGGGATATCT GCAGCCCAGAACCTTCCTGCTGAAATACAACGAGAACGGAACCATCACCGATGCCGTGGA CTGCGCCCTGGACCCTCTGT CTGAAACCAAGTGCACCCTGAAGAGCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCA ACTTTCGGGTGCAGCCCACC GAGAGCATCGTGAGATTTCCAAACATCACCAACCTGTGTCCTTTCGACGAGGTGTTTAAT GCCACAAGATTCGCCAGCGT GTACGCCTGGAATAGAAAAAGAATCTCCAACTGCGTGGCTGATTACTCAGTGCTTTACAA CCTGGCCCCATTCTTCACCT TCAAGTGCTACGGCGTTAGCCCTACCAAGCTCAATGATCTGTGCTTCACGAACGTGTACG CCGACAGCTTCGTGATCCGG GGCGACGAAGTCAGACAGATCGCCCCTGGACAGACCGGTAATATCGCCGACTACAATTAC AAGCTGCCTGATGATTTCAC AGGTTGCGTGATCGCCTGGAACTCCAACAAGCTGGACAGCAAGGTGTCCGGCAACTACAA CTACCTGTATAGACTTTTCA GAAAGTCCAACCTGAAGCCATTCGAGCGGGACATCAGCACTGAGATCTACCAGGCCGGCA ACAAACCCTGCAACGGAGTT GCCGGATTCAACTGCTATTTCCCTCTGAGATCTTACTCCTTCAGACCTACATACGGCGTG GGACACCAGCCTTACAGAGT AGTGGTGCTCAGCTTCGAGCTTCTGCACGCTCCTGCCACCGTGTGCGGCCCTAAGAAGAG CACGAACCTGGTGAAGAACA AATGTGTTAATTTTAACTTCAACGGCCTGAAGGGCACAGGAGTCCTGACCGAGAGCAATA AAAAATTCTTGCCCTTCCAG CAGTTCGGAAGAGACATCGCCGACACCACAGATGCTGTGAGAGACCCTCAGACCCTGGAA ATCCTCGACATCACCCCTTG CAGCTTCGGCGGCGTCAGCGTGATCACCCCGGGCACCAACACCTCTAACCAGGTGGCCGT GCTGTACCAGGGCGTGAATT GCACCGAGGTTCCTGTGGCCATCCACGCGGACCAGCTGACACCAACATGGCGGGTGTACA GCACCGGCTCCAACGTGTTT CAGACCAGAGCCGGCTGTCTGATCGGCGCCGAATATGTGAACAACAGCTACGAATGCGAC ATCCCAATCGGCGCCGGCAT TTGCGCCAGCTACCAGACACAGACCAAAAGTCACCGGAGAGCTCGGAGCGTGGCCTCTCA GAGCATTATCGCCTATACCA TGAGCCTGGGGGCCGAGAACAGCGTGGCCTATTCCAACAACAGCATCGCCATCCCTACCA ATTTCACCATCTCTGTGACC ACCGAGATCCTGCCAGTGTCCATGACAAAGACAAGCGTGGACTGCACCATGTACATCTGC GGCGACTCTACCGAGTGCAG CAACCTGCTGCTGCAGTACGGCAGCTTTTGCACACAGCTGAAACGGGCGCTGACAGGAAT TGCCGTTGAGCAGGACAAGA ACACTCAGGAGGTGTTTGCCCAAGTGAAGCAGATATATAAGACCCCTCCTATCAAATACT TCGGCGGCTTTAACTTCAGC CAGATCCTCCCTGATCCTTCTAAGCCTAGCAAGCGCAGCTTCATCGAGGACCTGCTGTTC AACAAGGTAACCCTGGCTGA CGCCGGCTTCATCAAGCAGTACGGTGATTGCCTGGGCGACATCGCAGCCCGGGACCTGAT CTGTGCCCAAAAATTCAAGG GCCTGACTGTTCTGCCTCCTCTGCTGACAGATGAAATGATCGCCCAGTACACCTCCGCCC TGCTGGCTGGCACAATCACC AGCGGCTGGACATTCGGCGCCGGCGCCGCGCTGCAGATCCCTTTCGCCATGCAGATGGCC TACAGATTCAACGGCATCGG AGTGACTCAGAACGTGCTGTACGAAAACCAGAAACTGATTGCAAATCAGTTTAACAGCGC AATCGGCAAGATCCAGGATA GCCTGTCCAGCACCGCCTCCGCTCTGGGCAAGCTGCAAGACGTGGTGAACCACAATGCCC AGGCTCTGAACACCTTGGTG AAGCAGCTGAGCAGCAAGTTCGGCGCCATTTCTTCCGTGCTGAACGACATCTTCAGCAGA CTCGATCCTCCCGAGGCCGA GGTGCAGATCGACAGACTGATCACGGGCAGACTGCAGTCTCTGCAGACATACGTGACACA GCAACTGATCAGAGCCGCTG AAATCAGGGCCTCTGCCAACCTGGCCGCCACCAAGATGTCTGAGTGCGTGCTCGGCCAGT CTAAAAGAGTGGACTTCTGC GGCAAAGGCTACCACCTGATGAGCTTCCCCCAGAGCGCCCCCCACGGCGTGGTGTTCCTA CACGTTACCTACGTGCCGGC

TCAAGAAAAGAACTTTACCACCGCCCCTGCCATCTGCCACGACGGAAAGGCCCACTT CCCTCGGGAGGGTGTGTTTGTCA

GCAACGGCACACACTGGTTCGTGACACAGCGGAACTTCTACGAGCCCCAAATCATCA CAACAGATAACACCTTCGTCAGC

GGCAACTGTGACGTGGTGATCGGCATCGTGAACAACACCGTGTATGACCCTCTGCAG CCTGAGCTGGACAGCTTTAAGGA

AGAGCTGGACAAGTACTTCAAGAATCACACAAGTCCTGACGTGGATCTGGGCGATAT CAGTGGCATCAACGCCTCTGTGG

TGAACATACAAAAGGAGATCGACAGACTGAACGAGGTGGCAAAGAACCTGAATGAAA GCCTGATCGACCTGCAAGAACTG

GGCAAGTACGAGCAGTACATCAAGTGGCCTTGGTACATTTGGCTGGGATTTATCGCA GGCCTCATCGCCATCGTGATGGT

GACAATCATGCTGTGTTGCATGACCAGCTGTTGCAGCTGCCTGAAAGGCTGTTGTAG CTGCGGCAGCTGCTGCAAGTTCG

ATGAGGACGACAGCGAGCCTGTCCTGAAGGGGGTGAAGCTGCACTACACATGA

SEQ ID NO: 19 is a codon-optimized nucleic acid sequence encoding a stabilized SARS-

CoV-2 Wuhan strain spike protein with a double proline substitution.

