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  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
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  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present disclosure relates to the field of vaccines, as well as preparations and methods of their use in the treatment and/or prevention of disease. Described are vaccines, pharmaceutical compositions containing the same (also referred to herein as immunogenic compositions), and uses thereof for treating or preventing coronavirus infections and related viral infections, such as those caused by sarbecoviruses and merbecoviruses.
• FIG. 3 shows select “beads on a string” formats in graphical representation, with examples provided for (A) RR-FN, (B) RN-FN, (C) RRN-FN, (D) RNRN-FN, (E) R-FN, (F) RR-FN, and (G) RR-FN.
• FIG. 23 shows pseudoneutralization titers elicited against Coronavirus Clade lb strains (A) WA-1, (B) Delta , (C) Beta, (D) BA5, (E) BQ.1.1, or (F) XBB.1.5 in sera from mice immunized with monovalent SpFN or SpFN antigenic distance mixes A-E (Groups 18-29). Titers from each group of mice and readout are represented as a box and whisker plot showing the mean (horizontal line), first standard deviation (box), and second standard deviation (vertical line). Any individual titers greater than 2 standard deviations away from the mean (i.e., outliers) are represented as circles. Further details of each group can be found in Table 8.
• FIG. 25 shows geometric mean pseudoneutralization titers elicited against Coronavirus strains WA-1, Delta, Beta, BA5, BQ.1.1, XBB.1.5, SARS1, or MERS in sera from mice immunized with Spike antigens presented as either monovalent or multivalent stabilized transmembrane (S-2P, monovalent groups 15-17, 37-40; multivalent groups 31, 41-44) and monovalent or multivalent spike conjugated to ferritin (SpFN, monovalent groups 18-24; multivalent groups 25-29).
• S-2P monovalent groups 15-17, 37-40; multivalent groups 31, 41-44
• SpFN monovalent or multivalent spike conjugated to ferritin
• FIG. 26 shows pseudoneutralization titers elicited against Coronavirus strains (A) WA-1, (B) Delta , (C) Beta, (D) SARS-1 in sera from mice immunized with Mix C SpFN (Group #), Mix D RFN (Group #), and Mix D RFN with empty ferritin (RFN+FN, Group #).
• the mRNAs were either administered as a co-administration of an admixture of LNPs separately encapsulating mRNA molecules encoding each construct (admin) or LNPs coencapsulating the mRNA molecules (encap).
• FIG. 27 shows pseudoneutralization titers elicited against Coronavirus strain pseudoviruses
• FIG. 28 shows that multivalent particles can be produced by either co-encapsulating multiple mRNAs, each expressing a different fusion protein (illustrated as RFNs of different strains), into a single LNP (as shown in the top panel), or by administering multiple LNPs, each encapsulating a different mRNA.
• FIG. 29 shows various mixes of strains used in experiments (Mixes A-E) and the relative antigenic distance of the strains, and schematic diagrams of resulting nanoparticles.
• FIG. 30 shows various examples of multivalent particles.
• the illustrated examples include a particle comprising two different RFN fusion proteins (A) and four different RFN fusion proteins
• the illustrated examples also include a multivalent particle that comprises a single RRFN fusion protein, in which each RBD (i.e., each “R” in the “RRFN”) is from a different strain (C), and a particle comprising two RRFN fusion proteins, wherein the order of the RBD domains is switched between the two different RRFN fusion proteins.
• RBD i.e., each “R” in the “RRFN”
• C different strain
• Other RRFN particles comprising RRFN fusion proteins with various RBD domains from different stains and in different orders could also be formed.
• FIG. 31 shows a dosing regimen for a study of multivalent antigens as described herein as a booster vaccine.
• the present disclosure provides immunogenic compositions including nanoparticle vaccines and mRNA molecules encoding them for treating or preventing coronavirus infections and coronavirus infectious diseases, and related infections and diseases caused by sarbecoviruses and merbecoviruses.
• the disclosed immunogenic compositions comprise at least two antigenic coronavirus peptides comprising at least a first antigenic coronavirus peptide and a second antigenic coronavirus peptide, or one or more messenger RNA (mRNA) molecules encoding them.
