WO2023212579A1

WO2023212579A1 - Recombinant polypeptides containing at least one immunogenic fragment and uses thereof

Info

Publication number: WO2023212579A1
Application number: PCT/US2023/066212
Authority: WO
Inventors: Fritz M. Schomburg; David M. RANCOUR
Original assignee: Boost Biopharma, Inc.
Priority date: 2022-04-25
Filing date: 2023-04-25
Publication date: 2023-11-02

Abstract

Provided is a recombinant polypeptide containing at least one immunogenic fragment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike glycoprotein, pharmaceutical compositions containing the same, and methods employing the same.

Description

RECOMBINANT POLYPEPTIDES CONTAINING AT LEAST ONE IMMUNOGENIC FRAGMENT AND USES THEREOF

CROSS-REFERENCE TO PRIOR APPLICATION

[0001] This application claims benefit to pending U. S. Patent Application No.

17,728,798. filed April 25, 2022, which is hereby incorporated by reference in its entirety⁷.

INCORPORATI0N-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleofide/amino acid sequence listing submited concurrently herewith and identified as follows: One 724,088 Byte XML file named 767220.xml, created April 18, 2023.

BACKGROUND OF THE INVENTION

[0003] Tire rapid evolution of new SARS-CoV-2 variants containing mutations that alter the amino acid sequence of the Spike protein resulting in altered function, and altered resistance to native immune defenses and to immune defenses elicited by currently marketed vaccines has led to a need for alternative platforms that allow for incorporation of new variant mutations hi a robust vaccine.

BRIEF SUMMARY OF THE INVENTION

[0004] The present invention relates to recombinant polypeptides that include at least one immunogenic fragment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV- 2) spike glycoprotein and, optionally, an antibody Fc region. In some embodiments, the recombinant polypeptide includes more than one immunogenic fragment, e.g. , two, three, four, five, or more immunogenic fragments. In some embodiments, the recombinant polypeptide includes one or more immunogenic fragments of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and/or Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) and optionally an antibody Fc region, optionally in combination with one or more SARS-CoV-2 immunogenic fragments,

[0005] The present invention further relates to pharmaceutical compositions, such a& vaccines, that include the recombinant polypeptide, la some embodiments, the pharmaceutical composition includes an adjuvant.

[0006] The present invention also relates to a method for preventing, inhibiting, reducing, eliminating, protecting, and/or delaying the onset of an infection or an infectious clinical condition caused by a beta coronavirus in a subject, wherein the method includes administering to the subject at least one recombinant polypeptide of the invention or a pharmaceutical composition including the same.

[0007] The present invention further relates to a method for inducing an immune response against a coronavirus in a subject, wherein the method includes administering to the subject at least one recombinant polypeptide of the invention or a pharmaceutical composition including the same.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG . 1 is a graph depicting Spike S I protein IgG response in Rhesus macaques at certain time points (in weeks) after initial injection with an exemplary construct,

[0009] FIG. 2 A is an image showing CHO cell expression of CTD_short_a-Fc (LS2330) after 5 days. Protein was affinity purified using Protein A agarose and analyzed by reducing SDS-PAGE and detected by Coomassie R-250- staining. M-Molecular weight markers. As can be seen in each of Figures 2A-2D, the constructs were resistant to proteolytic degradation during expression thus facilitating higher yields of intact soluble protein

[0010] FIG. 2B is an image showing CHO cell expression of CTD_long_a-Fc (LS3472) after 5 days. Protein was affinity purified using Protein A agarose and analyzed by reducing SDS-PAGE and detected by Coomassie R-250 staining. Protein loads per lane are indicated, [9011 ] FIG. 2C is an image showing CHO cell expression of CTD-deletioii series after 5 days. Protein was affinity purified using Protein A agarose from equal volumes of tr ansfected culture. Eluted protein was loaded based on volume, separated by reducing SDS-PAGE, and detected by Coomassie R-250 staining. Load volumes were twice as large for samples (left to

7 right) CTD_vs_a-Fc to RBD e-Fc compared to the samples on the left of the gel

(CT© long b-Fc to CTD_ short h-Fe).

[0012] FIG. 2D is an image showing CHO cell expression of CTD_shon_i-Fc (LS2371) after 5 days. Protein was affinity purified using Protein A agarose and analyzed by reducing SDS-PAGE and detected by Coomassie R-250 staining.

[0013] FIG. 3 is an image showing CHO cell expression of (CTD_short_d)2-Fc (clone 1 through 4 correspond to strains LS2397 through 2400; SEQ ID NO: 161) and (CTD_short_i)2-Fc (clone 1 through 4 correspond to strains LS2401 through 2404; SEQ ID NO; 163) after 4 or 7 days as indicated. Protein was affinity purified using Protein A agarose from equal volumes of transfected culture. Eluted protein was loaded based on volume, separated by reducing SDS-PAGE, and detected by Coomassie R-250 staining, M-Molecular weight markers.

|0(H 4| FIG. 4 is an image showing CHO cell expression of mutant (CTD_short_i)2-Fc constructs wherein amino acid mutations corresponding to newly identified SARS-CoV -2 variants have been added to both CTD domains (DI and D2) or second domain (D2) only of the CTD dimer. The mutants tested include (a) DI and D2 mutations for hybrid P.l and CAL.20C variants; K4I7T, L452R, E484K, N501Y (Strain 2435), (b) D2 mutations for 501.V2 variant / B.1.351, K417N, E484K, N501Y (Strains 2421), and (c) D2 mutations for hybrid P.l and CAL.20C variants; K417T, L452R, E484K, N501Y (Strain 2423). Protein was affinity purified using Protein A agarose from equal volumes of 4-day transfected culture.

Eluted protein was loaded based on volume, separated by reducing SDS-PAGE, and detected by Coomassie R-250 staining. M-Molecular weight markers. Protein loads were 3.48 (strain 2435), 4.52 (strain 2421), arid 3.14 pg (strain 2423).

[0615] FIGS. 5 A, 5B, and 5C are graphs showing the results of neutralization assays using serum samples from animals P0101(Fig 5A) and P0102 (Fig 5B) immunized with SEQ ID NO; 73 (LS2330 [CTD_short_a-Fc]. which were the subject of analysis discussed in Example 1. FIG 5C represents control samples including human serum from an individual prior to the emergence of SARS-CoV-2 (negative human serum sample) and the same serum spiked with a monoclonal antibody immunoreactive to the RBD portion of the Wuhan variant of the SARS-CoV-2 Spike protein. Infectivity of serum neutralized pseudotypes virus using 295T/ACE2 target cells was quantified by measuring NanoLuc luciferase activity (RLU) and graphed OH the y-axis. Reciprocal serum dilution is shown on the x-axis.

[0016] FIG. 6 is an image showing CHO cell expression of B A.X- 1 -rnonomer-Fc — BA.X-10-monomer-Fc after 4 days. Protein was affinity purified using Protein A agarose from equal volumes of transfected culture. E luted protein was loaded based on volume, separated by reducing SDS-PAGE, and detected by Coomassie R-250 staining. M-Molecular weight markers. ‘Transfect. Control’ is an ACE2-Fc transfection, expression control.

[0017] FIG. 7 is an image showing CHO cell expression of certain BXC-Fc constructs after 6 days. Protein was affinity purified using Protein A agarose from equal volumes of transfected culture. Eluted protein was loaded based on volume, separated by reducing SDS- PAGE, and detected by Coomassie R-250 staining. M-Molecular weight markers.

DETAILED DESCRIPTION OF THE INVENTION

[0018] U.S. Patent Application No. 17/535,309, filed November 24, 2021, which is a continuation application of International Patent Application No. PCT/US2021/040019, filed June 30. 2021, which claims benefit to U.S. Provisional Patent Applications No. 63/046,426, filed June 30, 2020, and 63/154,647, filed February 26, 2021, are each hereby incorporated by reference hi their entireties.

[0019] Recombinant polypeptides of the invention can include any suitable immunogenic fragment or fragments of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS- CoV-2) spike glycoprotein and optionally any suitable antibody Fc region. In some embodiments, an immunogenic fragment comprises, consist of, or consist essentially of, the N-temfinal domain of the S I subunit, the C-terminal domain of the S I subunit, or both. In certain embodiments, an immunogenic fragment can include the complete SARS-CoV-2 spike glycoprotein. Recombinant polypeptides of the invention include at least one immunogenic fragment, and can contain two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more such immunogenic fragments.

[0020] In some embodiments, one or more of the immunogenic fragments are identical to a wild-type SARS-CoV-2 spike glycoprotein, or any portion thereof. Wild-type spike glycoproteins include those of any SARS-CoV-2 strain that has been isolated from a subject. Examples include Wuhan-Hii-1, VOC 202012/01 / B. l.1.7 (Alpha or UK), VOC-202102/02 (B.l.1,7 with E4S4K) (UK), 501.V2 / B.1,351 (South Africa), B.1.429 / CAL.20C (California), B.1.525, and Lineage P.I (Gamma or Brazil), B.l .427 (Epsilon). B.1.429 (Epsilon), B.1,617.1, B.l.617.2 (Delta), B, 1.526 (Iota), B.1,617.3, B.l, A.2,5, €.36.3, B.1.I.318, B. I.35I, B.1.621, B.l.525, P.I.l, P.2 (Zeta), B.l.623, R.1, B.l.1.7, B.I.35I, B.l.351.3, B.l.351.3, BA.l, BA.1.1, BA.l.1.1, BA.l.1.2. BA.l.1.12. BA.l.1.13. BA.l.14, BA.1.15, BA.1.15.1, BA.1.16, BAI.17.2, BA.1.18, BA.2, BA.2+, BA.2.1, BA.2.10.1, BA.2.2, BA.2.3.2. BA.2.4, BA.2.5, BA2.6, BA.2.7, BA.2.8, BA.2.9, BA.2.10, BA2.12, BA.3. BA.4, BA.5, XA, XB, XC, XD, XE, XF, XG, XH, XI, XK, XL, XM, XN, XP, XQ, XR, XS, XT, B.l.617.2, BA.1.1.529, BF.ll, BA.5.2.6, BA.2.75, BA.4.6, BA.5.2.1, BF.7, BQ.l, BQ.1.1, BA.2.75.2, XBB, XBB.l, XBB.l.5, XBB.1.5.1, CH.1.1, CH.l.l.i, CH.1.1.2, XBB .1.16, XBB .1.9.1, and XBB .1.9.2 la some embodiments. one or more of the immunogenic fragments are identical to a wild-type S ARS-CoV or MERS spike glycoprotein, or any portion thereof. Wild-type SARS-CoV and MERS spike glycoproteins include those of any SARS-C’oV or MERS strain that has been isolated from a subject. Examples of such strains include SARS coronavirus Tor2 (GenBank accession number NC_004718.3) and MERS corona vims (GenBank accession number NC O 19843.3).

[0021] In some embodiments, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to a wild-type SARS- CoV- 2 spike glycoprotein, or any portion thereof. In some embodiments, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to a wild-type SARS-CoV or MERS spike glycoprotein, or any portion thereof.

[0022] In some embodiments, one or more of the immunogenic fragments are identical to a wild-type SARS-CoV-2 spike glycoprotein, or any portion thereof, except for at one or more of the following positions of the ammo acid sequence: L5, A67, H69. V70, D80, T95, G142, Y144, E154, F157, D253, G339, R346, L368, S371, S373, S375, T376. D405. R408,

K417, N440, K444, V445, G446, L452, N460, S477, T478, E484, F486, F490, Q493, G496, Q498, N501, T547, D614, Q677, P681, A701, T791, T859, F888, D950, and Q1071, wherein the positions of the listed amino acid residues correspond to the wild-type ammo acid sequence QHD43416 (ncbi.nlm.mh.goVz^rotein/QHD43416). In some embodiments, one or more of the immunogenic fragments are identical to a wild-type SARS-CoV-2 spike glycoprotein, or any portion thereof, except for at one or more of the following positions of the amino acid sequence: L5, A67, H69, V70, D80, T95, G142, Y144, El 54, Fl 57, D253, G339, R346, L368, S371, S373, S375, T376, D405, R408, K417, N440, K444, V445, G446, L452, N460, S477, T478, E484, F486, F490, Q493, G496, Q498, N501, Y505, T547, D614, Q677, P681, A701, T791. T859, F888, D950, and Q1071. wherein the positions of the listed ammo acid residues correspond to the wild-type amino acid sequence QHD43416 (ncbi.nhn.nih.gov/protein/QHD43416). In certain embodiments, one or more of the immunogenic fragments comprises, consists of, or consists essentially of an amino acid sequence selected from SEQ ID NOs: 245-254, except for at one or more of the following positions of the amino acid sequence: L5, A67, H69, V70, D80. T95, G142, Y144, El 54, FI57, D253, G339, R346, S371, S373, S375, T376, D405, R408, K417, N440, G446, L452, S477, T478, E484, Q493, G496, Q498, N501, Y5O5, T547, D614, Q677, P681, A701, T791, T859, F888, D950, and Q107I, wherein the positions of the listed amino acid residues correspond to the wild-type amino acid sequence QHD43416

(ncbi.nhn.nih.gov/protein/QHD43416). In certain embodiments, one or more of the immunogenic fragments comprises, consists of, or consists essentially of an amino acid sequence selected fr om SEQ ID NOs: 245-254, except for at one or more of the following positions of the amino acid sequence: G339, R346, S371, S373, S375. T376, D405, R408, K417, N440. G446. L452, S477, T478, E484, Q493, G496, Q498, N501, and Y5O5, wherein the positions of the listed amino acid residues correspond to the wild-type ammo acid sequence QHD43416 (ncbi.nlm.nih.gov/protem/QHD43416). The mutations at these positions can be any suitable mutation, including conservative and non-conservative amino acid mutations. For instance, a conservative substitution can replace one aliphatic amino acid (i.e., Glycine, Alanine, Valine. Leucine, Methionine or Isoleucine) for another, one polar, uncharged R group amino acid (i.e., Serine. Cysteine, Threonine, Proline, Asparagine, or Methionine) for another, one positively charged R group amino acid (i.e.. Histidine, Lysine, or Arginine) for another, one negatively charged R group amino acid (i.e.. Aspartate or Glutamate) for another, and one non-polar, aromatic R group amino acid (i.e., Phenylalanine, Tyrosine, or Tryptophan) for another. In some embodiments, one or more of the immunogenic fragments are identical to a wild-type SARS-CoV-2 spike glycoprotein, or any portion thereof, except for one or more of the following amino acid substitutions and deletions: L5F, A67V, 69del, 70del, D80G, T95I , G142D, 144del, E154K, F157S, D253G, L452R, S477N, E484K, E484Q. K417N, K417T, T478K, N501Y, D614G, Q677H, T791I, P681H, P681R, A701V, F888L, T859N, D950H, D950N, and Q1071H, wherein the listed amino acid substitutions and deletions are relative to the wild-type amino acid sequence QHD43416 (ncbi.idm.nih.gov/protein/QHD43416). In one embodiment, the one or more ammo acid substitutions and/or deletions is L452R. In another embodiment, the one or more amino acid substitutions and/or deletions is E484K. In a further embodiment, the one or more protein substitutions and/or deletions are K417N, E484K. and N501Y. In yet another embodiment, the one or more substitutions are and/or deletions are K417T, E484K, and N501Y. In another embodiment, the one or more amino acid substitutions and/or deletions are N501Y, 69del, 70del, and P681H. In another embodiment, the one or more amino acid substitutions and/or deletions are K417T, L452R, E484K, and N501Y.