ATGTTCGTCTTCCTGGTCCTGCTGCCCCTGGTCTCATCTCAGTGCGTGAATCTGACT ACAAGAACTCAGCTGCCTCCCGC CTACACCAATTCCTTCACCCGGGGCGTGTACTATCCTGACAAGGTGTTTAGAAGCTCCGT GCTGCACTCTACACAGGATC TGTTTCTGCCATTCTTTAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCA ATGGCACAAAGCGGTTCGAC AATCCCGTGCTGCCTTTTAACGATGGCGTGTACTTCGCCTCTACCGAGAAGAGCAACATC ATCAGAGGCTGGATCTTTGG CACCACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAACGTGGT CATCAAGGTGTGCGAGTTCC AGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAGAACAATAAGAGCTGGATGG AGTCCGAGTTTAGAGTGTAT TCTAGCGCCAACAATTGCACATTTGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAG GGCAAGCAGGGCAATTTCAA GAACCTGAGGGAGTTCGTGTTTAAGAATATCGATGGCTACTTCAAGATCTACTCTAAGCA CACCCCCATCAACCTGGTGC GCGACCTGCCTCAGGGCTTCAGCGCCCTGGAGCCACTGGTGGATCTGCCTATCGGCATCA ACATCACCCGGTTTCAGACA CTGCTGGCCCTGCACAGAAGCTACCTGACACCCGGCGACTCCTCTAGCGGATGGACCGCA GGAGCAGCAGCCTACTATGT GGGCTATCTGCAGCCTAGGACCTTCCTGCTGAAGTACAACGAGAATGGCACCATCACAGA CGCAGTGGATTGCGCCCTGG ACCCCCTGAGCGAGACAAAGTGTACACTGAAGTCCTTTACCGTGGAGAAGGGCATCTATC AGACATCCAATTTCAGGGTG CAGCCAACCGAGTCTATCGTGCGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAG GTGTTCAACGCAACCAGGTT CGCAAGCGTGTACGCATGGAATAGGAAGCGCATCTCTAACTGCGTGGCCGACTATAGCGT GCTGTACAACTCCGCCTCTT TCAGCACCTTTAAGTGCTATGGCGTGTCCCCCACAAAGCTGAATGACCTGTGCTTTACCA ACGTGTACGCCGATTCTTTC GTGATCAGGGGCGACGAGGTGCGCCAGATCGCACCTGGACAGACAGGCAAGATCGCCGAC TACAATTATAAGCTGCCAGA CGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGCGG CAACTACAATTATCTGTACC GGCTGTTTAGAAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCTCTACAGAGATCTACC AGGCCGGCAGCACCCCTTGC AATGGCGTGGAGGGCTTTAACTGTTATTTCCCACTGCAGTCCTACGGCTTCCAGCCCACA AACGGCGTGGGCTATCAGCC TTACCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCACCAGCAACAGTGTGCGGACC CAAGAAGTCCACCAATCTGG TGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGAACAGGCGTGCTGACCG AGTCCAACAAGAAGTTCCTG CCATTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTGCGCGACCCACAG ACCCTGGAGATCCTGGATAT CACACCCTGCTCTTTCGGCGGCGTGAGCGTGATCACACCAGGAACCAATACAAGCAACCA GGTGGCCGTGCTGTATCAGG ACGTGAATTGTACCGAGGTGCCTGTGGCCATCCACGCCGATCAGCTGACCCCAACATGGC GGGTGTACAGCACCGGCTCC AACGTGTTCCAGACAAGAGCAGGATGCCTGATCGGAGCAGAGCACGTGAACAATTCCTAT GAGTGCGACATCCCAATCGG CGCCGGCATCTGTGCCTCTTACCAGACCCAGACAAACTCTCCAAGGAGAGCACGGAGCGT GGCATCCCAGTCTATCATCG CCTATACCATGTCCCTGGGCGCCGAGAATTCTGTGGCCTACTCTAACAATAGCATCGCCA TCCCAACCAACTTCACAATC TCTGTGACCACAGAGATCCTGCCCGTGTCCATGACCAAGACATCTGTGGACTGCACAATG TATATCTGTGGCGATTCTAC CGAGTGCAGCAACCTGCTGCTGCAGTACGGCAGCTTTTGTACCCAGCTGAATAGAGCCCT GACAGGCATCGCCGTGGAGC AGGATAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCCCCTA TCAAGGACTTTGGCGGCTTC AATTTTTCCCAGATCCTGCCTGATCCATCCAAGCCTTCTAAGCGGAGCTTTATCGAGGAC CTGCTGTTCAACAAGGTGAC CCTGGCCGATGCCGGCTTCATCAAGCAGTATGGCGATTGCCTGGGCGACATCGCAGCACG GGACCTGATCTGTGCCCAGA AGTTTAATGGCCTGACCGTGCTGCCACCCCTGCTGACAGATGAGATGATCGCACAGTACA CAAGCGCCCTGCTGGCAGGA ACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATCCCCTTTGCCATG CAGATGGCCTATAGGTTCAA CGGCATCGGCGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAATCAGTT TAACTCCGCCATCGGCAAGA TCCAGGACAGCCTGTCCTCTACAGCCTCCGCCCTGGGCAAGCTGCAGGATGTGGTGAATC AGAACGCCCAGGCCCTGAAT ACCCTGGTGAAGCAGCTGAGCTCCAACTTCGGCGCCATCTCTAGCGTGCTGAATGATATC CTGAGCCGGCTGGACCCCCC CGAGGCAGAGGTGCAGATCGACCGGCTGATCACAGGCAGACTGCAGTCTCTGCAGACCTA TGTGACACAGCAGCTGATCA GGGCAGCAGAGATCAGGGCAAGCGCCAATCTGGCAGCAACCAAGATGTCCGAGTGCGTGC TGGGCCAGTCTAAGAGAGTG GACTTTTGTGGCAAGGGCTATCACCTGATGTCCTTCCCTCAGTCTGCCCCACACGGCGTG GTGTTTCTGCACGTGACCTA CGTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCCACGATGGCAAGGC CCACTTTCCAAGGGAGGGCG TGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCTACGAGCCCCAGA TCATCACCACAGACAATACC TTCGTGAGCGGCAACTGTGACGTGGTCATCGGCATCGTGAACAATACCGTGTATGATCCA CTGCAGCCCGAGCTGGACAG CTTTAAGGAGGAGCTGGATAAGTACTTCAAGAATCACACCTCCCCTGACGTGGATCTGGG CGACATCAGCGGCATCAATG CCTCCGTGGTGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTGGCCAAGAATCTGA ACGAGAGCCTGATCGATCTG CAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCATGGTACATCTGGCTGGGCTTC ATCGCCGGCCTGATCGCCAT CGTGATGGTGACCATCATGCTGTGCTGTATGACATCCTGCTGTTCTTGCCTGAAGGGCTG CTGTAGCTGTGGCTCCTGCT GTAAGTTTGATGAGGACGATTCCGAACCCGTGCTGAAGGGAGTGAAGCTGCATTACACCT GA DETAILED DESCRIPTION

I. Abbreviations

Ad adenovirus

CoV coronavirus

COVID-19 coronavirus disease 2019

Env envelope

GI gastrointestinal

HIV human immunodeficiency virus

IFU infection forming units

IM intramuscular

IN intranasal

OPV oral poliovirus

PP double protein substitution

S spike protein

SARS severe acute respiratory syndrome

TT tail truncated

URT upper respiratory tract

VOC variant of concern

Wu Wuhan strain

II. Terms

Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes single or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:

Adenovirus: A non-enveloped virus with a liner, double-stranded DNA genome and an icosahedral capsid. There are at least 68 known serotypes of human adenovirus, which are divided into seven species (species A, B, C, D, E, F and G). Different serotypes of adenovirus are associated with different types of disease, with some serotypes causing respiratory disease (primarily species B and C), conjunctivitis (species B and D) and/or gastroenteritis (species F and G). Adenovirus type 4 (Ad4) is a species E virus that can cause acute respiratory disease and ocular disease. Adenovirus-based vectors are commonly used for a variety of therapeutic applications, including vaccine and gene therapy vectors. In some embodiments herein, the adenovirus vector is a human replication-competent Ad4 with a complete or partial deletion in the E3 region.

Adjuvant: A component of an immunogenic composition used to enhance antigenicity. In some embodiments, an adjuvant can include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion, for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). In some embodiments, the adjuvant used in a disclosed immunogenic composition is a combination of lecithin and carbomer homopolymer (such as the ADJUPEEX™ adjuvant available from Advanced BioAdjuvants, EEC; see also Wegmann, Clin Vaccine Immunol 22(9): 1004-1012, 2015). Additional adjuvants for use in the disclosed immunogenic compositions include the QS21 purified plant extract, Matrix M, AS01, MF59, and ALFQ adjuvants. Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants. Adjuvants include biological molecules (a “biological adjuvant”), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM- CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL and toll-like receptor (TLR) agonists, such as TLR-9 agonists. The person of ordinary skill in the art is familiar with adjuvants (see, e.g., Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007).

Administration: The introduction of a composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, intranasal, inhalation, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical) and vaginal routes.

Codon-optimized: A nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (such as a particular species or group of species). For example, a nucleic acid sequence can be optimized for expression in mammalian cells or in a particular mammalian species (such as human cells). Codon optimization does not alter the amino acid sequence of the encoded protein.

Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or decrease the function of a protein, such as a coronavirus spike protein. “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to elicit an immune response when administered to a subject. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Furthermore, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some embodiments less than 1%) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.

The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Non-conservative substitutions are those that reduce an activity or function of a protein, such as a recombinant Env protein, such as the ability to elicit an immune response when administered to a subject. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest.

Coronavirus: A large family of positive-sense, single-stranded RNA viruses that can infect humans and non-human animals. Coronaviruses get their name from the crown-like spikes on their surface. The viral envelope is comprised of a lipid bilayer containing the viral membrane (M), envelope (E) and spike (S) proteins. Most coronaviruses cause mild to moderate upper respiratory tract illness, such as the common cold. However, three coronaviruses have emerged that can cause more serious illness and death: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Other coronaviruses that infect humans include human coronavirus HKU1 (HKUl-CoV), human coronavirus OC43 (OC43-CoV), human coronavirus 229E (229E-CoV), and human coronavirus NL63 (NL63-CoV).