• mRNA messenger RNA
• described herein are multivalent immunogenic compositions comprising two or more antigenic coronavirus peptides from different strains, or mRNA molecules encoding them.
• Heterologous antigens may focus the immune response to create additional breadth of recognized antigens. Further, immunization by multiple heterologous strains, even those across clades, may provide additional breadth of immune response.
• the nanoparticles disclosed herein are made up of fusion proteins that comprise a nanoparticle-forming peptide and an antigenic coronavirus peptide (e.g., at least two antigenic coronavirus peptides, optionally from different strains of coronavirus), which may be optionally joined together via a linker.
• the fusion proteins are capable of self-assembling into nanoparticles that are stable in solution and able to generate a protective neutralizing immune response (i.e., the production of neutralizing antibodies and/or defensive cytokines) when administered to a subject.
• a protective neutralizing immune response i.e., the production of neutralizing antibodies and/or defensive cytokines
• disclosed mRNA molecules when administered and expressed in vivo, result in the production of antigens that generate a protective neutralizing immune response.
• an immunogenic composition comprises one or more mRNA molecules encoding one or more fusion proteins that comprise a nanoparticle-forming peptide and an antigenic coronavirus peptide, and when administered and expressed in vivo, results in the production of a nanoparticle as disclosed herein that generates a protective neutralizing immune response.
• an immunogenic composition as disclosed herein may also comprise an adjuvant.
• the disclosed immunogenic compositions e.g., comprising mRNA molecules or nanoparticles
• the disclosed immunogenic compositions will provide protection against infection by coronaviruses, such as SARS-CoV-2 and other sarbecoviruses, MERS-CoV and other merbecoviruses, and other coronaviruses.
• the disclosed immunogenic compositions may also reduce illness caused by the coronaviruses.
• the disclosed immunogenic compositions may elicit protective immune responses in individuals that receive the vaccines.
• a phrase in the form “A/B” or in the form “A and/or B” means (A), (B), or (A and B); a phrase in the form “at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).
• a variant of a virus may comprise a genome sequence that shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to about 100% sequence identity or homology with the reference (or “parent”) genome sequence.
• SD in the context of the disclosed fusion proteins refers to a subdomain of a coronavirus spike protein.
• the spike protein there is a subdomain 1 and a subdomain 2 (see, e.g., Wrapp et al., Science 367, 1260-1263(2020)).
• SD could refer to either or both of SD1 and SD2 (i.e., subdomain 1 and 2) of the spike protein.
• Coronaviruses are a family of viruses (i.e., the Coronaviridae family) that cause respiratory infections in mammals and that comprise a genome that is roughly 30 kilobases in length.
• the Coronaviridae family is divided into four genera and the genome encodes 28 proteins across multiple open reading frames, including 16 non-structural proteins (nsp) that are post- translationally cleaved from a polyprotein. See, e.g., Letko et al., Nature Microbiology, 2020, 5(4):562-569.
• SARS-CoV-2 is a newly identified virus, it shares genetic and morphologic features with others in the Coronaviridae family, particularly those from the P— coronavirus genus.
• the genome of the recently isolated SARS-CoV-2 shares 82% nucleotide identity with human SARS-CoV (SARS-CoV-1) and 89% with bat SARS-like-CoVZXC21 (Lu et al., 2020).
• the spike (S) glycoprotein bears significant structural homology with SARS-CoV-1 compared to other coronaviruses such as MERS-CoV.
• S surface Spike glycoprotein of SARS-CoV-2 binds the same host receptor, ACE-2, to mediate cell entry (Letko et al., 2020; Yan et al., 2020a).
• coronavirus vaccine candidates developed to date are based on S or one of its sub-components.
• Coronavirus S glycoproteins contain three segments: a large ectodomain, a single-pass transmembrane anchor and a short intracellular tail.
• the ectodomain consists of a receptor-binding subunit, SI, which contains two sub-domains: one at the N-terminus and the other at the C- terminus.
• the latter comprises the receptor-binding domain (RBD), which serves the vital function of attaching the virus to the host receptor and triggering a conformational change in the protein that results in fusion with the host cell membrane through the S2 subunit.
• RBD receptor-binding domain
• Antibodies have been shown to neutralize viral entry by binding to the RBD of the spike protein. This region is also known to be the most variable part of the protein and is likely responsible for immune escape resulting in re-infection or lowered vaccine efficacy.