[0023] In certain embodiments, one or more of the immunogenic fragment comprises, consists of, or consists essentially of an ammo acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence comprising, consisting of, or consisting essentially of a wild-type SARS-CoV- 2 spike glycoprotein, or any portion thereof, wherein the amino acid sequence further comprises one or more of the following amino acid substitutions: G339D, R346K, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, L452R, S477N, T478K, E484A, E484K, Q493R, G496S, Q498R, N501Y, and Y5O5H to the extent the sequence does not already contain such residues. In some embodiments, one or more of the immunogenic fragments are identical to a wild-type SARS-CoV-2 spike glycoprotein, or any portion thereof, except for one or more of the following amino acid substitutions and deletions: L5F, A67V, 69del, 70del, D80G, T95I , G142D, 144del, E154K, F157S. D253G, G339D. R346K, S371F, S373P, S375F, T376A, D405N, R408S, N440K, G446S, L452R, S477N, E484A, E484K, E484Q, K417N, K417T, T478K, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, Q677H, T791I, P681H, P681R, A701V, F888L, T859N, D950H, D950N, and Q1071H, wherein the listed amino acid substitutions and deletions are relative to the wildtype amino acid sequence QHD434I6 (ncbimlm,mh.gov/protein/QHD43416). In some embodiments, one or more of the immunogenic fragments comprises, consists of or consists essentially of an amino acid sequence selected from SEQ ID NOs: 245-254, except for one or more of the following amino acid substitutions: G339D, R346K, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, L452R, S477N, T478K, E484A, E484K, Q493R, G496S, Q498R, N501Y, and Y505H, wherein the positions of the listed amino acid residues correspond to the wild-type amino acid sequence QHD43416 (ncbi.nhiimih.gov/protein/ In certain embodiments, one or more of the immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from SEQ ID NOs: 245-254, wherein the amino acid sequence further comprises one or more of the following amino acid substitutions: G339D, R346K, S371F, S373P, S375F, T376A, D405N, R408S, K417N. N440K, G446S, L452R, S477N, T478K, E484A, E484K, Q493R, G496S, Q498R, N501Y, and Y5O5H, to the extent the sequence does not already contain such residues.

[0024] In one embodiment, the one or more amino acid substitutions is G339D, R346K. T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, Q493R, Q498R, N501Y, and Y505H. In another embodiment, the one or more amino acid substitutions is G339D, S373P, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501 Y, and Y505H. In another embodiment, the one or more amino acid substitutions is G339D, S371F, S373P, S375F, T376A, D4O5N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, and Y5O5H. In another embodiment, the one or more ammo acid substitutions is G339D, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484K, Q493R, Q498R, N501Y, and Y5O5H. In another embodiment, the one or more amino acid substitutions is G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484K, Q493R, and N501Y. In another embodiment, the one or more amino acid substitutions is G339D, T376A, D405N, R4O8S, K417N, N440K, S477N, T478K, E484A, and N501Y, In another embodiment, the one or mote ammo acid substimtions is G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, and Y5O5H. In another embodiment, the one or more amino acid substitutions is G339D, S37IF, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N50IY, and Y505H. In another embodiment, the one or more amino acid substitutions is G339D, R346K, T376A, D405N, R408S, K417N, N440K, G446S, L452R, S477N, T478K, E484K, Q493R, G496S, Q498R, N501Y, and Y505H. In another embodiment, the one or more amino acid substitutions is G339D, S373P, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, and Y5O5H.

[0025] The subject can be mammalian, including human, non-human primate, horse, pig, cattle, cat, dog, sheep, mink, rodent, hamster, or bat. Subjects can further include western lowland gorilla, northern white-cheeked gibbon, Sumatran orangutan, crab-eating macaque, drill, proboscis monkey, bonobo, chimpanzee, Ugandan red colobus, red-shanked done, golden snub-nosed monkey, green monkey, patas monkey, rhesus macaque, olive baboon, gelade, sooty mangabey, southern pig-tailed macaque, angola colobus, coquerel’s sifaka, Gambian pouched rat, Chinese hamster, common gund, beluga whale, blue-eyed black lemur, indri, narwhal, narrow-ridged fmless porpoise, harbour porpoise, minke whale, Antarctic minke whale, gray whale, spalax, white-tailed deer, reindeer, southern tainandua, Stephens’s kangaroo rat, Pere David’s deer, iranscaucasian mole vole, long- finned pilot whale. Pacific white-sided dolphin, baiji, giant anteater, muskrat, killer whale, common bottlenose dolphin, aye-aye, fat-tailed dwarf lemur, thirteen-lined ground squirrel, yellow-bellied marmot, alpine marmot, golden hamster, sperm whale, dawian ground squirrel, gobi jerboa, barbry sheep , pronghorn, Nancy ma’s night monkey, hirola, American bison, zebu, wild yak, cattle, water buffalo, white-eared titi, common marmoset, wild goat, goat, Panamanian white-faced capuchin, cat, Masai giraffe, Nilgiri tahr, Candian lynx, coquerel’s giant mouse lemur, Siberian musk deer, sunda clouded leopard, clouded leopard, okapi, sheep, jaguar, leopard, Siberian tiger, Tibetan antelope, little pocket mouse, deer mouse, white- faced saki, cougar, black-capped squirrel monkey, tufted capuchin, arctic ground squirrel, bos indicus x bos Taurus, cheetah, mantled howler, Geoffioy’s spider monkey, Damaialand mole-rat, naked mole-rat hippopotamus, snowshoe hare, Dama gazelle, European rabbit, scimitar oryx, emperor tamarin, and alpaca.

[0026] In some embodiments, one or more of the immunogenic fragments contain one or more mutations, such that the one or more immunogenic fragments are not identical to a wild-type SARS-CbV-2 spike glycoprotein, or any portion thereof. The one or more mutations can be any suitable mutation and/or deletion, such as those disclosed herein. For example, an immunogenic fragment can contain sequences from two, three, four, five, six, seven, eight, nine, ten, or more strains, such that the resulting fragment is no longer identical to any of its parent strains. In this way, a single immunogenic fragment can present epitopes from multiple wild-type SARS-CoV-2 spike glycoproteins, or any portion thereof. This can lead to a more robust immune response and increased immune protection from a range of SARS-CoV-2 strains in a subject when a recombinant polypeptide of the invention containing one or more such immunogenic fragments, or a pharmaceutical composition containing the same, is administered to the subject.

[0027] hi some, embodiments, the nucleic acid sequence encoding an immunogenic fragment includes one, two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty, or more point mutations and/or deletions in comparison to the nucleic acid sequence encoding the corresponding fragment of a wild-type or mutant glycoprotein. In some embodiments, the amino acid sequence of an immunogenic fragment includes one, two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty, or more substitutions and/or deletions in comparison to the amino acid sequence encoding corresponding fragment of a wild-type or mutant glycoprotein.

[0028] In some embodiments, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (CTD_long_a), SEQ ID NO: 3 (CTD_long_a_D6I4G), SEQ ID NO: 5 (CTD_long_a-Dimer), SEQ ID NO: 7

(CTD long b), SEQ ID NO: 9 (CTD_long_c), SEQ ID NO: 11 (CTD_long_d), SEQ ID NO: 13 (CTD_long_e), SEQ ID NO: 15 (CTDJong_f), SEQ ID NO: 17 (CTD_long_g), SEQ ID NO: 19 (CTD_long_h), SEQ ID NO: 21 (CTDJongJ), SEQ ID NO: 23 (CTD_short_a), SEQ ID NO: 25 (CTD_short_bl SEQ ID NO: 27 (CTD_short_c), SEQ ID NO: 29 (CTD_shoit_d), SEQ ID NO: 31 (CTD_short_e), SEQ ID NO: 33 (CTD_short_f), SEQ ID NO: 35 (CTD_short_g). SEQ ID NO: 37 (CTD_short_h), SEQ ID NO: 39 (CTD_short_0_? SEQ ID NO: 41 (CTD._vs._a), SEQ ID NO: 43 (CTD._vs_ b), SEQ ID NO: 45 (CTD_ys _c), SEQ ID NO: 47 (CTD_vs_d), SEQ ID NO: 49 (CTD_vs_e), SEQ ID NO: 51 (RBD_a), SEQ ID NO: 53 (RBD_b), SEQ ID NO: 55 (RBD_c), SEQ ID NO: 57 (RBD d), SEQ ID NO: 59 (RBD_e), SEQ ID NO: 61 (NTD_long_a), SEQ ID NO: 63 (NTD_short_a), SEQ ID NO: 171 (RBD-tight), SEQ ID NO: 173 ((RBD-tight)?), SEQ ID NO: 175 (RBD-extended), SEQ ID NO: 177 ((RBD-extended)?), SEQ ID NO: 179 (RBD), SEQ ID NO: 181 ((RBD)?), SEQ ID NO: 183 ('(OTD_short_d)2), SEQ ID NO: 185 ((CTD_short_i)2), SEQ ID NO: 187 ((CTD_short_i)2- mod. I). SEQ ID NO: 189 ((CTD_short_i)?- mod. 2). SEQ ID NO: 191 ((CTD_short_i)₂ - mod. 3), SEQ ID NO: 199 (SARS-2003, SARS_short_h), SEQ ID NO: 201 (SARS-2003, SARS_short_i), SEQ ID NO: 203 (MERS_Lytic_a), SEQ ID NO: 205 (MERS_Lytic_b), SEQ ID NO: 207 (MERS_Lytic_c), SEQ ID NO: 209 (MERS_Lytic_d), SEQ ID NO: 211 (MERS_Lytic_e), SEQ ID NO: 213 (MERS LyticJ), SEQ ID NO: 215 (MERS_Lytic_g), SEQ ID NOs: 245-254 (BA.X-1 - BA.X-10), SEQ ID NOs: 265-274 (BA.X-l-dimer - BA.X-10-dimer), and SEQ ID NOs: 325-334 (BXC-1 - BXC-10). hi some embodiments, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 185 ((CTD_shoil_i)2), SEQ ID NO: 187 ((CTD_short_i)2- mod. 1), SEQ ID NO: 189 ((CTD_short_i)2- mod. 2), and SEQ ID NO: 191 ((CTD_short_i)2 - mod. 3). In further embodiments, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 185 ((CTD_short_i)2). In further embodiments, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 183 ((CTD_short_d)2), SEQ ID NO: 39 (CTD_short_i), SEQ ID NO: 29 (CTD_short_d), SEQ ID NO: 31 (CTD_short_e)_! SEQ ID NO: 37 (CTD_short_h), and SEQ ID NO: 23 (CTE)_ shori_a). In some embodiments, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least .91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 39. In some embodiments, an immunogenic- fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 185. In some embodiments, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 190. In some embodiments, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence selected from SEQ ID NOs: 245-254 (BA.X-1 - BA.X-10). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 245 (BA.X-1). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 246 (BA.X-2). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 247 (BA.X-3). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 248 (BA.X-4). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity' to the amino acid sequence represented by SEQ ID NO: 249 (BA.X-5). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 250 (BA.X-6). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 251 (BA.X-7). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 252 (BA.X-8). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 253 (BA.X-9). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 254 (BA.X-10). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 265 (BA.X-1 -dimer). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 266 (BA.X-2-dimer). In an embodiment, an hnniunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 267 (BA.X-3 -dimer). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 268 (BA.X-4-dfmer). In an embodiment, animmrmogemc fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 269 (BA.X-5-dimer). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an ammo acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 270 (BA.X-6-dimer). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 271 (BA.X-7-dimer). hi an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 272 (BA.X-8-dimer). hr an embodiment, an immunogenic fr agment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 273 (BA.X-9-dimer). In an embodiment, an immunogenic fr agment comprises, consists of, or consists essentially of an ammo acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 274 (BA.X-10-dimer).

[0029] In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 325 (BXC-1). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 326 (BXC-2). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 327 (BXC-3). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 328 (BXC-4). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 329 (BXC-5). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 330 (BXC-6). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 331 (BXC-7). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 332 (BXC-8). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 333 (BXC-9). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence represented by SEQ ID NO: 334 (BXC-10).

[0030] In some embodiments, the recombinant polypepride comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17. 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 199, 201, 203, 205, 207, 209, 211, 213, 215, 245-254, 265-274, and 325-334. In some embodiments, an immuiiogemc fragment comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 185 ((CTD_short_i)2), SEQ ID NO: 187 ((CTD_short_i)2 - mod. I), SEQ ID NO: 189 ((CTD_short_i)2- mod. 2), and SEQ ID NO: 191 ((CTD_short_i)2 - mod. 3). In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of the amino acid sequence represented by SEQ ID NO: 185. In an embodiment, an immunogenic fragment comprises, consists of, or consists essentially of the amino acid sequence represented by SEQ ID NO: 191. In further embodiments an hmnunogenic fra gment comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 185 ((CTD_short_i)?). In further embodiments, an immimogemc fragment comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 183 «CTD_short_d)₂), SEQ ID NO: 39 (CTD_short_i), SEQ ID NO: 29 (CTD_short_d), SEQ ID NO: 31 (CTD_short_e), SEQ ID NO: 37 (CTD_short_h), and SEQ ID NO: 23 (CTD short a). In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOS : 245-254 and 265-274. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOS: 245-254. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 245. hi some embodiments, the recombinant polypeptide comprises at least one immimogemc fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 246. hi some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO : 247. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 248. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 249. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 250. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 251. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 252. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 253. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 254. hi some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 265. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO : 266. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 267. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 268. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 269. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 270. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fr agment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 271 . In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 272, In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 273. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 274. In some embodiments, the recombinant polypeptide coinprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 325. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 326. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 327. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 328. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 329. hi some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 330. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 331. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 332. In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 333. In some embodiments, the recombinant polypeptide comprises at least one inimunogenic fragment that comprises, consists of. or consists essentially of an amino acid sequence represented by SEQ ID NO: 334.