COVID-19: The disease caused by the coronavirus SARS-CoV-2.

Degenerate variant: A polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is unchanged.

E3 region: Refers to the adenovirus early region 3 (E3) gene, which contains multiple open reading frames (ORFs). The E3 region of human adenovirus type 4 (Ad4) includes the following ORFs: 12. IK, 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K and 14.7K. In some embodiments herein, the deletion in the E3 region comprises a deletion of the 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K and 14.7K ORFs. In other embodiments, the deletion in the E3 region is a deletion of only the 24.8K, 6.3K and 29.7K ORFs.

Heterologous: Originating from a separate genetic source or species. For example, a heterologous polypeptide or polynucleotide refers to a polypeptide or polynucleotide derived from a different source or species.

Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In some embodiments, the response is specific for a particular antigen (an “antigen- specific response”), such as a SARS-CoV-2 spike protein. In some embodiments, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In other embodiments, the response is a B cell response, and results in the production of specific antibodies. “Priming an immune response” refers to treatment of a subject with a “prime” immunogen/immunogenic composition to induce an immune response that is subsequently “boosted” with a boost immunogen/immunogenic composition. Together, the prime and boost immunizations produce the desired immune response in the subject.

Immunogenic composition: A composition that includes an immunogen or a nucleic acid molecule or vector encoding an immunogen (such as SARS-CoV-2 spike protein), that elicits a measurable CTE response against the immunogen, and/or elicits a measurable B cell response (such as production of antibodies) against the immunogen, when administered to a subject. It further refers to isolated nucleic acids encoding an immunogen, such as a nucleic acid that can be used to express the immunogen (and thus be used to elicit an immune response against this immunogen). For in vivo use, the immunogenic composition can include the protein or nucleic acid molecule in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant.

Immunize: To render a subject protected from infection by a particular infectious agent, such as SARS-CoV-2. Immunization does not require 100% protection. In some examples, immunization provides at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% protection against infection compared to infection in the absence of immunization.

Isolated: An “isolated” biological component has been substantially separated or purified away from other biological components, such as other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides, nucleic acids, and viruses that have been “isolated” include those purified by standard purification methods. Isolated does not require absolute purity, and can include protein, peptide, nucleic acid, or virus molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

Neutralizing antibody: An antibody that reduces the infectious titer of an infectious agent by binding to a specific antigen on the infectious agent, such as a virus (e.g., a coronavirus). In some embodiments, an antibody that is specific for a SARS-CoV-2 spike protein neutralizes the infectious titer of SARS-CoV-2. For example, an antibody that neutralizes SARS-CoV-2 may interfere with the virus by binding it directly and limiting entry into cells. Alternately, a neutralizing antibody may interfere with one or more post-attachment interactions of the pathogen with a receptor, for example, by interfering with viral entry using the receptor. In some embodiments, a SARS-CoV-2 neutralizing antibody inhibits SARS-CoV-2 infection of cells, for example, by at least 50%, by at least 60%, by at least 70%, by at least 80% or by at least 90%, compared to a control antibody.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens (such as recombinant Ad4 expressing SARS- CoV-2 S protein) and immunogenic compositions.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate. In particular embodiments, suitable for administration to a subject the carrier may be sterile, and/or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to elicit the desired anti-SARS-CoV-2 immune response. It may also be accompanied by medications for its use for treatment purposes. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage.

Preventing, treating or ameliorating a disease: “Preventing” a disease refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in viral load. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as a coronavirus infection.

Recombinant: A recombinant nucleic acid, vector or virus is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished, for example, by the artificial manipulation of isolated segments of nucleic acids, for example, using genetic engineering techniques.

Replication-competent virus: A virus capable of undergoing genome replication and protein synthesis to produce progeny virus.

Sequence identity: The similarity between amino acid or nucleotide sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity; the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide or polynucleotide will possess a relatively high degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are known. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearson et al. , Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

Variants of a polypeptide or nucleic acid sequence are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid or nucleotide sequence of interest. Sequences with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids (or 30-60 nucleotides), and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet.

As used herein, reference to “at least 90% identity” (or similar language) refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.

SARS-CoV-2: A coronavirus of the genus betacoronavirus that first emerged in humans in 2019. This virus is also known as Wuhan coronavirus, 2019-nCoV, or 2019 novel coronavirus. The term “SARS-CoV-2” includes variants thereof, such as, but not limited to, alpha (B.1.1.7 and Q lineages); beta (B.1.351 and descendent lineages); delta (B.1.617.2 and AY lineages); gamma (P.l and descendent lineages); epsilon (B.1.427 and B.1.429); eta (B.1.525); iota (B.1.526); kappa (B.1.617.1); 1.617.3; mu (B.1.621, B.1.621.1), zeta (P.2) and omicron (B.1.1.529 and BA lineages). Symptoms of SARS-CoV-2 infection include fever, chills, dry cough, shortness of breath, fatigue, muscle/body aches, headache, new loss of taste or smell, sore throat, nausea or vomiting, and diarrhea. Patients with severe disease can develop pneumonia, multi-organ failure, and death. The time from exposure to onset of symptoms is approximately 2 to 14 days. The SARS-CoV-2 virion includes a viral envelope with large spike glycoproteins. The SARS-CoV-2 genome, like most coronaviruses, has a common genome organization with the replicase gene included in the 5'-two thirds of the genome, and structural genes included in the 3'-third of the genome. The SARS-CoV- 2 genome encodes the canonical set of structural protein genes in the order 5' - spike (S) - envelope (E) - membrane (M) and nucleocapsid (N) - 3'.

SARS Spike (S) protein: A class I fusion glycoprotein initially synthesized as a precursor protein of approximately 1256 amino acids for SARS-CoV, and 1273 amino acids for SARS-CoV- 2. Individual precursor S polypeptides form a homotrimer and undergo glycosylation within the Golgi apparatus as well as processing to remove the signal peptide, and cleavage by a cellular protease between approximately position 679/680 for SARS-CoV, and 685/686 for SARS-CoV-2, to generate separate SI and S2 polypeptide chains, which remain associated as S1/S2 protomers within the homotrimer, thereby forming a trimer of heterodimers. The SI subunit is distal to the virus membrane and contains the receptor-binding domain (RBD) that is believed to mediate virus attachment to its host receptor. The S2 subunit is believed to contain the fusion protein machinery, such as the fusion peptide. S2 also includes two heptad-repeat sequences (HR1 and HR2) and a central helix typical of fusion glycoproteins, a transmembrane domain, and a cytosolic tail domain. An exemplary wild-type (Wuhan strain) SARS-CoV-2 spike protein sequence is set forth herein as SEQ ID NO: 2. Exemplary modified Wuhan SARS-CoV-2 spike protein sequences are set forth herein as SEQ ID NOs: 3-5. In addition, exemplary SARS-CoV-2 variant spike protein sequences are set forth herein as SEQ ID NOs: 7-12.

Subject: Living multicellular vertebrate organisms, a category that includes human and non-human mammals. In some embodiments, the subject is a human. In some examples, a subject who is in need of inhibiting or preventing a SARS-CoV-2 infection is selected. For example, the subject can be uninfected and at risk of SARS-CoV-2 infection.

Therapeutically effective amount: A quantity of a specific substance, such as a disclosed immunogen (e.g., a recombinant Ad4 expressing SARS-CoV-2 S protein) or immunogenic composition, sufficient to achieve a desired effect in a subject being treated, such as a protective immune response. A “therapeutically effective amount” can be the amount necessary to inhibit SARS-CoV-2 replication or treat CO VID-19 in a subject with an existing SARS-CoV-2 infection. A “prophylactic ally effective amount” refers to administration of an agent or composition that inhibits or prevents establishment of an infection, such infection by SARS-CoV-2. It is understood that to obtain a protective immune response against an antigen of interest, multiple administrations of a disclosed immunogen/immunogenic composition can be required, and/or administration of a disclosed composition as the “prime” in a prime boost protocol wherein the boost immunogen can be different from the prime immunogenic composition. Accordingly, an effective amount of a disclosed immunogen/immunogenic composition can be the amount of the immunogen or immunogenic composition sufficient to elicit a priming immune response in a subject that can be subsequently boosted with the same or a different immunogen to elicit a protective immune response.