[0031] In some embodiments, the recombinant polypeptide comprises at least one immunogenic fragment that is encoded by a nucleotide sequence that comprises, consists of, or consists essenti ally of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 200, 202, 204, 206, 208, 210, 212, 214, 216, 285-294, 305-314, and 345-354. In some embodiments, the recombinant polypeptide includes at least two immunogenic fragments. For example, the recombinant polypeptide can include two, three, four, five, six, seven, eight, nine, ten, or more immunogenic fragments. In some embodiments, the recombinant polypeptide includes at least three immunogenic fragments. In some embodiments, the recombinant polypeptide includes at least three immunogenic fragments, of which at least one is a SARS-CoV-2 spike glycoprotein fragment as described herein, at least one is a SARS-C’oV spike glycoprotein fragment as described herein, and at least one is a MERS- CoV spike glycoprotein fragment as described herein. In some embodiments, the recombinant polypeptide includes at least three immunogenic fragments, of which at least one is a SARS-CoV-2 spike glycoprotein fragment, wherein the SARS-CoV-2 fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (CTD_long_a), SEQ ID NO: 3 (CTD_long_a_D614G), SEQ ID NO: 5 (CTD_long_a-Dimer), SEQ ID NO: 7 (CTD_long_b), SEQ ID NO: 9 (CTD_long_c), SEQ ID NO: 11 (OTD_long_d), SEQ ID NO: 13 (CTD_long_e), SEQ ID NO: 15 (CTDJong_f). SEQ ID NO: 17 (CTD_long_g), SEQ ID NO: 19 (CTD_long_h), SEQ ID NO: 21 (CTD_long_i), SEQ ID NO: 23 (CTD_short_a), SEQ ID NO: 25 (CTD_short_b), SEQ ID NO: 27 (CTD_short_c), SEQ ID NO: 29 (CTD_short_d), SEQ ID NO: 31 (CTD_short_e), SEQ ID NO: 33 (CTD_short_f), SEQ ID NO: 35 (CTD_short_g), SEQ ID NO: 37 (CTD_short_h), SEQ ID NO: 39 (CTD_shoit_i), SEQ ID NO: 41 (CTD ys.a), SEQ ID NO: 43 (CTD_vs_b), SEQ ID NO: 45 (CTD_vs_c), SEQ ID NO: 47 (CTD_vs_d), SEQ ID NO: 49 (CTD_vs_e), SEQ ID NO: 51 (RBD_a). SEQ ID NO: 53 (RBD b), SEQ ID NO: 55 (RBD_c), SEQ ID NO: 57 (RBD_d), SEQ ID NO: 59 (RBD_e), SEQ ID NO: 61 (NTDJong a), SEQ ID NO: 6.3 (NTD__short _a), SEQ ID NO: 171 (RBD-tight), SEQ ID NO: 173 ((RBD-tight)₂), SEQ ID NO: 175 (RBD-extended). SEQ ID NO: 177 ((RBD-extended)?), SEQ ID NO: 179 (RBD), SEQ ID NO: 181 ((RBD)?), SEQ ID NO: 183 ((CTD_short_d)₂), SEQ ID NO: 185 ((CTD_short_i)2), SEQ ID NO: 187 ((CTD_short_i)2- mod. I), SEQ ID NO: 189 ((CTD_short_i)2- mod. 2). SEQ ID NO: 191 ((CTD_short_i>2 - mod. 3), SEQ ID NOs: 245-254 (BA.X-1 - BA.X-10), SEQ ID NOs: 265- 274 (BA.X-l-dimer - BA.X-10-dimer), and SEQ ID NOs: 325-334 (BXC-1 - BXC-10); at least one is a SARS-CoV spike glycoprotein fragment, wherein the SARS-CoV fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 199 (SARS-2003, SARS_short_h), or SEQ ID NO: 201 (SARS-2003, SARS_sIioit_i); and at least one is a MERS-CoV spike glycoprotein fragment, wherein the MERS-CoV fragment comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 203 (MERS_Lytic_a). SEQ ID NO: 205 (MERS_Lytic_b), SEQ ID NO: 207 (MERS_Lydic_c), SEQ ID NO: 209 (MERS_Lytic_d), SEQ ID NO: 211 (MERS_Lytic_e), SEQ ID NO: 213 (MERS_Lytic_f), and SEQ ID NO: 215 (NfERS_Lytic_g).

[0032] In some embodiments, the recombinant polypeptide includes at least two immunogenic fr agments, wherein each of the at least two immunogenic fr agments comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1 (CTD_long_a), SEQ ID NO: 3 (CTD_long_a_D614G), SEQ ID NO: 5 (CTD_long_a-Dimer), SEQ ID NO: 7 (CTD_Iong_b), SEQ ID NO: 9 (CTD_long_c), SEQ ID NO: 11

(CTD long d), SEQ ID NO: 13 (CTD_long_e), SEQ ID NO: 15 (CTD_long_f), SEQ ID NO: 17 (CTD_long_g), SEQ ID NO: 19 (CTD_Iong_h), SEQ ID NO: 21 (CTD_long_i), SEQ ID NO: 23 (CTD_short_a), SEQ ID NO: 25 (CTD_short_b), SEQ ID NO: 27 (CTD_short_c), SEQ ID NO: 29 (CTD_short_d), SEQ ID NO: 31 (CTD_short_e), SEQ ID NO: 33 (CTE)_short_f), SEQ ID NO: 35 (CTD_ short_g), SEQ ID NO; 37 (CTD_ short _h), SEQ ID NO: 39 (CTD_:ShortJ), SEQ ID NO? 41 (CTD_vs_a), SEQ ID NO: 43 (CTD_vs_b), SEQ ID NO: 45 (CTD_vs _c), SEQ ID NO; 47 (CTD vs _d), SEQ ID NO: 49 (CTD_vs_e), SEQ ID NO: 51 (RBD_a), SEQ ID NO: 53 (RBD_b), SEQ ID NO: 55 (RBD_c), SEQ ID NO: 57 (RBD_d), SEQ ID NO: 59 (RBD_e), SEQ ID NO: 61 (NTD long a), SEQ ID NO: 63 (NTD short a), SEQ ID NO: 171 (RBD-tight), SEQ ID NO: 173 ((RBD-tight)₂), SEQ ID NO: 175 (RBD-extended), SEQ ID NO: 177 ((RBD-extended)₂), SEQ ID NO: 179 (RBD), SEQ ID NO: 181 ((RBD)₂), SEQ ID NO: 183 ((CTD_short_d)₂), SEQ ID NO: 185 ((CTD_short_i)₂), SEQ ID NO: 187 ('(CTD_shortJ)₂-mod. I ), SEQ ID NO: 189 ((CTD_short_i)2- mod. 2), SEQ ID NO: 191 ((CTD_short_i)₂ - mod. 3), SEQ ID NOs; 245- 254 (BA.X-1 - BA.X-10), SEQ ID NOs: 265-274 (BA.X-1-dimer - BA.X-10-dimer), and SEQ ID NOs: 325-334 (BXC-1 - BXC-10). In some embodiments, each of the at least two immunogenic fragments comprises, consists of, or consists essentially of an amino acid sequence with at least 90%. preferably at least 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence independently selected from the group consisting of SEQ ID NO: 183 ((CTO_short_d)₂), SEQ ID NO: 39 (CTD_short_i), SEQ ID NO: 29 (CTD_short_d), SEQ ID NO: 31 (CTD_short_e), SEQ ID NO: 37 (CTD_short_h), SEQ ID NO: 23 (CTD_short_a), SEQ ID NOs: 245-254 (BA.X-1 - BA.X-10), and SEQ ID NOs: 325-334 (BXC-1 - BXC-10). In some embodiments, each of the at least two immunogenic fragments comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 39 (CTD_short_i). In further embodiments, each of the at least two immunogenic fragments comprises, consists of, or consists essentially of an amino acid sequence independently selected from the group consisting of SEQ ID NO: 183 ((CTD_short_d)2), SEQ ID NO: 39 (CTD_short_i), SEQ ID NO: 29 (CTD_short_d), SEQ ID NO: 31 (CTD_short_e), SEQ ID NO: 37 (CTD_short_h), SEQ ID NO: 23 (CTD_short a), ), SEQ ID NOs: 245-254 (BA.X-1 - BA.X-10), SEQ ID NOs: 265-274 (BA.X-1 -dimer - BA.X-10-dimer), and SEQ ID NOs: 325-334 (BXC-1 - BXC-10). In further embodiments, each of the at least two immunogenic fragments comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 39 (CTD_short_i). In further embodiments, each of the at least two immunogenic fragments comprises, consists of, or consists essentially of an amino acid sequence independently selected fr om the group consisting of SEQ ID NOs: 245-254 (BA.X-1 - BA.X-10), and SEQ ID NOs: 265-274 (BA.X-1 -dimer - BA.X-10-dimer). In further embodiments, each of the at least two immunogenic fragments comprises, consists of, or consists essentially of an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 245-254 (BA.X-1 - BAX-10). In further embodiments, each of the at least two immunogenic fragments comprises, consists of, or consists essentially of an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 325-334 (BXC-1 - BXC- 10). The at least two immunogenic fragments can comprise, consist of, or consist essentially of. any suitable combination of amino acid sequences.

[0033] In certain embodiments, each of the at least two immunogenic fragments comprise, consist of, or consist essentially of the same ammo acid sequence selected from the group consisting of SEQ ID NOs: 245-254 (BA.X-1 - BA.X-10), e.g., the at least two immunogenic fragments each comprise, consist of. or consist essentially of an amino acid sequence represented by SEQ ID NO: 245. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of SEQ ID NO: 246. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 247. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 248. In another embodiment, the at least two inimunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 249. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 250. In another embodiment, the at least two inimunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 251. In another embodiment, the at least two immunogenic fragments each coinprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 252. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consistessentially of an amino acid sequence represented by SEQ ID NO: 253. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 254. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an arnino acid sequence represented by SEQ ID NO: 265. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 266. In another embodiment, the at least two inimunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 267. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 268. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 269. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 270. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 271. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an arnino acid sequence represented by SEQ ID NO: 272. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 273. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an arnino acid sequence represented by SEQ ID NO: 274. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 325. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an arnino acid sequence represented by SEQ ID NO: 326. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 327. In another embodiment, the at least two iimiiunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 328. In another embodiment, the at least two immunogenic fragments each comprise, consist of or consist essentially of an amino acid sequence represented by SEQ ID NO: 329. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 330. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 331. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 332. In another embodiment, the at least two immunogenic fragments each comprise, consist of, or consist essentially of an amino acid sequence represented by SEQ ID NO: 333. In another embodiment, the at least two immunogenic fragments each comprise, consist of or consist essentially of an ammo acid sequence represented by SEQ ID NO: 334.

[0034] hr certain embodiments, each of the at least two immunogenic fragments comprise. consist of or consist essentially of a different amino acid sequence independently selected from the group consisting of SEQ ID NOs: 245-254 (BA.X-1 - BA.X-10), e.g., when the recombinant polypeptide comprises, consists of, or consists essentially of two immunogenic fragments, one immunogenic fragment comprises, consists of, or consists essentially of SEQ ID NO: 245 and the second immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 246-254. hi certain embodiments, each of the at least two inmiunogenic fragments comprise, consist of, or consist essentially of a different amino acid sequence independently selected from the group consisting of SEQ ID NOs: 325-334 (BXC-1 - BXC-10), e.g., when the recombinant polypeptide comprises, consists of, or consists essentially of two inmiunogenic fragments, one immunogenic fragment comprises, consists of or consists essentially of SEQ ID NO: 325 and the second immunogenic fragment comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 325-334. [0035] In some embodiments, the recombinant polypeptide includes a plurality of identical immunogenic fragments. For example, a recombinant polypeptide can include two, three, four, five, six, seven, eight, nine, ten, or more immunogenic fragments, wherein eachfragment comprises, consists of, or consists essentially of the same amino acid sequence. In some embodiments, the recombinant polypeptide includes two, three, four or five identical immunogenic fragments. In yet further embodiments, the recombinant polypeptide includes two or three identical immunogenic fragments.

[0036] In some embodiments, the recombinant polypeptide includes a plurality of nonidentical immunogenic fragments. For example, a recombinant polypeptide can include two, three, four, five, six, seven, eight, nine, ten, or more immunogenic fragments, wherein each fra gment comprises, consists of, or consists essentially of a different amino acid sequence from each other fragment, i.e., each fragment is a different fr agment. In some embodiments, the recombinant polypeptide includes two, three, four or five different immunogenic fragments. In yet further embodiments, the recombinant polypeptide includes two or three different immunogenic fragments.

[0037] In some embodiments, the recombinant polypeptide includes a plurality⁷ of immunogenic fr agments, hi which some of the fr agments are identical, but not all. For example, a recombinant polypeptide can include two, three, four, five, six, seven, eight, nine, ten, or more immunogenic fragments, wherein each fragment comprises, consists of, or consists essentially of the same amino acid sequence, while also including one, two, three, four, five, six, seven, eight, nine, ten, or more immunogenic fragments, wherein each fragment comprises, consists of, or consists essentially of a different amino acid sequence from each other fragment. In some embodiments, the recombinant polypeptide includes a total of two, three, four or five immunogenic fragments. In yet further embodiments, the recombinant polypeptide includes a total of two or three immunogenic fragments.

[0038] In certain embodiments wherein the recombinant polypeptide comprises an antibody⁷ Fc region, the at least one immunogenic fragment can be arranged in any suitable serial orientation with respect to the Fc region. In some embodiments, the at least one immunogenic fragment is connected to the N-temiinus of the Fc region. This orientation can be depicted as [immunogenic fragment]* - [N-terminus - Fc region - C -terminus], wherein X is an integer I -10 representing the number of immunogenic fragments within the recombinant polypeptide. In some embodiments, the at least one immunogenic fragment is connected to the C-terminus of the Fc region. This orientation can be depicted as [N- terminus - Fc region - C-terminus] - [immunogenic fragment]x, wherein X is an integer 1-10 representing the number of immunogenic fragments within the recombinant polypeptide. In some embodiments, wherein the recombinant polypeptide includes at least two immunogenic fragments, at least one immunogenic fragment is connected to the N-tenninus of the Fc region, and at least one immunogenic fragment is connected to the C-terminus of the Fc region. This orientation can be depicted as [immunogenic fragment]x - [N-tenninus - Fc region - C-terminus] - [immunogenic fragment}*', wherein X and Y are independently an integer 1-10 representing the number of immunogenic fragments connected to each side of the Fc region. In some embodiments, wherein the recombinant polypeptide includes two immunogenic fragments, both immunogenic fragments are connected to the N-tenninus of the Fc region. Uris orientation can be depicted as [immunogenic fragment] - [immunogenic fragment] - [N-tenninus - Fc region - C-tenninus]. In other embodiments, wherein the recombinant polypeptide includes two immunogenic fragments, both immunogenic fragments are connected to the C-terminus of the Fc region. Tliis orientation can be depicted as [N- terminus - Fc region - C-terminus] - [immunogenic fragment] - [immunogenic fragment]. In some embodiments, wherein the recombinant polypeptide includes three immunogenic fra gments, each immunogenic fragment is connected to the N-tenninus of the Fc region. This orientation can be depicted as [immunogenic fragment] - [immunogenic fragment] - [immunogenic fragment] - [N-tenninus - Fc region - C-terminus], In other embodiments, wherein the recombinant polypeptide includes three immunogenic fragments, each immunogenic fragment is connected to the C-tenninus of the Fc region. Tliis orientation can be depicted as [N-tenninus - Fc region - C-terminus] - [immunogenic fragment] - [immunogenic fragment] - [immunogenic fragment].

Embodiments of the recombinant polypeptide that include a plurality of immunogenic fragments provide for a flexible expression platform with robust expression of full-length protein, modality to modify individual or multiple domains within one or more of the plurality⁷ of immunogenic fragments to reflect the most recent virus variant sequence(s), allow for single step affinity purification by use of an Fc region if present in the recombinant polypeptide, and provide high-level, long-term immune response as tested in Rhesus macaques,

[0040] In some embodiments, wherein the recombinant polypeptide includes a plurality of immunogenic fragments, the immunogenic fragments are connected to each other via a linker. The linker can be any suitable linker. Suitable linkers include a polypeptide comprising, consisting of, or consisting essentially of, an amino acid sequence of 1-35 residues, wherein each residue is independently serine, glycine, or aspartic acid, and further wherein the amino acid sequence contains zero or one aspartic acid residues. Other suitable linkers include a polypeptide comprising an ammo acid sequence selected from the group consisting of SEQ ID NO: 65 (Fcl), SEQ ID NO: 67 (Fci-TEV), SEQ ID NO: 69 (Fcl- Rv3C), SEQ ID NO: 193 (Short), SEQ ID NO: 195 (Medium), and SEQ ID NO: 197 (Long). When a recombinant polypeptide includes two or more such linkers, the amino acid sequence of the linkers can be identical or different. In some embodiments, wherein the recombinant polypeptide includes a plurality of immunogenic fragments, the immunogenic fragments are connected directly to each other without an intervening linker.

[0041] In some embodiments, the recombinant polypeptide comprising or consisting essentially of one or more immunogenic fragments further comprises an antibody Fc region. In some embodiments, the recombinant polypeptide comprising, consisting of, or consisting essentially of one or more immunogenic fragments does not comprise an antibody Fc region. In some embodiments, the recombinant polypeptide comprising or consisting essentially of the one or more immunogenic fragments initially comprises an antibody Fc region, e.g., the recombinant polypeptide comprises an antibody Fc region upon expression of the recombinant polypeptide, wherein the antibody Fc region is subsequently removed, for instance, enzymatically removed. Suitable enzymes include, but are not limited to, class I proteases such as IdeS and IdeZ, and class H enzymes such as the Tobacco Etch Virus (TEA⁷), Rhinovirus 3C (Rv3C), and Factor Xa. When a class II enzyme is employed, the recombinant polypeptide comprises the cleavage site for the employed enzyme, hi some embodiments, the cleavage site is within or adjacent to the linker region between the Spike protein subunit and the Fc domain. [0042] In some embodiments, removal occurs during a purification step or after the recombinant polypeptide has undergone at least one purification step. For instance, the purification is any suitable type of affinity purification, such as Protein A, Protein G, or Protein M purification. In some embodmients, subtractive chromatography steps are used to remove the enzyme. In subtractive chromatography, unwanted proteins are retained on the resin whilst desired proteins flow-tlirough and are collected for further uses. Examples of suitable subtractive chromatography methods include IMAC for Hiss-tagged enzymes, and Protein A resin, Protein G resin, or Protein M resin to remove Fc-fusion enzymes. In some embodiments, cut Fes and uncut Fc-fusions protein are removed by one or more of Protein A, Protein G. and Protein M resm chromatography.

[0643] In some embodiments, the one or more immunogenic fragments are connected to the antibody Fc region via a linker. The linker can be any suitable linker. Suitable linkers include a polypeptide comprising an amino acid sequence of 1-35 residues, wherein each residue is independently serine, glycine, or aspartic acid, and further wherein the amino acid sequence contains zero or one aspartic acid residues. Other suitable linkers include a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 65 (Fci), SEQ ID NO: 67 (Fcl-TEV), SEQ ID NO: 69 (Fcl-Rv3C), SEQ ID NO: 193 (Short), SEQ ID NO: 195 (Medium), and SEQ ID NO: 197 (Long). Adieu a recombinant polypeptide includes two or more such linkers, the amino acid sequence of the linkers can be identical or different from each other. In some embodiments, wherein the recombinant polypeptide includes at least one immunogenic fragment connected to the N-terminus of the Fc region, and/or at least one immunogenic fragment is connected to the C-terminus of the Fc region, the immunogenic fragment(s) nearest to the Fc region are connected directly to the Fc region without an intervening linker.

[0044] Tire antibody Fc region included in the recombinant polypeptide can be any⁷ suitable antibody Fc region. Suitable antibody Fc regions include wild-type human or other animal Iniunoglobulm Fc regions such as IgG, IgA, IgD, IgE, IgM, and their respective subclases, for example, human IgGl Fc regions and Fc regions derived therefrom. Other suitable antibody Fc regions include mutant Fc regions that enhance or diminish Fc-receptor binding affinity to speed up or slow down uptake, respectively, la some embodiments, the antibody Fc region comprises the amino acid sequence of SEQ ID NO: 71.

[0045] In some embodiments, the recombinant polypeptide comprises , consists of, or consists essentially of, an amino acid sequence with at feast 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 73 (LS2330 [CTD_short_a-Fc]), SEQ ID NO: 75 (LS3472, LS3473,LS3474 [CTD_long_a-Fc]), SEQ ID NO: 77 (LS3477

[CTD_short_a-TEV-Fc]), SEQ ID NO: 79 (LS3485 [CTD_long_a TEV-FcJ), SEQ ID NO: 81 (LS3489 [CTD_short_a_Rv3c-Fc]), SEQ ID NO: 83 (LS3497 [CTD_long_a-Rv3c-Fc], SEQ ID NO: 85 (LS2316, LS2317, LS2318, LS2319 [CTD_long_a-His8]), SEQ ID NO: 87 (LS3479 [NTD_short_a-TEV-Fc]), SEQ ID NO: 89 (LS3475 [NTD_long_a-TEV-Fc]), SEQ ID NO: 91 (LS3491 [NTD_short_a-Rv3c-Fc]), SEQ ID NO: 93 (ES3487 [NTD_long_a- Rv3C-Fc]), SEQ ID NO: 95 (LS2326 [NTD_long_a-Fc]), SEQ ID NO: 97 (LS2354 [C.'TD_long_a_D6I4G-Fc]), SEQ ID NO: 99 (LS2355 [CTDJong_a-Dimer-Fc]), SEQ ID NO:10I ( LS2356 [CTD_long_b-Fc]), SEQ ID NO: 103 (LS2357 [CTD_long_c-Fc])₃ SEQ ID NO: 105 (LS2358 [CTD_long_d-Fc]), SEQ ID NO: 107 (LS2359 [CTD_long_e-Fc]), SEQ ID NO: 109 (LS2360 [CTDJongJ-Fc]), SEQ ID NO: 111 (LS2361 [CTD long g- Fc]), SEQ ID NO: 113 (LS2362 [CTD_long_h-Fc]), SEQ ID NO: 115 (LS2363 [CTDJongJ-Fc]), SEQ ID NO: 117 (LS2364 [CTD_short_b-Fc]), SEQ ID NO: 119 (LS2365 [CTD_short_c-Fc]), SEQ ID NO: 121 (LS2366 [CTD_short_d-Fc]), SEQ ID NO: 123 (LS2367 [CTD_short_e-Fc]), SEQ ID NO: 125 (LS2368 [CTD_shortJ-Fc]), SEQ ID NO: 127 (LS2369 [CTD_short_g-Fc]), SEQ ID NO: 129 (LS2370 [CTD_short_h-Fc]), SEQ ID NO: 131 (LS2371 [CTD_short_i-Fc]), SEQ ID NO: 133 (LS2372 [CTD_vs_a-Fc]), SEQ ID NO: 135 (LS2373 [CTD_vs_b-Fc]), SEQ ID NO: 137 (LS2374 [CTD_vs_c-Fc]), SEQ ID NO: 139 (LS2375 [CTD_vs_d-Fc]), SEQ ID NO: 141 (LS2376 [CTD_vs_e-Fc]), SEQ ID NO: 143 (LS2377 [RBD_a-Fc]). SEQ ID NO: 145 (LS2378 [RBD_b-Fc]), SEQ ID NO: 147 (LS2379 [RBD_c-Fc]), SEQ ID NO: 149 (LS2380 [RBD_d-Fc]), SEQ ID NO: 151 (LS2381 [RBD_e-Fc]), SEQ ID NO: 153 (LS2382 [NTD_short_a-Fc]), SEQ ID NO: 155 (LS2393 [(RBD-tighfh-Fc]), SEQ ID NO: 157 (LS2394 [(RBD-extended)₂-FcJ), SEQ ID NO: 159 (LS2395 [(RBD)₂-FC]), SEQ ID NO: 161 (LS2397-2400 [(CTD_short_d)2-Fc]),. SEQ ID NO: 163 (LS2401.-2404 [(C'TD_shortJ)₂-Fc]), SEQ ID NO? 165 (LS2421 and LS2422 [(CTD_short_i)2-Fc)-mod. 1], SEQ ID NO: 167 (LS2423 [(CTD_short j)2-Fe)-mod. 2], SEQ ID NO? 169 (LS2435 [(CTD_shorU)₂-Fc)-mod. 3], SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221 SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO? 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NOs: 255-264 (BA.X-1- monomer-Fc - BA.X-10-monomer-Fc), and SEQ ID NOs: 275-284 (BA.X-1-dimer-Fc - BA.X-10-dimer-Fc). In some embodiments, the recombinant polypeptide comprises, consists of, or consists essentially of, an ammo acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 163 (LS2401-2404 [(CTD_short_i)2-Fc]), SEQ ID NO: 165 (LS2421 and LS2422 [(CTD_shortJ>Fc)-mod. I], SEQ ID NO: 167 (LS2423 [(CTD_short_i)2-Fc)-mod. 2], and SEQ ID NO: 169 (LS2435 [(CTD_short_i)2-Fc)-niod. 3]. In some embodiments, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 163 (LS2401-2404 [(CTD_shoit_i)₂-Fc]). In some embodiments, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 161 (ES2397-2400 [(CTD_short_d)2-Fc]), SEQ ID NO: 121 (LS2366 [CTD_slioit_d-Fc]), SEQ ID NO: 123 (LS2367 [CTD_short_e-Fc]), SEQ ID NO: 129 (LS2370 [CTD_short_h-Fc]), SEQ ID NO: 131 (LS2371 [CTD_short_i-Fc]), and SEQ ID NO: 73 (LS2330 [CTD_short_a-Fc]), In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the ammo acid sequence represented by SEQ ID NO: 163. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the ammo acid sequence represented by SEQ ID NO: 169. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting erf SEQ ID NOs: 255-264 (BA.X-1-monomer-Fc - BA.X- 10-monomer-Fc). In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 275-284 (BA.X-1-dimer-Fc - BA.X-10-dimer-Fc). In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 255. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 256. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence, with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 257. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 258. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 259. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 260. In another embodiment, the recombinant polypeptide comprises, consists of. or consists essentially of. an ammo acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 261, In another embodiment the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 262. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 263. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 264. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 275. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 276. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 277. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 278. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 279. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an ammo acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 280, In another embodiment the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 281. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 282. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 283. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 284. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 335-344 (BXC-l-Fc - BXC-10-Fc). In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 335. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 336. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 337. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least .91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 338. In another embodiment, the recombinant polypeptide comprises, consists of. or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 339. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an ammo acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 340. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 341. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 342. hi another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an ammo acid sequence represented by SEQ ID NO: 343. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence with at least 90%. preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence represented by SEQ ID NO: 344.

[0046] bi some embodiments, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 255-264, 275-284, and 335-344. In some embodiments, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence selected from the group consisting of SEQ ID NO: 163 (LS2401-2404 [(CTD_short_i)j-Fc]), SEQ ID NO: 165 (LS242I and LS2422 [(CTD_short_i)j-Fc), SEQ ID NO: 167 (LS2423 [(CTD_shoitJ)₂-Fc), and SEQ ID NO: 169 (1S2435 [(CTD_short_i)₂-Fc). In some embodiments, the recombinant polypeptide comprises, consists of, or consists essentially o£ the amino acid sequence of SEQ ID NO: 163 (LS2401-2404 [(CTD short i)z- Fc]). In some embodiments, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence selected from the group consisting of SEQ ID NO: 161 (LS2397-2400 [(CTD_short_d)2-FcJ), SEQ ID NO: 121 (LS2366 [CTD_short_d-Fc]), SEQ ID NO: 123 (LS2367 [CTD_short_e-Fc]), SEQ ID NO: 129 (LS2370 [CTD_short_h-Fc]), SEQ ID NO: 131 (LS237I [CTD_sliort_i-Fc]), and SEQ ID NO: 73 (LS2330 [CTD_short_a- Fc]). In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, the amino acid sequence represented by SEQ ID NO: 163. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, the amino acid sequence represented by SEQ ID NO: 169. In some embodiments, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 255-264 and SEQ ID NOs: 275-284. In farther embodiments, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 255-264. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 255. In another embodiment, the recombinant polypeptide comprises, consists of. or consists essentially of, an ammo acid sequence represented by SEQ ID NO: 256. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 257. In another embodiment, the recombinant polypeptide comprises, consists of or consists essentially of, an amino acid sequence represented by SEQ ID NO: 258. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 259. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of an amino acid sequence represented by SEQ ID NO: 260. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 261. In another embodiment, the reconibhiant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 262, In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of. an amino acid sequence represented by SEQ ID NO: 263. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 264, In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 275. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 276. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 277, In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 278. hi another embodiment, the reconibinaiit polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 279. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 280. hi another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 281. In another embodiment, the reconibinaiit polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 282. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 283. hi another embodiment, the reconibinaiit polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 284. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 335. In another embodiment, the reconibinaiit polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 336. In another embodiment, the reconibinaiit polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 337. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 338. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 339. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 340. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 341. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 342. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 343. In another embodiment, the recombinant polypeptide comprises, consists of, or consists essentially of, an amino acid sequence represented by SEQ ID NO: 344.

[0047] In some embodiments, the recombinant polypeptide is encoded by a recombinant polynucleotide. In certain embodiments, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 74 (LS2330 [CTD_short_a-Fc]), SEQ ID NO: 76 (LS3472, LS3473,LS3474 [CTD_long_a-Fc]), SEQ ID NO: 78 (LS3477 [CTD_short_a- TEV-Fc]), SEQ ID NO: 80 (LS3485 [CTD_long_a TEV-Fc]), SEQ ID NO: 82 (LS3489 [CTD_short_a_Rv3c-Fc]), SEQ ID NO: 84 (LS3497 [CTD_long_a-Rv3c-Fc], SEQ ID NO: 86 (LS2316, LS2317, LS2318, LS2319 [CTD_long_a-His8]), SEQ ID NO: 88 (LS3479 [NTD_sliort_a-TEV-Fc]), SEQ ID NO: 90 (LS3475 [NTD_long_a-TEV-Fc]), SEQ ID NO: 92 (LS3491 [NTD_short_a-Rv3c-Fc]), SEQ ID NO: 94 (LS3487 [NTD_long_a-Rv3C-Fc]), SEQ ID NO: 96 (LS2326 [NTD_long_a-Fc]), SEQ ID NO: 98 (LS2354 [CTD_long_a_D614G-Fc]), SEQ ID NO: 100 (LS2355 [CTD_long_a-Dimer-Fc]), SEQ ID NO: 102 ( LS2356 [CTD_long_b-Fc]), SEQ ID NO: 104 (LS2357 [CTD_long_c-Fc]), SEQ ID NO: 106 (L-S2358 [CTD_long_d-Fc]), SEQ ID NO: 108 (LS2359 [CTD_long_e-Fc]), SEQ ID NO: 110 (LS2360 [CTD_long_f-Fc]), SEQ ID NO: 112 (LS2361 [CTD_long_g- FcJ), SEQ ID NO: 114 (LS2362 [CTDJongJi-Fc]), SEQ ID NO: 116 (LS2363 [CTD_long_i-Fc]), SEQ ID NO: 118 (LS2364 [CTD_short_b-Fc]), SEQ ID NO: 120 (LS2365 [CTD_short_c-Fc]), SEQ ID NO: 122 (LS2366 [CTD_short_d-Fc]), SEQ ID NO: 124 (1S2367 [CTD_short_e-Fc]), SEQ ID NO: 126 (LS2368 [CTD_short_f-Fc]), SEQ ID NO: 128 (LS2369 [CTD_short_g-FcJ), SEQ ID NO: 130 (LS2370 [CTD_short_h-Fc]), SEQ ID NO: 132 (LS2371 [CTD_short __i-Fc]), SEQ ID NO: 134 (LS2372 [CTD_vs_a-Fc]), SEQ ID NO: 136 (LS2373 [CTIWsJb-Fc]), SEQ ID NO: 138 (LS2374 [CTIWs_c-Fc]), SEQ ID NO: 140 (LS2375 [CTD_vs_d-Fc]), SEQ ID NO: 142 (LS2376 [CTD_vs_e-Fc]), SEQ ID NO: 144 (LS2377 [RBD_a-Fc]), SEQ ID NO: 146 (LS2378 [RBD_b-Fc])> SEQ ID NO: 148 (LS2379 [RBD_c-Fc]), SEQ ID NO: 150 (LS2380 [RBD_d-Fc]), SEQ ID NO: 152 (LS2381 [RBD_e-Fc]), SEQ ID NO: 154 (LS2382 [NTD_short_a-Fc]), SEQ ID NO: 156 (LS2393 [(RBD-tight)2-Fc]), SEQ ID NO: 158 (LS2394 [(RBD-extended)₂-Fc]), SEQ ID NO: 160 (LS2395 [(RBD)2-FC]), SEQ ID NO: 162 (LS2397-2400 [(CTD_sliort_d)₂-Fc]), SEQ ID NO: 164 (LS2401-2404 [(CTD_shoit_i)2-Fc]), SEQ ID NO: 166 (LS2421 and LS2422 [(CTD_short_i)₂-Fc), SEQ ID NO: 168 (LS2423 [(CTD_short_i)₂-Fc), SEQ ID NO: 170 (LS2435 [(CTD_short_i)2-Fc), SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NOs: 295-304, SEQ ID NOs: 315-324, and SEQ ID NOs: 355-364.