In one example, a desired response is to elicit an immune response that inhibits or prevents SARS-CoV-2 infection. The SARS-CoV-2 infected cells do not need to be completely eliminated or prevented for the composition to be effective. For example, administration of an effective amount of an immunogen or immunogenic composition can elicit an immune response that decreases the number of SARS-CoV-2 infected cells (or prevents the infection of cells) by a desired amount, for example, by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SARS-CoV-2 infected cells), as compared to the number of SARS-CoV-2 infected cells in the absence of the immunization.

Unit dosage form: A physically discrete unit, such as a capsule, tablet, or solution, that is suitable as a unitary dosage for a human patient, each unit containing a predetermined quantity of one or more active ingredient(s) calculated to produce a therapeutic effect, in association with at least one pharmaceutically acceptable diluent or carrier, or combination thereof.

Vaccine: A pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen- specific immune response to an antigen of a pathogen, for example a viral pathogen, or to a cellular constituent correlated with a pathological condition. A vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell or one or more cellular constituents. In one specific, non-limiting example, a vaccine reduces the severity of the symptoms associated with SARS-CoV-2 infection and/or decreases the viral load compared to a control. In another non- limiting example, a vaccine reduces SARS-CoV-2 infection and/or transmission compared to a control.

Vector: An entity containing a DNA or RNA molecule bearing a promoter(s) that is operationally linked to the coding sequence of a protein (such as an immunogenic protein) of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses. Non-limiting examples of viral vectors include adenovirus vectors, adeno- associated virus (AAV) vectors, and poxvirus vectors (e.g., vaccinia, fowlpox). III. Introduction

Of the available vaccine platforms for presenting viral glycoproteins to the immune system, replicating vectors have several important advantages over most non-replicating vectors (Robert- Guroff, Curr Opin Biotechnol 18(6):546-556, 2007). Replication-competent vectors can express viral surface proteins such that the total dose of antigen vastly exceeds those of non-replicating vectors. Replicating mucosal vaccines induce mucosal immunity, including IgA and IgG antibodies, and a balanced T cell response including resident memory T cells. In addition, replicating vectors, such as replication-competent adenovirus (Ad) vectors, express viral glycoproteins over a prolonged period of time, similar to live virus infections. This feature is thought to be important for the loading of dendritic cells in the lymph node and the induction of a durable antibody response (Cirelli et al., Cell 177(5): 1153-1171, 2019; Tam et al., Proc Natl Acad Sci USA 113(43): E6639-E6648, 2016; Mueller et al., Mol Pharm 12(5): 1356-1365, 2015). Each of these features contributes to the magnitude and durability of immune responses observed after replicating viral vaccinations.

The vaccine constructs disclosed herein are replication-competent Ad4 encoding a SARS- CoV-2 spike (S) protein. In the disclosed Ad4 vector, which is derived from an Ad4 vaccine strain, the gene encoding a SARS-CoV-2 spike protein is cloned into an E3 region having a deletion of multiple E3 ORFs. The parent Ad4 vaccine vector has been given to over 10 million people with an excellent safety record. Ad4-recombinants have been developed for both influenza virus H5 and human immunodeficiency virus (HIV) envelope (Env) and Gag proteins. These Ad4-based vaccines have been through pre-clinical testing in rabbits for immunogenicity and human testing in phase 1 clinical trials.

The replication-competent Ad4-based vaccine platform has several distinct advantages compared to other proposed and licensed SARS-CoV-2 vaccines. For example, the efficacy of Ad4 vaccines has already been established as they have been administered routinely as a single dose enteric capsule in the U.S. military and found to prevent respiratory disease with an efficacy of greater than 95%. In addition, when administered intranasally or onto the tonsils, replication- competent Ad4-based vaccines induce a neutralizing antibody response in human subjects. Upper respiratory tract administration also bypasses pre-existing Ad4 immunity in most people. By inducing mucosal immunity, the Ad4-based vaccine platform not only provides protection for vaccinated subjects, but also has the potential to interrupt transmission of SARS-CoV-2 to others. In contrast to non-replicating viral vaccines, the replication-competent Ad4-based system produces a durable immune response. Furthermore, unlike mRNA-based SARS-CoV-2 vaccines, Ad4 vaccines can be stored long term at 4-8°C. Moreover, the disclosed vaccine platform is unmatched in terms of scalability and cost. It is estimated that the disclosed SARS-CoV-2 vaccine can be produced for less than 1 cent per dose.

IV. Overview of Embodiments

Disclosed herein is a recombinant adenovirus type 4 (Ad4) expressing a SARS-CoV-2 spike (S) protein (in some embodiments, referred to herein as “Ad4-SARS-CoV-2-spike” or “Add- Spike”), a recombinant Ad4 nucleic acid vector encoding the recombinant Ad4-Spike, and immunogenic compositions thereof.

In one aspect, provided herein is a recombinant Ad4 expressing a SARS-CoV-2 S protein. The recombinant Ad4 is replication-competent and the genome of the Ad4 includes a deletion in the adenovirus E3 region and an insertion of a coding sequence for the SARS-CoV-2 S protein. In some embodiments, the amino acid sequence of the S protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of a native S protein, such as the S protein of the Wuhan SARS-CoV-2 strain set forth herein as SEQ ID NO: 2. In specific examples, the amino acid sequence of the S protein comprises or consists of SEQ ID NO: 2.

The amino acid numbering used herein for residues of the SARS-CoV-2 S protein is with reference to the wild-type Wuhan strain SARS-CoV-2 S sequence provided as SEQ ID NO: 2. With reference to the SARS-CoV-2 S protein sequence provided as SEQ ID NO: 2, the ectodomain of the SARS-CoV-2 S protein includes about residues 16-1208. Residues 1-15 are the signal peptide, which is removed during cellular processing. The S1/S2 cleavage site is located at position 685/686. The HR1 is located at about residues 915-983. The central helix is located at about residues 988-1029. The HR2 is located at about 1162-1194. The C-terminal end of the S2 ectodomain is located at about residue 1208. The position numbering of the S protein may vary between SARS-CoV-2 stains, but the sequences can be aligned to determine relevant structural domains and cleavage sites (see, e.g., FIG. 4).

In some embodiments, the recombinant Ad4 comprises a coding sequence for a SARS- CoV-2 S protein comprising one or more (such as two, for example two consecutive) proline substitutions at or near the boundary between a HR1 domain and a central helix domain that stabilize the S protein in the prefusion conformation. In some such embodiments, the one or more (such as two, for example two consecutive) proline substitutions that stabilize the S protein in the prefusion conformation are located between a position 15 amino acids N-terminal of a C-terminal residue of the HR1 and a position 5 amino acids C-terminal of a N-terminal residue of the central helix. In some embodiments, the one or more (such as two, for example two consecutive) proline substitutions that stabilize the SARS-CoV-2 S protein in the prefusion conformation are located between residues 975 to 995 (such as 981-992). In some embodiments, the SARS-CoV-2 S protein is stabilized in the prefusion conformation by K986P and V987P substitutions (“PP” or “2P”). In some embodiments, the SARS-CoV-2 S protein is stabilized in the prefusion conformation by one or two proline substitutions at positions D985, K986, or V987 of the S ectodomain protomers in the trimer. In some examples, the SARS-CoV-2 S protein stabilized in the prefusion conformation by the one or more proline substitutions (such as K986P and V987P substitutions) comprises one or more additional modifications for stabilization in the prefusion conformation.

In some embodiments, the SARS-CoV-2 S protein encoded by the recombinant Ad4 genome comprises an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 3 (Wuhan-PP), wherein the SARS- CoV-2 S protein is stabilized in the prefusion conformation with one or more of the modifications provided herein (such as the K986P and V987P substitutions). In other embodiments, the stabilized, proline substituted S protein is derived from a SARS-CoV-2 variant. In some examples, stabilized S protein derived from a SARS-CoV-2 variant comprises an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 7 (beta-PP), SEQ ID NO: 8 (Wuhan/RDB-beta-PP), SEQ ID NO: 9 (delta-PP), SEQ ID NO: 10 (gamma-PP), SEQ ID NO: 11 (delta plus-PP) or SEQ ID NO: 12 (omicron-PP). In particular examples, the amino acid sequence of the stabilized SARS-CoV-2 S protein comprises or consists of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

In other embodiments, the SARS-CoV-2 S protein encoded by the recombinant Ad4 genome comprises a C-terminal truncation, such as a truncation of the cytoplasmic tail or a truncation of the endocytosis motif. In specific examples, the truncated SARS-CoV-2 S protein comprises or consists of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

An exemplary nucleic acid sequence encoding a SARS-CoV-2 S protein is provided as SEQ ID NO: 6. In some examples, the nucleic acid sequence encoding the S protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 6. In specific non-limiting examples, the nucleic acid sequence encoding the S protein comprises or consists of SEQ ID NO: 6.

The DNA sequence of the exemplary SARS-CoV-2 S protein provided above can be modified to introduce the amino acid substitutions and deletions disclosed herein for prefusion stabilization. In some embodiments, this DNA sequence (with or without modification to introduce amino acid substitutions) can be included in the recombinant Ad4 vector as the sequence encoding the SARS-CoV-2 S protein. In some embodiments, the S protein is encoded by a codon-optimized nucleic acid sequence. In some examples, the nucleic acid sequence encoding the S protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 13 (beta-PP), SEQ ID NO: 14 (Wuhan/RBD beta-PP), SEQ ID NO: 15 (delta-PP), SEQ ID NO: 16 (gamma-PP), SEQ ID NO: 17 (delta plus-PP), SEQ ID NO: 18 (omicron-PP) or SEQ ID NO: 19 (Wuhan-PP). In specific examples, the nucleic acid sequence encoding the S protein comprises or consists of any one of SEQ ID NOs: 13-19.

In some embodiments, the deletion in the E3 region is a deletion of at least two, at least three, at least four, at least five, at least six, or at least seven E3 open reading frame (ORFs). In some examples, the deletion includes at least two, at least three, at least four, at least five, at least six, or at least seven of the 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K and 14.7K ORFs. In particular non- limiting examples, the deletion in the E3 region includes a deletion of each of the 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K and 14.7K ORFs.

In some embodiments, the coding sequence for the SARS-CoV-2 S protein is inserted in place of the deleted portion of the E3 region.

In some embodiments, the nucleotide sequence of the genome of the recombinant Ad4 is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1. In some examples, the nucleotide sequence of the genome of the recombinant Ad4 comprises or consists of SEQ ID NO: 1.

Also provided herein is a recombinant, replication-competent Ad4 nucleic acid vector. In some embodiments, the recombinant Ad4 vector includes a deletion in the adenovirus E3 region and an insertion of a coding sequence for the SARS-CoV-2 S protein. In some embodiments, the amino acid sequence of the S protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of a native S protein, such as the S protein of the Wuhan SARS-CoV-2 strain set forth herein as SEQ ID NO: 2. In specific examples, the amino acid sequence of the S protein comprises or consists of SEQ ID NO: 2.

In some embodiments, the SARS-CoV-2 S protein is stabilized in the prefusion conformation by K986P and V987P substitutions (“PP” or “2P”). In some embodiments, the SARS-CoV-2 S protein is stabilized in the prefusion conformation by one or two proline substitutions at positions D985, K986, or V987 of the S ectodomain protomers in the trimer. In some examples, the SARS-CoV-2 S protein stabilized in the prefusion conformation by the one or more proline substitutions (such as K986P and V987P substitutions) comprises one or more additional modifications for stabilization in the prefusion conformation. In some embodiments, the SARS-CoV-2 S protein encoded by the recombinant Ad4 nucleic acid vector comprises an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 3 (Wuhan-PP), wherein the SARS-CoV-2 S protein is stabilized in the prefusion conformation with one or more of the modifications provided herein (such as the K986P and V987P substitutions). In other embodiments, the stabilized, proline substituted S protein is derived from a SARS-CoV-2 variant. In some embodiments, the S protein is encoded by a codon-optimized nucleic acid sequence. In some examples, stabilized S protein derived from a SARS-CoV-2 variant comprises an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 7 (beta-PP), SEQ ID NO: 8 (Wuhan/RDB-beta-PP), SEQ ID NO: 9 (delta-PP), SEQ ID NO: 10 (gamma-PP), SEQ ID NO: 11 (delta plus-PP) or SEQ ID NO: 12 (omicron-PP). In particular examples, the amino acid sequence of the stabilized SARS-CoV-2 S protein comprises or consists of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

In other embodiments, the SARS-CoV-2 S protein encoded by the recombinant Ad4 nucleic acid vector comprises a C-terminal truncation, such as a truncation of the cytoplasmic tail or a truncation of the endocytosis motif. In specific examples, the truncated SARS-CoV-2 S protein comprises or consist of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

In some embodiments of the disclosed Ad4 vector, the deletion in the E3 region is a deletion of at least two, at least three, at least four, at least five, at least six, or at least seven E3 ORFs. In some examples, the deletion includes at least two, at least three, at least four, at least five, at least six, or at least seven of the 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K and 14.7K ORFs. In particular non-limiting examples, the deletion in the E3 region includes a deletion of each of the 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K and 14.7K ORFs.

In some embodiments of the disclosed Ad4 vector, the coding sequence for the SARS-CoV- 2 S protein is inserted in place of the deleted portion of the E3 region. In some examples, the coding sequence for the S protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 2-5 and 7-12. In specific non-limiting examples, the coding sequence for the S protein comprises or consists of any one of SEQ ID NOs: 2-5 and 7-12.

In some embodiments, the nucleotide sequence of the Ad4 vector is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1. In some examples, the nucleotide sequence of the Ad4 vector comprises or consists of SEQ ID NO: 1. Further provided herein are immunogenic compositions that include a recombinant Ad4 or a recombinant Ad4 vector, and a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition further includes an adjuvant. In other embodiments, the immunogenic composition does not include an adjuvant.

Methods of eliciting an immune response against SARS-CoV-2 in a subject are also provided. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a recombinant Ad4, a recombinant Ad4 (nucleic acid) vector, or an immunogenic composition disclosed herein. Also provided are methods of immunizing a subject against SARS-CoV-2 infection. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a recombinant Ad4, a recombinant Ad4 vector, or an immunogenic composition disclosed herein.

In some embodiments of the disclosed methods, the recombinant Ad4, recombinant Ad4 vector, or immunogenic composition is administered intranasally or onto the tonsils. In some examples, intranasal administration includes administration of an aerosol. The particle size of the aerosol should allow for delivery to the upper respiratory tract, but not the lower respiratory tract. In specific examples, the aerosol contains particles greater than 10 microns in diameter, such as greater than 20 microns, greater than 30 microns, greater than 40 microns or greater than 50 microns. In particular examples, the aerosol contains particles of about 10 to about 150 microns, such as about 20 to about 125 microns or about 30 to about 100 microns. One of skill in the art is capable of selecting an appropriate device for intranasal delivery of the disclosed recombinant Ad4, recombinant Ad4 vector, or immunogenic composition to the upper respiratory tract. Non-limiting examples of devices include Accuspray™ (Becton-Dickinson) and the MAD Nasal™ (Teleflex ®) atomizer.

In some embodiments, the method includes administering a dose of about 10 ⁴ to about 10 ⁶ recombinant Ad4 particles, such as about 5 x 10 ⁴ to about 5 x 10 ⁵ viral particles or about 1 x 10 ⁵ viral particles. In some examples, the dose is about 1 x 10 ⁴ , 2 x 10 ⁴ , 3 x 10 ⁴ , 4 x 10 ⁴ , 5 x 10 ⁴ , 6 x

10 ⁴ , 7 x 10 ⁴ , 8 x 10 ⁴ , 9 x 10 ⁴ , 1 x 10 ⁵ , 2 x 10 ⁵ , 3 x 10 ⁵ , 4 x 10 ⁵ , 5 x 10 ⁵ , 6 x 10 ⁵ , 7 x 10 ⁵ , 8 x 10 ⁵ , 9 x

10 ⁵ , or 1 x 10 ⁶ recombinant Ad4 particles.

In some embodiments, the recombinant Ad4, the recombinant Ad4 vector, or the immunogenic composition is administered in a single dose.

In some embodiments, the recombinant Ad4, the recombinant Ad4 vector, or the immunogenic composition is administered as part of a prime-boost immunization protocol. In some examples, the recombinant Ad4, the recombinant Ad4 vector, or the immunogenic composition is the prime dose. In other examples, the recombinant Ad4, the recombinant Ad4 vector, or the immunogenic composition is the boost dose.