In some embodiments, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 164 (LS2401-2404 [(CTD_short_i)2-Fc]), SEQ ID NO: 166 (LS2421 and LS2422 [(CTD_short_i>2-Fc>mod.I], SEQ ID NO: 168 (LS2423 [(CTD_short_i)2-Fc)-niod. 2]), and SEQ ID NO: 170 (LS2435 [CCTD_short_i>2-Fc-mod. 3). In some embodiments, the polynucleotide comprises, consists of, or consists essentially of, an nucleic acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the nucleic acid sequence of SEQ ID NO: 164 (LS2401-2404 [(CTD_short_i)₂-Fc]). In some embodiments, the polynucleotide comprises, consists of, or consists essentially of, an nucleic acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an nucleic acid sequence selected from the group consisting of SEQ ID NO: 162 (LS2397-2400 [(CTD_short_d)2-FG])_s SEQ ID NO: 122 (LS2366 [CTD_short_d-Fc]), SEQ ID NO: 124 (LS2367 [CTD_short_e-Fc]), SEQ ID NO: 130 (LS2370 [CTD_short_h-Fc]), SEQ ID NO: 132 (LS2371 [CTD_short_i-Fc]), and SEQ ID NO: 74 (LS2330 [CTD_short_a-Fc]). In an embodiment, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the nucleic acid sequence represented by SEQ ID NO: 164. In an embodiment, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence with at least 90%, preferably at least 95%, 96%, 97%, 98%, or 99%, sequence identity to the nucleic acid sequence represented by SEQ ID NO: 170. In certain embodiments, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 295-304 and SEQ ID NOs: 315-324. In certain embodiments, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 295-304. In certain embodiments, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence with at least 90%, preferably at least 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 355-364.

[0049] In some embodiments, the polynucleotide comprises, consists of or consists essentially of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,

118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,

154, 156, 158, 160, 162, 164, 166, 168, 170, 218, 220, 222, 224, 226, 228, 230, 232, 234,

236, 238, 240, 242, 244, 295-304, 315-324, and 355-364. In some embodiments, the polynucleotide comprises, consists of. or consists essentially of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 164 (LS2401-2404 [(CTD_short_i)2-FcJ), SEQ ID NO: 166 (LS2421 and LS2422 [(CTD_short_i)2-Fc)-mod, 1], SEQ ID NO: 168 (LS2423 [(C.'TD_shon_i)2-Fc)-mod.2], and SEQ ID NO: 170 (LS2435 [(CTD_short_i>2-Fc)- mod. 3], In some embodiments, the polynucleotide comprises, consists of. or consists essentially of, the nucleic acid sequence of SEQ ID NO: 164 (LS 2401-2404 [(CTD_ short_i)2- Fc]). In some embodiments, polynucleotide comprises, consists of, or consists essentially of, an nucleic acid sequence selected from the group consisting of SEQ ID NO: 162 (LS2397- 2400 [(CTD_short_d)2-Fc]), SEQ ID NO; 122 (LS2366 [CTD_short_d-Fc]), SEQ ID NO: 124 (LS2367 [CTD_short_e-Fc]), SEQ ID NO: 130 (LS2370 [CTD_short_h-Fc]), SEQ ID NO: 132 (LS2371 [CTD_sliort_i-Fc]), and SEQ ID NO: 74 (LS2330 [CTD_shoit_a-Fc]). In an embodiment, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence represented by SEQ ID NO: 164. In an embodiment, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence represented by SEQ ID NO: 170. In certain embodiments, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 295-304 and SEQ ID NOs: 315-324. In certain embodiments, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 295-304. In certain embodiments, the polynucleotide comprises, consists of, or consists essentially of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 355-364.

[0050] Nucleic acid or amino acid sequence “identity,” as referenced herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence, Hie percent identity is the number of nucleotides or amino acid residues that are the same (i.e., that are identical) as between the optimally aligned sequence of interest and the reference sequence divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer). Alignment of sequences and calculation of percent identity can be performed using available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, BLASTp, BLASTn, and the like) and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algoritlmis also are disclosed in, for example, Altschul et al., J. Molecular Biol. , 215(3): 403-410 (1990), Beigert et al.. Proc. Natl. Acad. Set. USA, IQ6(lff): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids. Cambridge University Press, Cambridge, UK (2009), Soding, Biainformatics, 21(7): 951-960 (2005), Altschul et al.. Nucleic Acids Res., 25(17): 3389-3402 (1997), aad Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)). Percent (%) identity of sequences can be also calculated, for example, as 100 x [(identical positions)/min(TGA, TGs)], where TGA and TGB are the sum of the number of residues and internal gap positions in peptide sequences A and B in the alignment that minimizes TGA and TGB. See, e.g., Russell et al., J. Mol Biol., 244: 332-350 (1994).

[0051] In some embodiments, the nucleotide sequence of the polynucleotide is codon optimized and/or codon pair optimized.

[0052] Another embodiment is a recombinant vector that comprises, consists of, or consists essentially of, a polynucleotide that encodes the recombinant polypeptide described herein. The recombinant vector can be any suitable vector. Examples of suitable recombinant vectors include but are not limited to a pcDNA3.1, a pSV. a pCMV, a pBApo- CMV, or a pBApo-EFl alpha expression vector.

[0053[ Yet another embodiment is an isolated cell that includes the recombinant polypeptide described herein or a recombinant polynucleotide that contains a nucleic acid sequence that encodes the r ecombinant polypeptide.

[0054] Another embodiment is a pharmaceutical composition that contains the recombinant polypeptide described herein and at least one pharmaceutically acceptable carrier. Hie at least one pharmaceutically acceptable earner can be any suitable carrier. Examples of suitable earners include water and any suitable buffer. Suitable buffers include HEPES-bufifered saline and phosphate-buffered saline. In certain embodiments, the pharmaceutical composition further contains at least one adjuvant. The at least one adjuvant can be any suitable adjuvant. Examples of suitable adjuvants include alum adjuvants, emulsion adjuvants, and pattern recognition receptor agonist adjuvants. Further examples include AS03, MF59, Squalene Emulsion, Alum, aluminum hydroxide gels, calcium phosphate hydroxide, paraffin oil, cytokines (IL-1, IL-2, IL- 12), killed bacterial products such as Bordetella and Mycobacterium bacteria, bacterial toxoids, squalene and DL-n- tocopherol emulsions, squalene-oil-in-water emulsion, aluminum phosphate gels, saponins, cyclic dinucleotides, and TLR agonists, preferably TERI, TLR2, TLR4, TLR5, TLR7, TLR8, TLR9 etc,, and combinations thereof. In certain embodiments, the adjuvant is AS03. In certain other embodiments, the adjuvant is Alum, In yet further embodiments, the pharmaceutical composition does not contain an adjuvant.

[0055] The pharmaceutical composition can contain any therapeutically effective amount of the recombinant polypeptide described herein. A therapeutically effective amount is an amount sufficient to induce an immune response against the target virus or viruses, for instance, SARS-CoV-2, SARS-CoV, and/or MERS-CoV, preferably SARS-CoV-2.

Typically, a dosage is therapeutically effective if it prevents, inhibits, reduces, eliminates, protects against, and/or delays the onset of an infection or an infections clinical condition caused by a beta coronavirus in a subject. Infectious clinical conditions include, for example, fever or chills, cough, shortness of breath or difficult breathing, fatigue, muscle or body aches, headache, loss of taste or smell, sore throat, congestion, ninny nose, nausea, vomiting, and diarrhea. In some embodiments, a single dose of the pharmaceutical composition contains 10 nanograms to 1 milligram, 0.1-250 micrograms, 10-100 micrograms, or 12.5 - 50 micrograms of the recombinant polypeptide. hi some embodiments, a single dose of the pharmaceutical composition contains 0.01-1, 0.1-1, 0.5-5, 1-20, or 5-15, 1-50, or 10-50 micrograms of the recombinant polypeptide. In some embodiments, wherein the pharmaceutical composition does not contain an adjuvant, a single dose of the pharmaceutical composition could contain.01 -1, 0.1-1, 0.5-5, 1-20, or 5-15, 1-50, or 10-50 micrograms of the recombinant polypeptide. However, a single dose of the pharmaceutical composition could also contain increased amounts of the recombinant polypeptide, such as 100-1000, 100-250, or 250-500 micrograms of recombinant polypeptide.

[0056] A further embodiment is a method for preventing, inhibiting, reducing, eliminating, protecting against, or delaying the onset of an infection or an infectious clinical condition caused by a beta corona virus in a subject comprising administering to the subject the recombinant polypeptide described herein, the polypeptide encoded by the recombinant polynucleotide described herein, or a dose of the pharmaceutical composition described herein. Examples of beta coronaviruses include SARS-CoV, MERS-CoV, and SARS-CoV-2. [0057] Another embodiment is a method for inducing an immune response against a beta coronavirus in a subject comprising administering to the subject the recombinant polypeptide described herein, the polypeptide encoded by the recombinant polynucleotide described herein, or a dose of the pharmaceutical composition described herein. Examples of beta corona viruses include SARS-CoV, MERS-CoV, and SARS-CoV-2.

[0058] bi the methods described herein, administration can be achieved by any suitable administration method. Suitable administration methods include oral, topical (including sublingual, buccal, and transdermal), rectal, parenteral, cutaneous, subcutaneous, intravenous, intramuscular, intrapuhnonary, intranasal, intraarterial, intrathecal, and intraperitoneal administration.

[0059] bi the methods described herein, the dosage form may be any suitable dosage form for the intended route of administration. Suitable dosage forms for oral and topical use include tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, pastilles, such as “gummies” including those containing gelatin and/or glycerin, elixirs, and transdermal patches. Suitable dosage forms for parenteral, e.g., subcutaneous, intramuscular, intraarterial, intrathecal, and intraperitoneal administration include aqueous and non-aqueous sterile injection solutions. Suitable dosage forms for intrapulmonary or intranasal administration include aqueous or oil solutions.

[0060] bi the methods described herein, the subject can be any suitable animal that is capable of being infected by a beta coronavirus, such as SARS-CoV, MERS-CoV, and SARS-CoV-2. The subject can be a human, non-hunian primate, horse, pig, cattle, cat, dog, sheep, mink, rodent, hamster, or bat, preferably human.

Examples of NomLimiEng Aspects of /lie Disclosure

|0l)61.J Aspects, including embodiments, of the invention described herein may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered (l)-(36) are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below.

[0062] ( I ) A recombinant polypeptide comprising at least one immunogenic fragment of

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike glycoprotein and optionally an antibody Fc region.

[0063] (2) The recombinant polypeptide of aspect 1, wherein the at least one fragment of the SARS-CoV-2 spike glycoprotein comprises the N-tenninal domain of the SI subunit, the C -terminal domain of the SI subunit, or both.

[0064] (3) The recombinant polypeptide of aspect I or 2, wherein the at least one fragment of the SARS-CoV-2 spike glycoprotein comprises an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1

(CTD long a), SEQ ID NO: 3 (CTD_long_a_D614G). SEQ ID NO: 5 (CTD_long_a-Dimer), SEQ ID NO: 7 (CTD_long_b), SEQ ID NO: 9 (CTD_long_c), SEQ ID NO: 11

(CTD_long_d), SEQ ID NO: 13 (CTD_long_e), SEQ ID NO: 15 (CTDJong i), SEQ ID NO: 17 (CTD_long_g), SEQ ID NO: 19 (CTD_long_h), SEQ ID NO: 21 (CTDJongJ), SEQ ID NO: 23 (CTD_short_a), SEQ ID NO: 25 (CTD_short_b). SEQ ID NO: 27 (CTD_short_c), SEQ ID NO: 29 (CTD_short_d), SEQ ID NO: 31 (CTD_short_e), SEQ ID NO: 33 (CTD_short_f), SEQ ID NO: 35 (CTD_short_g), SEQ ID NO: 37 (CTD_short_h), SEQ ID NO: 39 (CTD_short_i), SEQ ID NO: 41 (CTD_vs_a), SEQ ID NO: 43 (CTD_vs_b), SEQ ID NO: 45 (CTD_vs_c), SEQ ID NO: 47 (CTD_vs_d), SEQ ID NO: 49 (CTD_vs_e). SEQ ID NO: 51 (RBD_a), SEQ ID NO: 53 (RBD_b), SEQ ID NO: 55 (RBD_c). SEQ ID NO: 57 (RBD_d), SEQ ID NO: 59 (RBD_e), SEQ ID NO: 61 (NTD_long_a), SEQ ID NO: 63 (NTI)_short_a), SEQ ID NO: 171 (RBD-tighf), SEQ ID NO: 173 f(RBD-tight)₂), SEQ ID NO: 175 (RBD-extended), SEQ ID NO: 177 ((RBD-extended)2), SEQ ID NO: 179 (RBD), SEQ ID NO: 181 ((RBDfr), SEQ ID NO: 183 ((CTD_short_d)2), SEQ ID NO: 185 ((CTD__shoit i)s), SEQ ID NO: 187 ((CTD shcut J)₂ - mod. 1), SEQ ID NO: 189 ((CTD_short_i)2- mod. 2), SEQ ID NO: 191 ('(OTD_short_i>2 - mod. 3), SEQ ID NOs: 245- 254 (BA.X-1 - BAX-10), SEQ ID NOs: 265-274 (BA. X-l -dimer - BA.X-10-dimer), and SEQ ID NOs: 325-334 (BXC-1 - BXC-iO).

[0065] (4) The recombinant polypeptide of any one of aspects 1-3, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 199, 201, 203, 205, 207, 209, 211, 213, 215, 245-254, 265-274, and 325-334.

[0066] (5) The recombinant polypeptide of any of one aspects 1 -4, wherein the polypeptide comprises at least two immunogenic fragments.

[0067] (6) The recombinant polypeptide of aspect 5, wherein each of the at least two immunogenic fragments comprises an amino acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: I (CTD_long_a), SEQ ID NO: 3 (CTD_long_a_D614G), SEQ ID NO: 5 (CTD_long_a-Dimer), SEQ ID NO: 7

(CTD long b), SEQ ID NO: 9 (CTD_long_c), SEQ ID NO: 11 (CTD_long_d), SEQ ID NO: 13 (CTD_long_e), SEQ ID NO: 15 (CTD_long_f), SEQ ID NO: 17 (CTD_long_g), SEQ ID NO: 19 (CTD_long_h). SEQ ID NO: 21 (CTD_long_i), SEQ ID NO: 23 (CTD_short_a), SEQ ID NO: 25 (CTD_short_b), SEQ ID NO: 27 (CTD_short_c), SEQ ID NO: 29 (CTD_short_d), SEQ ID NO: 31 (CTD_short_e), SEQ ID NO: 33 (CTD_short_f). SEQ ID NO: 35 (CTD_short_g), SEQ ID NO: 37 (CTD_short_h), SEQ ID NO: 39 (CTD_sIiort_i), SEQ ID NO: 41 (CTD_vs_a), SEQ ID NO: 43 (CTD_vs_b). SEQ ID NO: 45 (CTD_vs_c). SEQ ID NO: 47 (CTD_vs_d), SEQ ID NO; 49 (CTD_vs_e), SEQ ID NO; 51 (RBD_a), SEQ ID NO: 53 (RBD_b), SEQ ID NO: 55 (RBD_c), SEQ ID NO: 57 (RBD d), SEQ ID NO: 59 (RBD_e), SEQ ID NO: 61 (NTDJong a), SEQ ID NO: 63 (NTD_short_a), SEQ ID NO: 171 (RBD-tight), SEQ ID NO: 173 ((RBD-tight)₂), SEQ ID NO: 175 (RBD-extended), SEQ ID NO: 177 ((RBD-extended)?), SEQ ID NO: 179 (RBD), SEQIDNO: 181 ((RBD)₂)/SEQ ID NO: 183 ((CTD_sfaort_d)₂), SEQ ID NO: 185 ((CTD_shortJM SEQ ID NO: 187 ((CTD_shortJ)2- mod. 1), SEQ IDNO: 189 ((CTD_short_i}₂- mod. 2), SEQ ID NO: 191 ((CTD_shoit i)₂ - mod. 3), SEQ ID NO: 199 (SARS-2OQ3, SAKS _short_h), SEQ ID NO: 201 (SARS-2003, SARS_short _i), SEQ ^ID NO: 203 (MERS_Lytic_a), SEQ ID NO: 205 (MERS_Lytic_b), SEQ ID NO: 207 (MERS_Lytic_c), SEQ ID NO: 209 (NIERS_Lytic_d), SEQ ID NO: 211 (MERS_Lytic_e), SEQ ID NO: 213 (MERS_Lytic_f), SEQ ID NO: 215 (MERS_Lytic_g), SEQ ID NOs: 245-254 (BA.X-1 - BA.X-10), SEQ ID NOs: 265-274 (BA.X-1-dimer - BA.X-10-dimer), and SEQ ID NOs: 325-334 (BXC-1 - BXC-I0).

[0068] (7) The recombinant polypeptide of aspect 5, wherein each of the at least two immunogenic fragments comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 199, 201, 203, 205, 207, 209, 211, 213, 215, 245-254, 265-274, and 325-334.

[0069] (8) The recombinant polypeptide of any one of aspects 5-7, wherein each immunogenic fragment of the at least two immunogenic fragments comprises the same amino acid sequence.

[0070] (9) The recombinant polypeptide of any one of aspects 5-8, wherein each immunogenic fragment of the at least two immunogenic fragments comprises a different amino acid sequence from the other immunogenic fragments.

[0071] (10) The recombinant polypeptide of any one of aspects 1-9, wherein the polypeptide comprises two, three, ibur, or five immunogenic fragments.

[0072] (11) The recombinant polypeptide of any one of aspects 5-10, wherein the at least two immunogenic fragments are connected to each other via a linker.

[0073] (12) The recombinant polypeptide of aspect 11, wherein the linker is a polypeptide comprising an amino acid sequence of 1-35 residues, wherein each residue is independently serine or glycine.

[0074] (13) The recombinant polypeptide of any one of aspects 1-12, wherein the at least one immunogenic fragment of the SARS-CoV-2 spike glycoprotein is connected to the antibody Fc region via a linker. [0075] (14) The recombinant polypeptide of aspect 13, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65 (Fcl), SEQ ID NO? 67 (Fcl-TEV), SEQ ID NO: 69 (FcI-Rv3C), SEQ ID NO: 193 (Short), SEQ ID NO? 195 (Medium), and SEQ ID NO: 197 (Long).

[0076] (15) The recombinant polypeptide of any one of aspects 1-14, wherein the antibody Fc region is from a human IgG I antibody or derived therefrom.

[0077] (16) The recombinant polypeptide of aspect 15, wherein the antibody Fc region comprises the amino acid sequence of SEQ ID NO: 71.

[0078] ( 17) The recombinant polypeptide of any one of aspects 1 - 16, wherein the polypeptide comprises an amino acid sequence with at least 90%, preferably at least 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 73 (LS2330 [CTD_short_a-Fc]), SEQ ID NO: 75 (LS3472, LS3473,LS3474 [CTD_long_a-Fe]), SEQ ID NO: 77 (LS3477 [CTD_short_a-TEV-Fc]), SEQ ID NO: 79 (LS3485 [CTD_long_a TEV-FcJ), SEQ ID NO: 81 (LS3489

[CTD_short_a_Rv3c-Fc]), SEQ ID NO: 83 (LS3497 [CTD_long_a-Rv3c-Fc], SEQ ID NO: 85 (LS2316, LS2317, LS2318, LS2319 [CTD_long_a-His₈]), SEQ ID NO: 87 (LS3479 [NTD_short_a-TEV-Fc]), SEQ ID NO: 89 (LS3475 [NTD_long_a-TEV-Fc]), SEQ ID NO? 91 (LS3491 [NTD_short_a-Rv3c-Fc]), SEQ ID NO: 93 (LS3487 [NTDJong_a-Rv3C-Fc]), SEQ ID NO: 95 (LS2326 [NTD_long_a-Fc]), SEQ ID NO? 97 (LS2354

[CTD_long_a_D614G-Fc]), SEQ ID NO: 99 (LS2355 [CTD_long_a-Dimer-Fc]), SEQ ID NO.TOl ( LS2356 [CTD_long_b-Fc]), SEQ ID NO: 103 (LS2357 [CTD_long_c-Fc]), SEQ ID NO: 105 (LS2358 [CTD_long_d-Fc]), SEQ ID NO: 107 (LS2359 (CTD_long_e-FcJ), SEQ ID NO: 109 (LS2360 [CTD_long_f-Fc]), SEQ ID NO: 111 (LS2361 [CTD long g- FcJ), SEQ ID NO: 113 (LS2362 [CTDJongJi-Fc]), SEQ ID NO: 115 (LS2363 [CTD_long_i-Fc]), SEQ ID NO: 117 (LS2364 [CTD_short_b-Fc]), SEQ ID NO: 119 (LS2365 [CTD_shoit_c-Fc]), SEQ ID NO: 121 (LS2366 [CTD_short_d-Fc]), SEQ ID NO: 123 (LS2367 [CTD_short_e-Fc]), SEQ ID NO: 125 (LS2368 [CTD_sliort_f-Fc]), SEQ ID NO: 127 (LS2369 [CTD_short_g-Fc]), SEQ ID NO: 129 (LS2370 [CTD_short_h-Fc]), SEQ ID NO: 131 (LS2371 [CTD_short_i-Fc]), SEQ ID NO: 133 (LS2372 [CTD_vs_a-Fc]), SEQ ID NO: 135 (LS2373 [CTD_vs_b-Fc]), SEQ ID NO: 137 (LS2374 [CTD_vs_c-Fc]), SEQ ID NO: 139 (LS2375 [CTD_vs_d-Fc.]), SEQ ID NO: 141 (LS2376 [C'TD_vs_e-Fc]), SEQ ID NO: 143 (LS2377 [RBD_a-Fc]), SEQ ID NO: 145 (LS2378 [RBD b-Fc|), SEQ ID NO: 147 (LS2379 [RBD_c-Fc]), SEQ ID NO: 149 (LS2380 [RBD_d-Fc]), SEQ ID NO: 151 (LS2381 [RBD _e-Fc]), SEQ ID NO: 153 (LS2382 [NTD_short_a-FcJ), SEQ ID NO: 155 (LS2393 [(RBD-tight)2-Fc]), SEQ ID NO: 157 (LS2394 [(RBD-extendedh-Fc]), SEQ ID NO: 159 (LS2395 [(RBD)₂-Fc]), SEQ ID NO: 161 (LS2397-24OO [(CTD_sliort_d)2-Fc]), SEQ ID NO: 163 (LS2401-2404 [(CTD_shoit_i)2-Fc]), SEQ ID NO: 165 (LS2421 and LS2422 [(CTD_short_i)2-Fc)-mod. 1], SEQ ID NO: 167 (LS2423 [(CTD_short_i)2-Fc)-mod. 2], SEQ ID NO: 169 (LS2435 [(CTD_short_i)₂-Fc)-mod. 3], SEQ ID NOs: 255-264, SEQ ID NOs: 275-284, and SEQ ID NOs: 335-344.

[0079] (18) The recombinant polypeptide of aspect 17, wherein the polypeptide coinprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 73,75, 77, 79, 81, 83, 85. 87, 89, 91, 91, 93, 95, 97, 99. 101, 103. 105, 107. 109, 111. 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151. 153, 155. 157, 159. 161, 163. 165, 167. 169, 217. 219, 221. 223, 225. 227, 229. 231, 233, 235, 237, 239, 241, 243, 255-264, 275-284, and SEQ ID NOs: 335-344.

[0080] (19) A recombinant polynucleotide encoding the recombinant polypeptide of any one of aspects 1-18.

[0081] (20) The recombinant polynucleotide of aspect 19, wherein the polynucleotide comprises a nucleic acid sequence with at least 90%, preferably at least 95%, 96%, 97%, 98%, or 99%, sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 74 (LS2330 [CTD_short_a-Fc]), SEQ ID NO: 76 (LS3472, LS3473.LS3474 [CTD_lon^_a-Fc]), SEQ ID NO: 78 (LS3477 [CTD_short_a-TEV-Fc]), SEQ ID NO: 80 (LS3485 [CTD_long_a TEV-Fc]), SEQ ID NO: 82 (LS3489 [CTD_short_a_Rv3c-Fc]). SEQ ID NO: 84 (LS3497 [CTD_Iong_a-Rv3c-Fc], SEQ ID NO: 86 (LS2316, LS2317, LS2318, LS2319 [CTD_long_a-Hiss]), SEQ ID NO: 88 (LS3479 [NTD_short_a-TEV-Fc]), SEQ ID NO: 90 (LS3475 [NTD_long_a-TEV-Fc]), SEQ ID NO: 92 (LS3491 [NTD_short_a-Rv3c- Fc]). SEQ ID NO: 94 (LS3487 [NTD_long_a-Rv3C-Fc]), SEQ ID NO: 96 (LS2326 [NTD_long_a-Fc]), SEQ ID NO: 98 (LS2354 [CTD_Iong_a_D614G-Fc]). SEQ ID NO: 100 (LS2355 [CTD_long_a-Dimer-Fc]), SEQ ID NO: 102 ( LS2356 [CTD_Iong_b-Fc]), SEQ ID NO: 104 (LS2357 [CTD_long_c-Fc])₃ SEQ ID NO: 106 (LS2358 [CTDjong_d-Fe]), SEQ ID NO: 108 (LS2359 [CTD_fong_e-Fc])_? SEQ ID NO: 110 (ES2360 [CTDJoirg. f-Fc])₃ SEQ ID NO: 112 (LS2361 [CTD_longjg-Fc])₃ SEQ ID NO: 114 (LS2362

SEQ ID NO; 116 (LS2363 [CTDJong_i-Fc])_f SEQ ID NO: 118 (ES2364 [CTD_sbort_b- Fc]), SEQ ID NO: 120 (LS2365 [CTD_short_c-Fc]), SEQ ID NO: 122 (ES2366 [CTD_short_d-Fc]), SEQ ID NO: 124 (LS2367 [CTD_short_e-Fc])₃ SEQ ID NO: 126 (LS2368 [CTD_short_f-Fc-])₃ SEQ ID NO: 128 (LS2369 [CTD_short_g-Fc]), SEQ ID NO: 130 (LS237O [CTD_short_h-Fc]). SEQ ID NO: 132 (LS2371 [CTD_short_i-Fc])₃ SEQ ID NO: 134 (ES2372 [CTD_vs_a-Fc]), SEQ ID NO: 136 (LS2373 [CTD_vs_b-Fc]), SEQ ID NO: 138 (ES2374 [CTD_vs_c-Fc]), SEQ ID NO: 140 (LS2375 [CTD_vs_d-Fc])» SEQ ID NO: 142 (LS2376 [CTD_vs_e-Fc]), SEQ ID NO: 144 (LS2377 [RBD_a-Fc]), SEQ ID NO: 146 (LS2378 [RBD_b-Fc])> SEQ ID NO: 148 (LS2379 [RBD_c-Fc]), SEQ ID NO: 150 (LS238O [RBD_d-Fc]), SEQ ID NO: 152 (LS2381 [RBD e-Fc]), SEQ ID NO: 154 (LS2382 [NTD_short_a-Fc]), SEQ ID NO: 156 (LS2393 [(RBD-tight)2-FcJ), SEQ ID NO: 158 (LS2394 [(RBD-exteiided)₂-Fc]), SEQ ID NO: 160 (LS2395 [(RBD)₂-Fc])₃ SEQ ID NO: 162 (LS2397-2400 [(CTD_short_d)2-Fc])_, SEQ ID NO: 164 (LS2401-2404 [(CTD_short_i)2-Fc]), SEQ ID NO: 166 (LS242I and LS2422 [(CTD_short_i)₂-Fc)-mod. 1], SEQ ID NO: 168 (LS2423 [(CTD_short_i)2-Fc)-mod.. 1], and SEQ ID NO: 170 (LS2435 [(CTD_shoit_i)2-Fc)- mod. 3], SEQ ID NOs: 295-304, SEQ ID NOs: 315-324, and SEQ ID NOs: 355-364.

[0082] (21) The recombinant polynucleotide of aspect 20, wherein the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 295-304, 315-324, and 355-364.

[0083] (22) The recombinant polynucleotide of any one of aspects 19-21, wherein the nucleic acid sequence has been codon optimized.

[0084] (23) A pharmaceutical composition comprising the recombinant polypeptide of any one of aspects I- IS or the polypeptide encoded by the recombinant polynucleotide of any one of aspects 19-22, and a pharmaceutically acceptable carrier. |(I085| (24) The pharmaceutical composition of aspect 23, wherein the pharmaceutical composition further comprises at least one adjuvant

[0086] (25) The pharmaceutical composition of aspect 24, wherein the at least one adjuvant is selected from the group consisting of alum adjuvants, emulsion adjuvants, and pattern recognition receptor agonist adjuvants.

[0087] (26) The pharmaceutical composition of aspect 25. wherein the at least one adjuvant is MF 59, Squalene Emulsion, Alum, aluminum hydroxide gels, calcium phosphate hydroxide, paraffin oil, cytokines (IL-1, IL-2, IL- 12), killed bacterial products such as Bordetella and Mycobacterium bacteria, bacterial toxoids, squalene and DL-o-tocopherol emulsions, aluminum phosphate gels, saponins, cyclic dinucleotides, and TLR agonists, preferably TLR1, TLR2, TLR.4, TLR5, TLR7, TLR8, TLR9 etc,, and combinations thereof [0088] (27) The pharmaceutical composition of aspect 24, wherein the at least one adjuvant is a squalene-oil-in-waier emulsion adjuvant.