V. Preclinical and Clinical Studies Relevant to COVID-19 Vaccine Development

By studying the vaccine-induced mucosal neutralizing antibody responses in a series of live oral poliovirus (OPV) challenge studies, investigators have robustly demonstrated the remarkable separation of the systemic and mucosal antibody systems (Brickley et al. , Clin Infect Dis. 2018;67(suppl_l):S42-S50). This research demonstrates that, despite inducing high levels of serum antibody and providing individual protection from paralytic polio, inactivated Salk vaccines fail to induce the intestinal IgA responses that are critical for inhibiting enteric poliovirus replication and preventing fecal-oral transmission. In contrast, primary vaccination with live attenuated Sabin OPV induces robust mucosal IgA responses and sterilizing immunity upon challenge with live OPV. This observation emphasizes the critical nature of inducing mucosal immunity to prevent infection and transmission of COVID-19. It is believed that the lack of mucosal immunogenicity seen with OPV will be echoed by subunit or replication-incompetent systemically administered SARS-CoV-2 vaccines.

In pre-clinical testing of SARS-CoV-2 vaccines, a similar advantage to mucosal immunization in blocking infection has been observed. In ferrets, IM or mucosal immunization with a replication-defective Ad5-spike recombinant induced similar levels of spike- specific antibodies in the serum, yet only mucosal immunization induced sterilizing protection of the upper respiratory tract (URT) (Wu et al., Nat Commun 11(1): 4081, 2020). A similar advantage of intranasal administration over intramuscular administration in inducing mucosal immunity and sterilizing protection of the URT has been observed using lentiviral- or chimp adenoviral- spike recombinants in mouse models permissive to SARS-CoV-2 infection (Ku et al., Cell Host Microbe 81931-3128(20)30672-7, 2020; Hassan et al., Cell 183(1): 169-184, 2020; King et al., King et al., bioRxiv 2020.10.10.331348, 2020). It has been observed that local specific IgA is highly associated with terminating viral shedding in humans after challenge with coronavirus 229E (Callow et al., J Hyg 95(1): 173-189, 1985).

Prior attempts to protect against a viral mucosal infection for which the host is naive using a parenterally administered non-replicating vaccine have failed or produced enhanced disease. Examples include respiratory syncytial virus (RSV), parainfluenza virus (PIV)-3, Ad4, rotavirus, and measles virus. The reasons for these failures lie in part in the difficulty in protecting mucosal surfaces coated on their apical surfaces with viral receptors, 100-1000-fold lower antibodies on these surfaces compared to serum, and distorted and short-lived immune responses generated by non-replicating vectors. Clinical trials of the disclosed Ad4-SARS-CoV-2-spike vaccine will evaluate in detail the humoral and mucosal responses to the SARS-CoV-2 spike protein and the adenovirus vector. It is expected that the disclosed Ad4-SARS-CoV-2-spike vaccine will produce mucosal antibodies in the respiratory tract and most closely mimic the immune profile observed following natural SARS-CoV-2 infection. Furthermore, it is believed that the disclosed vaccine offers the best possibility for durably interrupting transmission during the COVID-19 pandemic.

Among the recombinant viral vectors available for human use, replicating adenoviruses offer several important advantages. Replicating Ad4 has been given to more than 10 million people in the military as a vaccine against Ad4 respiratory disease and has an extraordinary safety and efficacy record (Gaydos and Gaydos, Mil Med. 1995;160(6):300-304). This recombinant Ad4 is attenuated by administration to the gastrointestinal tract in the form of an enteric coated tablet, and does not cause respiratory disease (Choudhry et al., Vaccine 2016:34(38) 4558-4564). Using an enteric capsule delivery, a phase 3 study was undertaken with 4,000 volunteers entering basic military training. The results demonstrated a vaccine efficacy of 99.3% and seroconversion in 94.5% against respiratory disease caused by Ad4 (Kuschner et al., Vaccine 2013:31 2963-2971).

In one trial in humans, replicating recombinant adenoviral vectors expressing influenza virus H5 delivered enterically were only modestly immunogenic. This is most likely related to the attenuation of replication by administration to the gastrointestinal tract (Gurwith et al. , Lancet Infect Dis. 2013;13(3):238-50) coupled with the E3 deletion. The introduction of a large gene such as that coding for the coronavirus spike protein into an adenovirus vector involves the removal of most early (in this case E3) genes and conveys at least a 10-fold attenuation to the parent adenovirus in tissue culture, chimpanzees, and humans (Lubeck et al., Nat Med. 1997;3(6):651-8).

In another clinical trial, high and remarkably durable levels of influenza- specific neutralizing antibodies were observed when a replication-competent Ad4 expressing the influenza virus hemagglutinin type 5 Vietnam (Ad4-H5-Vtn) was administered to the URT compared to the gastrointestinal (GI) tract (Matsuda et al., Sci Immunol. 2019;4(34):eaau2710; Matsuda et al., J Clin Invest 131(5):el40794, 2021). The vaccine delivered into the URT was very safe (nasal congestion or throat discomfort in 25% of participants, none above grade 2) up to a dose of 10 ⁸ . This level of reactogenicity is at approximately the same level as seen in placebos, and with some parenterally administered non-replicating platforms now being tested against SARS-CoV-2, and below that of a currently licensed varicella zoster (Shingrix) vaccine. URT administration of adenoviruses to Ad4-seropositive humans did result in reinfection. URT administration uses the difficulties in protecting the upper respiratory tree to its advantage to overcome vector-specific immunity. An example of that is the ability of an adenovirus expressing Ebola glycoprotein to induce protective immunity on Ebola challenge by the intranasal route in adeno-immune primates while no protection was observed after IM administration of the Ebola construct in previously adeno immune animals.

Prior results with Ad4-H5-Vtn and Ad4-HIV recombinants indicated that nearly all human participants developed a response to the transgene. After a single intranasal or tonsillar administration of the vaccine, increases in H5-specific B cells, H5-specific antibody somatic hypermutation, and potency were observed. The vaccines also induced a very durable response. The response to the licensed split influenza vaccine typically wanes by 5-10-fold within 2-6 months following immunization. However, when Ad4-H5-Vtn participants were asked to return for boosting 3-5 years later, neutralizing antibodies were still at the level that one observes at the peak response after immunization with the licensed vaccine. The Ad4-SARS-CoV-2-spike vaccine construct disclosed herein could be used to generate mucosal immunity after a systemic vaccination. Alternatively, a subunit vaccine could be administered following immunization with the disclosed vaccine to boost mucosal and systemic antibody, which has been shown to occur with the H5-Vtn vaccine construct.

VI. Immunogenic Compositions

Immunogenic compositions that include a disclosed immunogen (e.g., a recombinant Ad expressing a SARS-CoV-2 S protein, or a recombinant Ad4 nucleic acid vector comprising a SARS-CoV-2 S protein coding sequence), and a pharmaceutically acceptable carrier are also provided. Such compositions can be administered to subjects by a variety of administration modes, for example, intranasal, onto the tonsils, inhalation, oral, intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes. Methods for preparing administrable compositions are described in more detail in such publications as Remingtons Pharmaceutical Sciences, 19 ^th Ed., Mack Publishing Company, Easton, Pennsylvania, 1995.

Thus, an immunogen described herein can be formulated with pharmaceutically acceptable carriers to help retain biological activity while also promoting increased stability during storage within an acceptable temperature range. Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffer saline solution, water, emulsions (e.g., oil/water or water/oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. The resulting aqueous solutions may be packaged for use as is or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple dosing. Formulated compositions, especially liquid formulations, may contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually ^1% w/v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and/or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.

The immunogenic compositions of the disclosure can contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

The pharmaceutical composition may optionally include an adjuvant to enhance an immune response of the host. Suitable adjuvants are, for example, toll-like receptor agonists, alum, AIPO4, alhydrogel, Lipid-A and derivatives or variants thereof, oil-emulsions, saponins, neutral liposomes, liposomes containing the vaccine and cytokines, non-ionic block copolymers, and chemokines. Non- ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL- 12 (Genetics Institute, Cambridge, MA), may be used as an adjuvant (Newman et al. , 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage in that they help to stimulate the immune system in a non-specific way, thus enhancing the immune response to a pharmaceutical product. In some embodiments, an adjuvant is not required and is thus not administered with the Ad4-Spike vaccine.

In some embodiments, the composition can be provided as a sterile composition. The pharmaceutical composition typically contains an effective amount of a disclosed immunogen and can be prepared by conventional techniques. Typically, the amount of immunogen in each dose of the immunogenic composition is selected as an amount which elicits an immune response without significant, adverse side effects. In some examples, the dose is about 1 x 10 ⁴ to about 10 ⁶ viral particles, such as about 5 x 10 ⁴ to about 5 x 10 ⁵ viral particles or about 1 x 10 ⁵ viral particles.