[0089] (28) A vector comprising the recombinant polynucleotide of any one of aspects

19-22.

|009(lj (29) An isolated cell comprising the recombinant polypeptide of any one of aspects 1-18, or the polypeptide encoded by the recombinant polynucleotide of any one of aspects 19-22.

[0091] (30) A method for preventing, inhibiting, reducing, eliminating, protecting, or delaying the onset of an infection or an infections clinical condition caused by a beta coronavirus in a subject comprising administering to the subject the recombinant polypeptide of any one of aspects 1-18, the polypeptide encoded by the recombinant polynucleotide of any one of aspects 19-22, or the pharmaceutical composition of any one of aspects 23-27.

[0092] (31) A method for inducing an immune response against a beta coronavirus in a subject comprising administering to the subject the recombinant polypeptide of any one of aspects 1-18, the polypeptide encoded by the recombinant polynucleotide of any one of aspects 19-22, or the pharmaceutical composition of any one of aspects 23-27.

[0093] (32) The method of aspect 30 or 3 L wherein the recombinant polypeptide of any one of aspects 1-18, the polypeptide encoded by the recombinant polynucleotide of any one of aspects 19-22, or the pharmaceutical composition of any one of aspects 23-27 is administered by oral, topical, rectal, parenteral, cutaneous, subcutaneous, intravenous, intramuscular, mtranasal^ intrapulmonary, intra arterial, intrathecal, or mteiperitoiieal administration.

[0094] (3.3) The method of any one ofaspects 30-32, wherein the recombinant polypeptide of any one of aspects 1-18, the polypeptide encoded by the recombinant polynucleotide of any one of aspects 19-22, or the pharmaceutical composition of any one of aspects 23-27 is administered in a dosage form, wherein the dosage form is a tablet, troche, lozenge, aqueous or oil suspension, dispersible powder or granules, emulsion, hard or soft capsule, syrup, pastille, elixir, transdermal patch, aqueous sterile injection solution, aqueous solution, or oil solution.

[0095] (34) The method of any one of aspects 30-33, wherein the coronavirus is selected from the group consisting of SARS-CoV-2, SARS-CoV, and MERS-CoV.

[0(!96] (3.5) The method of any one of aspects 30-34, wherein the subject is a mammal, preferably a human or mm-human primate.

[0097] (36) The use of the recombinant polypeptide of any one of aspects 1-18, the polypeptide encoded by the recombinant polynucleotide of any one of aspects 19-22, or the pharmaceutical composition of any one of aspects 23-27 for the preparation of a medicament for the treatment or prevention of illness caused by SARS-CoV-2.

[0098] (37) The recombinant polypeptide of any one of aspects 1-18, the polypeptide encoded by the recombinant .polynucleotide of any one of aspects 19-22, or the pharmaceutical composition of any one. of aspects 23-27 for use as a medicament.

EXAMPLES

[0099] The following descriptions of cloning and protein expression apply to each example.

[0100] Cloning : Inserts encoding a certain fragment of SARS CoV-2 vims Spike protein

SI were designed based prediction and previous Lytic Solutions expression data. Codon optimized cDNA was synthesized. The insert was cloned using either (a) restriction digests and DNA ligations or (b) NEB HiFi DNA assembly builder mix into a pcDNA3.1 vector containing appropriate secretion signal sequences, linkers, and tags for secreted fusion protein expression, as well as sequence encoding a human IgGl Fc region. The resulting clone encoding a recombinant SARS CoV-2 Spike protein fragment - Fc region fusion protein (“recombinant CoV-2 fusion protein”) was verified by either colony PCR and/or restriction digests, DNA sequencing was also used.

[0101] Protein Expression : Supercoiled plasmid of the verified clone was transiently transfected into CHO-S cells and expressed under the control of a constitutive promoter within cell culture conditions ranging from 30-37°C and 3-10 days in CO? (8%) incubators with rotary shaking agitation at speeds of 150RPM. Cells were removed by centrifugation and culture medium containing the recombinant CoV-2 fusion protein were passed over Protein A agarose to bind the Fc region-containing CoV-2 protein. Filtration could also have been used to remove cells. The column containing bound recombinant CoV-2 fusion protein was washed with phosphate buffered saline. The recombinant CoV-2 fusion protein bound to the Protein A column was eluted with low pH glycine followed by neutralization in pH8.0 Tris. The recombinant CoV-2 fusion protein was dialyzed to remove glycine/tris and placed into HEPES-buffered saline (10 mM HEPES. 150 mM NaCl, pH adjusted with NaOH to pH 7.2-7.5). No additional purification was employed in this case. However, additional purification by any chromatography method such as HIC or ion exchange can optionally be used.

[0102] Example !:

[0103] SARS CoV-2 antigens were selected from the genomic sequence (ncbi.nlm.nih.gov/nuccorez'MN908947) to use in generating antibody and T-cell responses to the receptor binding domains of SARS CoV-2 virus Spike protein. The selected domain encoding regions were codon optimized for CHO cell expression using IDTDNA codon optimization algorithms. Template DNA was synthesized at Twist Bio. A modified pcDNA3.1 vector was used for protein expression that included human IgGl Fc for translational fusion generation, i.e., a fusion protein containing a SARS CoV-2 virus Spike protein fragment and an antibody Fc region. The vector encoding the fusion protein was expressed in CHO cells, in which the fusion protein was secreted to the media, cells and cell debris were removed by centrifugation, the recombinant protein was captured with Protein A resin, and eluted with low pH glycine buffer. The resulting fusion protein was buffer exchanged by dialysis and mixed with adjuvant. The resulting vaccine composition was injected mtxamuscularly into Cynomolgus monkeys. The vaccine composition generated an unexpectedly strong immune response in Cynomolgus monkeys.

[0104] Vaccination: Following protein, expression in accordance with the description above, the purified recombinant CoV-2 fusion protein (LS2330, CTD_short-a-Fc; SEQ ID NO: 73) was mixed with Titermax Gold adjuvant, a modified squalene in water emulsion adjuvant, according to manufacturer directions, and injected intramuscularly into the thigh of Cynomolgus monkeys. Injections were performed at day 0 and day 14. Dosages were 250ug of antigen of CTD_short-a-Fc (Seq ID NO:73). Prior to the first injection on day 0, a baseline sample was collected from each test subject.

[0105] The immune response was monitored every two weeks following the injection on day 0, with the earliest sample taken on day 14. Accordingly, samples were collected on day 14 and day 28. The samples were analyzed, and the results are shown hi Figure 1. Venous blood was obtained from Cynomolgus monkeys pre immunization in EDTA-containmg vacutubes (value shown as Day 0 serum sample hi FIG 1). Immediately after the preimmunization blood sample was taken, monkeys were immunized with CTD_short-a-Fc (Seq ID NO: 73). Serum was isolated by centrifugation of the non-coagulated blood. Serum was diluted 1:100 and analyzed on Intuitive Biosciences ELISA platform for anti-Sl spike binding antibodies. Additional time points were taken at 2 weeks post Day 0, 4 weeks post Day 0, and 6 weeks post Day 0. The single immune boost (CTD_short-a-Fc (Seq ID NO:73)) was performed immediately after the 2 week blood sample was taken. Samples were processed similar to Day 0 samples and immune response was recorded as Relative Intensity Units (RIU) on Intuitive Bioscience ELISA platform. Capture of anti-SARS CoV-2 antibodies was performed by spotting SARS CoV-2 S I spike protein (Sino Biologicals) onto the wells of Intuitive Bioscience ELISA platform 96 well plates, adding diluted serum — full concentration serum and dilutions to 1:200 serum:buffer were used, but higher concentrations of serum surprisingly resulted in a signal too strong to read on the platform, thus requiring a 1:200 fold dilution for the leadings shown in FIG 1 — and incubating for 10-120 minutes to allow binding of anti-CTD antibodies to the SI spike protein attached to the plate wells. Serum was washed 3X form the wells, and followed by application of anti-cynomolgus detection antibody to the well incubated for 10-1'20 minutes, then washed 3X from the well. Detection reagents were added and the signal was quantified compared to control spots of unrelated proteins .

[0106] Figure 1 depicts an unexpectedly strong immune response in Cyiioaiolgus monkeys to the tested construct, particularly in comparison to the immune response generated by other known SARS-CoV-2 constructs. See, for instance, Graham et al., “Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOxl nCoV-19,” bioRxiv preprint doi: doi.org/10.1101/2020.06.20.159715 (posted June 20, 2020)

[0107] Neutralization Assays: Figures 5A-C depict the results of neutralization assays done on the tested Cynomolgus monkeys P0101 and P0102 (respectively depicted as “Animal 101” and “Animal 102” in Figure 1). Regarding Figures 5A and 5B, “wpi” in the legend denotes weeks post injection/inmnmization. SARS-CoV-2 pseudotyped particles were generated as previously described (see Schmidt, F., et al. Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses. J Exp Med, v. 217, n. I l , 11 2020). Briefly, 293T cells were transfected with pHIV-INLGagPol, pCCNG/nLuc and pSARS-CoV-2-SA19. Particles were harvested 48 hours after transfection, filtered and stored at -80°C. Fourfold serially diluted serum from the immunized monkeys was incubated with SARS-CoV-2 pseudotyped virus for 1 h at 37 °C. The mixture was subsequently incubated with 293T/ACE2cl.22 cells (plated on Poly-D-Lysine-coated 96-well plates) with the final stalling dilution of serum being 1:50. At 48 h later the cells were washed with PBS and lysed with Luciferase Cell Culture Lysis 5x reagent (Promega). Nanoluc Luciferase activity in lysates v/as measured using the Nano-Gio Luciferase Assay System (Promega) with the Modulus II Microplate Reader (Turner BioSystems). The raw nanoluc luciferase activity values (relative luminescence units) were normalized to those derived from cells infected with SARS-CoV-2 pseudotyped vims in the absence of serum or a rabbit monoclonal antibody diluted in normal human serum at 0.105 mg/mL (40592-R001, Sinobiologic-al, Wayne, PA). The half-maximal inhibitory concentration for serum (NT50) was determined using four-parameter nonlinear regression (GraphPad Prism). [0108 J Immunization of the two Cynomolgus macaques (IDs: P0101 and P0102) with

SEQ ID NO: 73 (ES233O [CTD_ _short_a-Fc] produced robust neutralizing antibody response. Neutralization assays were performed using a replication-defective single-cycle pseudotyped virus carrying SARS-CoV-2 spikes and the NanoLuc luciferase reporter. This assay has been previously shown to accurately predict serum neutralizing activity against authentic SARS- CoV-2 (see Schmidt, F., et al. referenced above). As a control for neutralization sensitivity, human serum obtained from a SARS-CoV -2 negative individual was used alone or spiked with a monoclonal neutralizing antibody (Figure 5C). Serum samples collected at the various timepoints from 0 to 20 weeks post-immunization were evaluated for neutralizing activity. Sera from animals immunized with SEQ ID NO: 73 (LS2330 [CTD_short_a-Fc] had readily detectable neutralization activity, as early as 2 weeks post-immunization that significantly increased until weeks 4 to 8 of the study. Indeed, neutralizing titers were exceptionally high at 4-8 weeks after immunization, in the range of 10,000 to 100,000 and were maintained in the 1000 to 10,000 range at 20 weeks after immunization.

[0109] Example 2:

[0110] Overview: SARS CoV-2 antigens were selected from the SARS CoV -2 genomic sequence (acbijrim.aih.gov/nuccore7vfN908947) to use in generating antibody and T-cell responses to the receptor binding domains of SARS CoV-2 virus Spike protein . Preliminary data identified select individual regions of the SARS CoV-2 virus Spike protein that would be amenable to high-level expression as Fc-fiisions, that are resistant to proteolysis when expressed in CHO cells, and that generate strong immune response in Cynomolgus macaques. We further determined that in-series multimerization of select SARS CoV -2 virus Spike protein domains can be used to create proteins that retain high-level expression without significant proteolysis sensitivity whilst doubling the theoretical antigenicity of the molecule to be used for immune stimulation. Hie design of the multiple domain molecules provides a scaffold for straight-forward modification to incorporate amino acid mutations identified in new and emerging variants of the SARS CoV-2 vims Spike protein. The resulting vaccine composition of the Wuhan variant was injected intramuscularly into Rhesus macaques and elicit stronger immune responses using simple adjuvants at doses at mere fractions of that needed with single domain Fc-fiisions. [0111] Following protein expression in accordance with the description above, resulting protein was analyzed by reducing SDS-PAGE to determine protein integrity for monomeric CTD-Fes (Figures 2A-D), in-series dimer CTD-Fcs (Figure 3) and in-series dimer mutant CTD-Fcs (Figure 4). Protein yields were calculated using absorbance of 280 nm light and protein-specific extinction coefficients determined in silico-according to their expected amino acid composition. Representative protein yields are given in Table 1.

[0112] TABLE 1

[0113] Based on the desire to develop methods to increase the relative antigen content of a protein molecule, in-series concatemers of RBD-containiiig protein fragments trans la tionally fused to an Fc. CTD_short_d and CTD_short_i ('SEQ ID NOs: 29 and 39, respectively) were prepared and selected for in-series expression given their high-level, protease resistant expression as monomers. When conjoined with a serine-glycine linker and expressed in CHO cells, the resulting double-domain proteins expressed at similar levels as the monomer constructs. In addition, the in-series design retained the molecular resistance to proteolysis during expression and purification. Therefore, by combining intramolecular dimerization driven by Fc interactions with in-series domain expression, it was possible to go from a single SARS-CoV-2 antigen fragment per molecule to having 4 (and potentially more) per molecule. The higher antigen content in conjunction with robust expression levels and protein stability⁷ provides a suitable framework for an immunogen to be used for vaccine and boost applications.

[0114] As an example of this approach, double-domain Fc-fusion constructs (i.e., recombinant polypeptides containing two immunogenic fragments connected to an antibody Fc region) containing amino acid mutations within one or both of the domains were prepared. The mutations used were either from a single virus variant or a hybrid of two vims variants. Incorporation of the mutations into the double-domain-Fc wild-type molecule (strain 2401) depicted in Figure 3 followed by expression hi CHO cells resulted in intact, soluble protein with similar yields to the wild-type amino acid sequence. These results support the concept that this multi-domain Fc-fiision platform provides a robust scaffold for the incorporation and expression molecules that reflect new variant mutations. This provides a robust, timely and cost-effective system to adapt vaccine composition to meet the needs to mitigate evolving variants, A vaccine containing strain 2401 was subsequently tested in Rhesus macque monkeys.

[0115] Vaccination: Vaccination and boosters to nCoV-2 double dimer (Lytic Solutions Strain #2401, SEQ ID NO: 163, administered to all animals except for those indicated to be administered the nCoV-2 quadruple mutant vaccine) or nCoV -2 quadrapie mutant vaccine (Lytic Solutions Strain #2435, SEQ ID NO: 169, referred to in Table 2C as “Variant COVID Vaccine” in Adjuvant & Dose column) were performed on Rhesus macaque monkeys by the following methodology. 50, 25, or 12.5 micrograms (these numbers are used to define dosage) of nCoV-2 protein was mixed with either AS03 (Invivogen catalog vac-as03-10) or alum (Invivogen catalog vac-alu-250) as a 1 : 1 volume mixture protein: adjuvant. For 50ug doses 500ul of each protein and adjuvant were used, for 25ug and 12.5 ug 250ul of each protein and adjuvant were used. Dosages were split and injected intramuscularly into left and right thighs at the time of initial vaccination and at booster vaccination. Initial vaccination day was designated day 0. Boosters were given 28 day post day 0 unless otherwise noted. Serum samples were taken prior to initial vaccination on day 0, day 14, day 28 prior to booster vaccination, and day 42 and day 56 were 2/4 weeks post booster injection. Therefore data from 14 and 28 (immunization plus 2 weeks and immunization plus 4 weeks, respectively) days are specific to a single dose of vaccine whereas samples from day 42 and day 56 are 2 doses of vaccine (booster plus 2 weeks, and booster plus 4 weeks respectively). Each animal received the same dosage of protein and adjuvant for the booster dose that they received in the primary dose.