In some embodiments, the composition can be provided in unit dosage form for use to elicit an immune response in a subject, for example, to prevent SARS-CoV-2 infection in the subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and/or a metering mechanism for administering the unit dose or multiples thereof. In some examples, the unit dosage is about 1 x 10 ⁴ to about 10 ⁶ viral particles, such as about 5 x 10 ⁴ to about 5 x 10 ⁵ viral particles. In specific examples, the unit dosage is about 1 x 10 ⁵ viral particles.

VII. Methods of Eliciting an Immune Response

The disclosed immunogens (e.g., a recombinant replication-competent adenovirus expressing a SARS-CoV-2 spike protein), polynucleotides and vectors encoding the disclosed immunogens, and compositions including same, can be used in methods of inducing an immune response to SARS-CoV-2 to prevent, inhibit (including inhibiting transmission), and/or treat a SARS-CoV-2 infection.

Provided herein are methods of eliciting an immune response against SARS-CoV-2 in a subject. In some embodiments, the method includes administering to the subject an effective amount of a recombinant adenovirus, adenovirus vector or immunogenic composition disclosed herein. In some examples, the recombinant adenovirus, vector or immunogenic composition is administered intranasally (such as in a spray) or orally (such as by using enteric-coated tablets).

When inhibiting, treating, or preventing SARS-CoV-2 infection, the methods can be used either to avoid infection in an SARS-CoV-2 seronegative subject (e.g., by inducing an immune response that protects against SARS-CoV-2 infection), or to treat existing infection in a SARS- CoV-2 seropositive subject.

To identify subjects for prophylaxis or treatment according to the methods of the disclosure, accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease or condition, or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, conventional work-ups to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods, such as various ELISA and other immunoassay methods to detect and/or characterize SARS-CoV-2 infection. These and other routine methods allow the clinician to select patients in need of therapy using the methods and immunogenic compositions of the disclosure. In accordance with these methods and principles, a composition can be administered according to the teachings herein, or other conventional methods, as an independent prophylaxis or treatment program, or as a follow-up, adjunct or coordinate treatment regimen to other treatments.

The disclosed immunogens can be used in coordinate (or prime-boost) immunization protocols or combinatorial formulations. In certain embodiments, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate immunogens or formulations, each directed toward eliciting an anti- SARS-CoV-2 immune response, such as an immune response to SARS-CoV-2 spike protein. Separate immunogenic compositions that elicit the anti- SARS-CoV-2 immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate immunization protocol.

In one embodiment, a suitable immunization regimen includes at least two separate inoculations with one or more immunogenic compositions including a disclosed Ad4-Spike with a second inoculation being administered more than about two, about three to eight, or about four weeks following the first inoculation. A third inoculation can be administered several months after the second inoculation, and in specific embodiments, more than about five months after the first inoculation, more than about six months to about two years after the first inoculation, or about eight months to about one year after the first inoculation. Periodic inoculations beyond the third are also desirable to enhance the subject's “immune memory.” The adequacy of the vaccination parameters chosen, e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. Alternatively, the T cell populations can be monitored by conventional methods. In addition, the clinical condition of the subject can be monitored for the desired effect, e.g., prevention of SARS- CoV-2 infection, improvement in disease state (e.g., reduction in viral load), or reduction in transmission frequency. If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response. Thus, for example, a dose of a disclosed immunogen can be increased or the route of administration can be changed.

It is contemplated that there can be several boosts, and that each boost can be a different immunogen. It is also contemplated in some examples that the boost may be the same immunogen as another boost, or the prime.

The prime and the boost can be administered as a single dose or multiple doses, for example, two doses, three doses, four doses, five doses, six doses or more can be administered to a subject over days, weeks or months. Multiple boosts can also be given, such one to five, or more. Different dosages can be used in a series of sequential inoculations. For example, a relatively large dose in a primary inoculation and then a boost with relatively smaller doses. The immune response against the selected antigenic surface can be elicited by one or more inoculations of a subject.

In several embodiments, a disclosed immunogen can be administered to the subject simultaneously with the administration of an adjuvant. In other embodiments, the immunogen can be administered to the subject after the administration of an adjuvant and within a sufficient amount of time to elicit the immune response. In other embodiments, no adjuvant is administered.

SARS-CoV-2 infection does not need to be completely inhibited for the methods to be effective. For example, elicitation of an immune response to SARS-CoV-2 can reduce or inhibit SARS-CoV-2 infection by a desired amount, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SARS-CoV-2 infected cells), as compared to SARS-CoV-2 infection in the absence of immunization. In additional examples, SARS-CoV-2 replication can be reduced or inhibited by the disclosed methods. SARS- CoV-2 replication does not need to be completely eliminated for the method to be effective. For example, the immune response elicited using one or more of the disclosed immunogens can reduce SARS-CoV-2 replication by a desired amount, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SARS-CoV-2 replication), as compared to SARS-CoV-2 replication in the absence of the immune response.

Following immunization of a subject, serum can be collected from the subject at appropriate time points, frozen, and stored for neutralization testing. Methods to assay for neutralization activity, include, but are not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry based assays, single-cycle infection assays, and pseudovirus neutralization assays.

In some embodiments, immunization is achieved by administration of recombinant Ad4 vector DNA. Immunization by nucleic acid constructs is taught, for example, in U.S. Patent No. 5,643,578 (which describes methods of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or a humoral response), U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637 (which describe operably linking a nucleic acid sequence encoding an antigen to regulatory sequences enabling expression), and broadly described in Janeway & Travers, Immunobiology: The Immune System In Health and Disease, page 13.25, Garland Publishing, Inc., New York, 1997; and McDonnell & Askari, N. Engl. J. Med. 334:42-45, 1996.

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described. EXAMPLES

Example 1: Expression of Wild-Type and Modified SARS-CoV-2 Spike Proteins

The following studies evaluated cell-surface expression of wild-type Wuhan strain SARS- CoV-2 spike protein (SEQ ID NO: 2) and three modified versions of the Wuhan strain spike protein: stabilized (PP), tail truncated (TT), and endocytosis motif truncated (no-Endo). PP contains double proline stabilization substitutions at amino acid positions 986 and 987 (SEQ ID NO: 3); TT includes a deletion of the terminal 24 amino acids of the cytoplasmic tail (SEQ ID NO: 4); and no-Endo contains a deletion of the C-terminal endocytosis signaling motif (SEQ ID NO: 5) (see FIG. 4).

Expression of SARS-CoV-2 WT, PP, TT and no-Endo spike proteins was evaluated in A549 cells. Cells were transfected with a shuttle vector plasmid containing the gene for a WT or modified SARS-CoV-2 spike protein. Untransfected cells served as negative controls and cells transfected with a plasmid expressing an HIV-1 Env protein was used as a positive control for transfection. Expression of spike and Env was measured by flow cytometry using a SARS-CoV-2 spike protein- specific antibody and an HIV Env-specific antibody (VRC01), respectively. As shown in FIG. 1, SARS-CoV-2 spike protein expression in transfected A549 cells diminished with truncation of the tail, and truncation of the endocytosis motif, relative to wild-type spike protein.

Nucleic acid sequence encoding the WT, PP or TT SARS-CoV-2 spike protein was inserted into the E3 region of a replication-competent Ad4 vector having a deletion of the E3 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K and 14.7K ORFs. The nucleotide sequence of the recombinant Ad4 containing the WT spike protein coding sequence is set forth herein as SEQ ID NO: 1. Expression of the WT, stabilized and truncated spike protein in recombinant Ad4-infected A549 cells was evaluated. Replicating Ad4 carrying the WT spike nucleic acid sequence (nCoV-WT), the PP-stabilized spike nucleic acid sequence (nCov-PP) or the tail-truncated spike nucleic acid sequence (nCov-TT) was used to infect A549 cells. A replicating adenovirus expressing an HIV-1 Env protein (FDE3) was used as a positive control of infection and uninfected (unIF) cells were used as a negative control. Expression of spike protein was measured by flow cytometry using a SARS-CoV-2 spike protein- specific antibody. Antibody VRC01 was used to detect expression of HIV-1 Env. Spike protein expression from the Ad4-Spike after 2 days of infection is shown in FIG. 2 A. In FIG. 2B, expression of the PP-stabilized and truncated Spike proteins is shown. As shown in FIGS. 2A-2B, expression of spike protein was high from both the nCoV-WT and nCoV- PP constructs. Example 2: Immunogenicity of Ad4-Spike (WT) in rabbits

Immunogenicity of Ad4-Spike (expressing the WT spike protein sequence of SEQ ID NO: 2) was tested in New Zealand white rabbits. Rabbits and other experimental animals do not replicate the Ad4 virus, however intramuscular administration (IM) is commonly used as a screen for immunogenicity. Rabbits were immunized IM on day 0 and day 28 with 1.29 x 10 ⁹ infectious units (IFU) of purified replicating Ad4-Spike. Using a luciferase assay, serum neutralization against Wuhan SARS-CoV-2 pseudovirus was detected at 4 weeks (prior to the second immunization), and continued to increase through the 12-week study period.