[0116] Analysis of antibody titers were performed at Intuitive Biosciences, Madison WI on their proprietary ELISA system (918 Deming Way, Suite 100, Madison WI 53719 USA). Serum was serially diluted in CSA buffer (Intuitive product no. 7-1037) to dilutions of 1 TOO, 1:1000, 1:10,000, 1:100,000, and 1:1,000,000. Analysis was performed at and by Intuitive Biosciences. ELISA units are measured as density on their platform with a maximum signal of approximately 45,000-50,000 counts. Titer signals were determined from dilutions that yielded signals less than l/3rd maximal signal. Fifteen animals were used to determine vaccine/booster efficacy. Animal names are codes generated for each animal at the primate facility at UW-Madison. All animals were assayed through day 56 post initial vaccination. 50, 25, and 12.5 ug doses gave similar titers for day 28. Some animals were followed past the study design point of 56 days to various time points up to 23 weeks post initial vaccine (due to continued potency of vaccine response). Data is summarized in Tables 2 A, 2B, and 2C. It is noted that, although some of the Plate Sample ID numbers are overlapping, e.g.. Tables 2 A and 2B both have a row with a plate sample ID of 59, this is merely an artifact of the data collection process, such that rows with the same Plate Sample ID represent independently collected data points.

[0117] 50ug, 25ug, 12.5ug: These data show that relatively low dosages of the tested constructs, including 12.5 ug. still elicited sufficient immune stimulation and/or boosting. Such low amounts of protein per vaccine dose allow for an increased number of active doses to be produced per liter of cell culture. As production levels increase, not only do cost of goods go down, but the timeframe to produce large numbers of doses can be decreased in comparison to vaccine compositions requiring higher amounts of protein per dose to provide sufficient immune protection. This is particularly relevant to providing immune protection against any newly-arising SARS-CoV-2 strains.

[0118] The assembly of four point-mutations into a single RBD polypeptide was undertaken prior to public disclosure of the Delta mutant isolated first in India. Vaccine LS2435 (SEQ ID NO: 169) contains mutations in four sites that reflect mutation that evolved from new variants identified from the UK. South Africa, Brazil and southern California.

Convergent evolution of mutations in new variants lead this to be an attractive approach of stacking multiple mutations in one construct to represent multiple variants, such as seen in the Delta variant. Each RBD point mutation was determined to add virulency through either immune system avoidance or enhanced viral entry or production of higher viral loads (or a combination thereof). The identified mutations were combined to develop a vaccine that could address each mutation and immune epitope singly or in combination. When evolutionary boundaries are considered, it became clear that mutational stacking was likely to take place through either recombination of previous viral strains, or additional mutations stacked onto previous viral strains that enhanced vinilence/transimssion. Considering the mutation-stacked SARS CoV-2 strains can evade the immune response as well as generate a more potent viral titer, having a vaccine that displays high levels of antibody and T-cell potency is a major advantage over previous COVID- 19 vaccines which can provide less potent immune responses to these mutants. The SARS CoV-2 mutant-containing vaccine L S2435 demonstrates highly potent immune stimulation and antibody production in Rhesus macaques, hi fact, the levels of antibodies achieved with the mutant vaccine are similar to responses seen from the tested wild-type vaccine, i.e,, LS2401. [0119] Table 2A

[0122] Example 3:

[0123] Further constructs containing certain SARS-CoV-2 immunogenic fragments were prepared and expressed in accordance with the methods disclosed herein. See, e.g., the first three paragraphs of the Example section. The protein expression of the constructs was subsequently measured after four days of expression. See Table 3; FIG. 6. All tested constructs expressed at a concentration of at least about 50 mg protein L culture.

|0124] Table 3 : Protein yields for Protein A affinity-purified BA.X-monomer-Fc proteins after 4-days of expression.

[0126] Table 4 (above) depicts the amino acid substitutions, and corresponding positions thereof, within immunogenic fragments BA.X-1 - BA.X-10 (SEQ ID NOs: 245-254) in comparison to the Wuhan wild-type variant. Table 5 (below) depicts the amino acid substitutions, arid corresponding positions thereof, within the listed natural SARS-CoV-2 variants. As revealed by comparison of Tables 4 and 5, the BA.X-1 - BA.X-10 immunogenic fragments each contain a combination of mutations not present in any shown natural SARS-CoV-2 strain. The exemplary particular combination of mutations selected for each BA.X-1 - BA.X-10 immunogenic fragment provides broad antigenic coverage over existing SAR.S-CoV-2 Spike protein variants. Without being bound to any theory, it is expected that the broad antigenic coverage should also provide full or partial protection against later-arising SARS-CoV -2 Spike protein variants as well. The constructs containing the BA.X-I - BA.X-10 immunogenic fragments are capable of being expressed at levels suitable for commercial vaccine production. See Table 3, FIG. 6.

[0127] Table 5

[0128] Example 4

[0129] Further synthetic constructs containing certain SARS-CoV -2-based immunogenic fragments were prepared and expressed in accordance with the methods disclosed herein.

S ee, e.g., the first three paragraphs of the Example section. The protein expression of the constructs was subsequently measured after six days of expression. See FIG. 7; Table 6. All tested constructs expressed at a concentration of at least about 90 nig protein/L culture.

Accordingly, these constructs are all capable of being expressed at levels suitable for commercial vaccine production.

[0130] Table 6: Protein yields for Protein A affinity-purified BXC-monoiner-Fc proteins after 6-days of expression.

[0131] Table 7 below depicts the amino acid substitutions, and corresponding positions thereof, within immunogenic fragments BXC-l-BXC-10 (SEQ ID NOs: 325-334) in comparison to the Wuhan wild-type variant. Table 5 (above) depicts the amino acid substitutions, and corresponding positions thereof within certain listed natural SARS-CoV -2 variants. As revealed by comparison of Tables 7 and 5, the BXC-l-BXC-10 immunogenic fragments each contain a combination of mutations not present in any shown natural SARS- CoV-2 strain. Table 7: Mutations within BXC Monomer (with corresponding SEQ ID NO) Comparison to Wuhan wild-type variant

[0132] Table 8; Selected Sequences

[0133] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplar,⁷ and not restrictive. It will be understood that changes and modifications may be made by those of ordinary skill within the scope of the following claims. In particular, the present invention covers further embodiments with any combination of features from different embodiments described above and below. Additionally, statements made herein characterizing the invention refer to an embodiment of the invention and not necessarily all embodiments.

[0134] The terms used in the claims should be construed to have the broadest reasonable interpretation consistent with the foregoing description. For example, the use of the article “a” or “the” in introducing an element should not be interpreted as being exclusive of a plurality of elements. Likewise, the recitation of' “or” should be interpreted as being inclusive, such that the recitation of “A or B” is not exclusive of “A and B,” unless it is clear from the context or the foregoing description that only one of A and B is intended. Further, the recitation of “at least one of A, B and C” should be interpreted as one or more of a group of elements consisting of A. B and C, and should not be interpreted as requiring at least one of each of the listed elements A, B and C, regardless of whether A, B and C are related as categories or otherwise. 'Moreover, the recitation of “A, B and/or C” or “at least one of A, B or C” should be interpreted as including any singular entity from the listed elements, e.g., A, any subset from the listed elements, e.g. , A and B, or the entire list of elements A, B and C.

Claims

Crisis

1 A recombinant polypeptide comprising at least one immunogenic fragment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike glycoprotein and optionally an antibody Fc region.

2. The recombinant polypeptide of claim 1. wherein the at least one fragment of the SARS- CoV-2 spike glycoprotein comprises the N-terminal domain of the SI subunit, the C- terminal domain of the S 1 subunit, or both.

3. The recombinant polypeptide of claim 1 or 2, wherein the at least one fragment of the SARS-CoV-2 spike glycoprotein comprises an amino acid sequence with at least 90%. preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 245-254 (BA.X-1 - BA.X-10), SEQ ID NOs: 265-274 (BA.X-l-dimer - BA.X-10- dimer), and SEQ ID NOs: 325-334 (BXC-1 - BXC-10).

4. The recombinant polypeptide of any one of claims 1-3, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 245-254, 265-274, and 325-334.

5. The recombinant polypeptide of any of one claims 1-4, wherein the polypeptide comprises at least two immunogenic fragments.

6. The recombinant polypeptide of claim 5, wherein each of the at least two immunogenic fragments comprises an amnio acid sequence with at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 245-254 (BA.X-1 - BA.X- 10), SEQ ID NOs: 265-274 (BA.X-l-dimer - BA.X-10-dimer), and SEQ ID NOs: 325- 334 (BXC-1 - BXC-10).

7. The recombinant polypeptide of claim 5, wherein each of the at least two immunogenic fragments comprises an ammo acid sequence selected from the group consisting of SEQ ID NOS: 245-254, 265-274, and 325-334.

8. The recombinant polypeptide of any one of claims 5-7, wherein each immunogenic fragment of the at least two immunogenic fragments comprises the same amino acid sequence.

9. The recombinant polypeptide of any one of claims 5-8, wherein each immunogenic fragment of the at least two immunogenic fragments comprises a. different amino acid sequence from the other immunogenic fragments.

10 , The recombinant polypeptide of any one of claims 1-9, wherein the polypeptide comprises two, three, four, or five immunogenic fragments.

11. The recombinant polypeptide of any one of claims 5- 10, wherein the at least two immunogenic fragments are connected to each other via a linker.

12. The recombinant polypeptide of claim 11, wherein the linker is a polypeptide comprising an amino acid sequence of 1-35 residues, wherein each residue is independently serine or glycine.

13. The recombinant polypeptide of any one of claims 1-12, wherein the at least one immunogenic fragment of the SARS-CoV-2 spike glycoprotein is connected to the antibody Fc region via a linker.

14. The recombinant polypeptide of claim 13, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65 (Fcl), SEQ ID NO: 67 (Fcl-TEV), SEQ ID NO: 69 (Fcl-Rv3C), SEQ ID NO: 193 (Short), SEQ ID NO: 195 (Medium), and SEQ ID NO: 197 (Long).

15. The recombinant polypeptide of any one of claims 1-14, wherein the antibody Fc region is from a human IgGl antibody or derived therefrom.

16. The recombinant polypeptide of claim 15, wherein the antibody Fc region comprises the amino acid sequence of SEQ ID NO: 71.

17. The recombinant polypeptide of any one of claims 1-16, wherein the polypeptide comprises an amino acid sequence with at least 90%, preferably at least 95%, 96%, 97%, 98%, or 99%, sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 255-264, SEQ ID NOs: 275-284, and SEQ ID NOs: 335-344.

18. The recombinant polypeptide of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 255-264, 275-284, and 335-344.

19. A recombinant polynucleotide encoding the recombinant polypeptide of any one of claims 1-18.

20. The recombinant polynucleotide of claim 19, wherein the polynucleotide comprises a nucleic acid sequence with at least 90%, preferably at least 95%, 96%, 97%. 98%, or 99%, sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOsr 295-304, 315-324, and 355-364,

21. The recombinant polynucleotide of claim 20, wherein the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 295-304, 315- 324, and 355-364.

22. The recombinant polynucleotide of any one of claims 19-21, wherein the nucleic acid sequence has been codon optimized.

23. A phannaceutical composition comprising the recombinant polypeptide of any one of claims 1-18 or the polypeptide encoded by the recombinant polynucleotide of any one of claims 19-22, and a pharmaceutically acceptable carrier.

24. The pharmaceutical composition of claim 23, wherein the phannaceutical composition further comprises at least one adjuvant.

25. The phannaceutical composition of claim 24, wherein the at. least, one adjuvant is selected from the group consisting of alum adjuvants, emulsion adjuvants, and pattern recognition receptor agonist adjuvants.

26. The pharmaceutical composition of claim 25, wherein the at least one adjuvant is MF'59, Squalene Emulsion, Alum, aluminum hydroxide gels, calcium phosphate hydroxide, paraffin oil, cytokines (IL-1, IL-2, IL- 12), killed bacterial products such as Bordetella and Mycobacterium bacteria, bacterial toxoids, squalene and DL-a-tocopherol emulsions, aluminum phosphate gels, saponins, cyclic diimcleotides, and TLR agonists, preferably TLR1, TLR2, TLR4, TLR5, TLR7, TLR8, TLR9 etc., and combinations thereof.

27. The phannaceutical composition of claim 24, wherein the at. least, one adjuvant is a squalene-oil-in-water emulsion adjuvant.

28. A vector comprising the recombinant polynucleotide of any one of clahns 19-22.

29. An isolated cell comprising the recombinant polypeptide of any one of claims 1-18, or the polypeptide encoded by the recombinant polynucleotide of any one of clahns 19-22,

30. A method for preventing, inhibiting, reducing, eliminating, protecting, or delaying the onset of an infection or an infectious clinical condition caused by a beta coronavirus in a subject comprising administering to the subject the recombinant polypeptide of any one of claims 1-18. die polypeptide encoded by the recombinant polynucleotide of any one of claims 19-22, or the pharmaceutical composition of any one of claims 23-27.

31. A method for inducing an immune response against a beta coronavirus in a subject comprising administering to the subject the recombinant polypeptide of any one of claims 1-18, the polypeptide encoded by the recombinant polynucleotide of any one of claims 19-22, or the pharmaceutical composition of any one of claims 23-27.

32. The method of claim 30 or 31, wherein the recombinant polypeptide of any one of clamis 1-18, the polypeptide encoded by the recombinant polynucleotide of any one of claims 19-22, or the pharmaceutical composition of any one of claims 23-27 is administered by oral, topical, rectal, parenteral, subcutaneous, intravenous, intramuscular, intranasal, intrapulmonaiy, intraarterial, intrathecal, or interperitoneal administration.

33. The method of any one of aspects 30-32, wherein the recombinant polypeptide of any one of aspects 1-18 , the polypeptide encoded by the recombinant polynucleotide of any one of aspects 19-22, or the phannaceutical composition of any one of aspects 23-27 is administered in a dosage form, wherein the dosage form is a tablet, troche, lozenge, aqueous or oil suspension, dispersible powder or granules, emulsion, hard or soft capsule, syrup, pastille, elixir, transdeiinal patch, aqueous sterile injection solution, aqueous solution, or oil solution.

34. The method of any one of claims 30-33, wherein the coronavirus is selected from the group consisting of SARS-CoV-2, SARS-CoV, and MERS-CoV.

35. The method of any one of claims 30-34, wherein die subject is a mammal, preferably a human or non-human primate.

36. The use of the recombinant polypeptide of any one of claims 1-18, the polypeptide encoded by the recombinant polynucleotide of any one of claims 19-22, or the phannaceutical composition of any one of claims 23-27 for the preparation of a medicament for the treatment or prevention of illness caused by SARS-CoV-2.

37. The recombinant polypeptide of any one of claims 1-18, tire polypeptide encoded by the recombinant polynucleotide of any one of claims 19-22, or the phannaceutical composition of any one of claims 23-27 for use as a medicament .