Example 3: Immunogenicity studies in hamsters

Human adenoviruses are capable of infecting Syrian golden hamsters (van der Lubbe et al. , NPJ Vaccines 6(1):39, 2021). Thus, immunogenicity studies were performed in these animals. A dose titration from 10 ² -10 ⁷ infection forming units (IFU) of intranasal Ad4-SARS-CoV-2 Wuhan spike with PP stabilization (Ad4-SARS-CoV-2w _u pp) was conducted. Strong serum neutralization was observed at week 4 (FIG. 5A) and week 8 (FIG. 5B) in a lentivirus pseudotype assay at the highest doses of Ad4-SARS-CoV-2w _u pp.

These results suggested that the hamster is semi-permissive for Ad4, but replicates the virus sufficiently to induce serum neutralizing antibodies. Spike-specific IgA and IgG were also observed in the nasal wash on day 60.

Hamsters were then immunized with intranasal Ad4 expressing stabilized (double proline substituted - PP) spike proteins from variants of concern (VOC). Included in this study were Ad4- CoV2-Wuhan, Ad4-CoV2-SA (beta), Ad-CoV2-Wu/RBD-SA, Ad4-CoV2-Indian (delta) and Ad4- CoV2-Brazil (gamma). An Ad4 expressing an influenza virus H5 hemagglutinin (Ad4-H5) and sham inoculation were included as negative controls.

Serum neutralization against Wuhan, delta and omicron pseudovirus was determined 28 days and 56 days following intranasal administration. The results are shown in FIGS. 6A-6E. Ad4 expressing the Wuhan-PP (SEQ ID NO: 3) or Delta-PP (SEQ ID NO: 9) were the most immunogenic.

Example 4: Challenge study in hamsters

This example describes a study to test candidate vaccines in the Syrian golden hamster model.

In this study, Syrian golden hamsters are intranasally administered an immunogenic candidate identified in Example 3 (Candidate 1 or Candidate 2) at a dose of 10 ⁷ IFU and subsequently challenged with SARS-CoV-2 by co-habitation with SARS-CoV-2 Delta- or SARS- CoV-2 Omicron-infected animals (van Doremalen et al., Sci Transl Med 13(607):eabh0755, 2021). Table 1 shows the groups of animals that are used. Animals in Group A are challenged at day 60, while animals in Group B are challenged 6 months after immunization. Hamsters receiving intranasal administration of Ad4-H5-Vtn are included as negative controls. Pfizer mRNA or Ad26- Spike is administered intramuscularly as a comparator.

Table 1. Challenge study in hamsters It is expected that intranasal Ad4-Spike vaccine will give systemic neutralizing antibodies that are of the same order of magnitude as mRNA or Ad26 but is more durable. It is also expected that the Ad4-Spike will cause greater restriction of the challenge virus compared to parenterally administered vaccines. Example 4: Human Clinical Study

A Phase 1/2 open-label study of a single dose of intranasally administered Ad4-Spike in healthy volunteers is conducted. Enrollment begins with volunteers who may or may not have had prior coronavirus disease 2019 (CO VID- 19) or vaccination. The international setting chosen is one where supplies of CO VID- 19 vaccines are limited and SARS-CoV-2-naive volunteers may be more easily enrolled. All SARS-CoV-2-naive participants are offered an emergency use authorization (EUA) vaccine at the completion of the study or following the 6-month timepoint if their neutralization titer is below ~40 (which is the lower boundary of the interquartile range for the Modema mRNA 1272 vaccine). Each study participant receives a single dose of an intranasal Ad4- SARS-CoV-2 vaccine or an intramuscular (IM) immunization with an authorized or licensed booster. Study participants are monitored for adverse events (AEs), and blood and respiratory secretions are collected for immunogenicity and safety testing periodically throughout the study period. Nasal swabs are collected to monitor adenovirus shedding, and nasal washes are collected to monitor mucosal immune responses. Household and intimate contacts willing to participate are also enrolled and monitored for transmission of the vaccine virus by serology.

The primary endpoints are for safety measured by the frequency and grade of solicited and unsolicited adverse events in the first 28 days after vaccination. Safety is evaluated by separately assessing the incidence, severity, and type of adverse events in the candidate vaccine arms of the trial over the duration of follow-up. It is expected that 21% (N= 10/48) of vaccine recipients may experience vaccine-related signs and symptoms (e.g., headache, fatigue, myalgia, rhinorrhea, nausea, diarrhea). Vaccine virus shedding is evaluated by describing the presence, quantity, and duration of shed virus in serially collected nasal wash samples.

A second endpoint is immunogenicity. Immunogenicity is evaluated in serially collected serum, nasal, and stool samples. Immunogenicity is determined by a lentivirus-based pseudovirus neutralization assay. The assay includes functional antibodies as measured by characterization of B-cell clones, complement-enhancement and antibody dependent enhancement, mucosal and T cell immunity. Respiratory mucosal responses are being seen after CO VID-19 infection and are thus expected to be a distinguishing hallmark of the Ad4-Spike vaccine. If the Ad4- vectored SARS- CoV-2 vaccine ‘takes’ in 95% of recipients and is immunogenic to adenovirus 4 and SARS-CoV-2 spike protein in 90% of these recipients, it is expected that systemic immune responses will be induced in 85% (N=44/52) of vaccine recipients and mucosal responses will be induced in 90- 100% of volunteers.

A second dose at 60 days is administered in the rare instance of no evidence of vaccine take at 30 days. However, the primary analysis is after 1 dose as this vaccine is expected to be a single dose regimen. Most participants in prior Ad4-based vaccine trials did not develop a higher response after a second immunization, a second dose would only induce a response in the infrequent case that a participant is not infected on the first dose.

As volunteers will not be pre-screened for serum antibodies, a subset of the volunteers will be seropositive at baseline for Ad4 (-30%, N=20/60) as a result of exposure to circulating wildtype adenoviruses. The response of those with pre-existing Ad4 immunity in the previous vectored vaccine trials has suggested that Ad4 immunity may modulate the response to the vector and limit virus shedding, but vector specific immunity will still be induced.

Participants are monitored for safety and immunogenicity for one year. The Phase 1 trial optionally includes parallel exploratory arms designed into the clinical trial to permit using Add- Spike in conjunction with other SARS-CoV-2 Spike immunogens such as DNA, mRNA, or protein vaccines. It is expected that Ad4-Spike will contribute greater durability and mucosal T and B cell responses compared to non-replicating, parenterally administered protein or nucleic acid vaccines.

The target study population excludes only those who may be negatively impacted by respiratory viral infections, such as pregnant women or those with severe immunodeficiencies. The symptoms of recombinant Ad4 vaccination, when they occur, tend to be mild and self-limited. Those persons without difficulties in handling upper respiratory infections should not experience severe symptoms with the Ad4-Spike vaccine. Although pre-existing immunity to Ad4 is not uncommon (30%), it is largely overcome by intranasal vaccination. The degree to which vectorspecific immunity is overcome will be assessed and is expected to be a function of the replication of the vaccine virus and the immunogenicity of the spike protein. The prevalence of Addantibodies in persons under 16 is extremely low, making this vaccine a very attractive mode to induce durable immunity in school aged children. The primary endpoints are safety and immunogenicity. Safety is definitively addressed in phase 2 of the trial if the primary endpoint is reached.

When prior Ad4 recombinant virus vaccines were given intranasally, the virus replicated at a low level for 2-4 weeks. However, shedding of the virus detected by viral culture was at a low level and for a median of one day. Participants are counselled to avoid intimate contact for 14 days after vaccination. For these reasons, transmission of the vaccine virus to household or intimate contacts has not been observed. Most vaccinees are asymptomatic. However, the most common adverse events (AEs) are throat discomfort and nasal congestion in 25% of participants, none above grade 2. It is expected that a recombinant Ad4 that includes the SARS-CoV-2 Spike protein will yield results similar to prior Ad4-based, intranasally administered vaccines.

A phase 3 study and/or challenge study is conducted following phase 2. In view of the many possible embodiments to which the principles of the disclosed subject matter may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the disclosure and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim all that comes within the scope and spirit of these claims.