• ferritin is a small protein expressed by many organisms that can form into a homotypic 24-mer “nanoparticle.” It has been shown in previous work to serve as an antigen presentation system by decorating the N-terminal region with an antigen of interest. An antigen can be conjugated to the ferritin moiety without detracting from nanoparticle formation. Ferritin with an antigen conjugated via a sufficiently long linker can form quaternary structures such as (8) CoV trimers.
• Antigen display systems with multiple antigens (multivalent) have been demonstrated to increase breadth of responses against coronavirus (see, e.g., Cohen at al., Science, 371 : 735-741 (Feb. 2021); Cohen at al., Science 377, eabq0839(2022) (DOI:10.1126/science.abq0839) and influenza (see, e.g., Kanekiyo et al., Nat. Immunol., 20: 367-72 (Apr 2019).
• Boosting or sequential vaccination with heterologous strains has been shown to improve effectiveness of coverage against heterologous strains from the original strain. See, e.g., Tan et al., N. Eng. J. Med., 385: 1401-06 (Aug. 2021).
• the coronavirus that is treated or prevented by the disclosed immunogenic compositions is a 0-coronavirus.
• the 0-coronavirus is selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (also known by the provisional name 2019 novel coronavirus, or 2019-nCoV or COVID-19), human coronavirus OC43 (hCoV-OC43), Middle East respiratory syndrome-related coronavirus (MERS-CoV, also known by the provisional name 2012 novel coronavirus, or 2012-nCoV), severe acute respiratory syndrome-related coronavirus (SARS- CoV, also known as SARS-CoV-1), HKU-1, 229E, and NL63.
• SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
• hCoV-OC43 human coronavirus OC43
• MERS-CoV Middle East respiratory syndrome-related coronavirus
• SARS- CoV severe acute respiratory syndrome-related coronavirus
• the disclosed immunogenic compositions comprise a fusion protein comprising a nanoparticle-forming peptide and an antigenic coronavirus peptide, which may optionally be connected by a linker (i.e., a “linker domain”).
• the antigenic coronavirus peptide may comprise one or more fragments or full-length proteins derived from a coronavirus (e.g., SARS-CoV-2 or SARS-CoV-1 or MERS-CoV), as described in more detail below.
• nanoparticle-forming peptide may comprise a substitution of the glutamic acid residue (E) at position 13 of SEQ ID NO: 1 and a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the N-terminal domain of SEQ ID NO: 1, such as in the following sequences:
• the nanoparticle-forming peptide may comprise a variant of any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, which may comprise an amino acid sequence that shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to 100% sequence identity or homology with any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
• the nanoparticle-forming peptide may be a non- ferritin-based peptide, such as a peptide that comprises the following sequence or a fragment or variant thereof:
• the nanoparticle-forming peptide may comprise a variant of SEQ ID NO: 4, which may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations in SEQ ID NO: 4.
• the disclosed fusion proteins generally comprise a flexible amino acid linker; however, the linker domain (i.e. linker) is optional and in some embodiments the nanoparticle-forming peptide may be directly joined with the antigenic coronavirus peptide.
• the linker may be about 3 to about 50 amino acids in length, or more particularly about 4 to about 42 amino acids in length. In some embodiments, the linker may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 ammo acids in length.
• the linker domain may comprise glycine (G) repeats and or a combination of glycine (G) and serine (S) residues.
• the linker domain may comprise 1, 2, or 3 repeats of any one of SEQ ID NOs: 5-17 or 583.
• the linker domain comprises a variant of any one of SEQ ID NOs: 5- 17 or 583 that may comprise an amino acid sequence that shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to 100% sequence identity or homology with any one of SEQ ID NOs: 5- 17 or 583.
• linker sequences are not intended to be limiting, and those of skill in the art will understand that other flexible peptide linkers may also be suitable for connecting the nanoparticle-forming peptide and the antigenic coronavirus peptide, based on the guidance provided herein.
• the antigenic coronavirus peptide of the disclosed immunogenic compositions and fusion proteins comprises a coronavirus spike protein (also known as “S protein” or “glycoprotein S”), which is generally responsible for viral entry into a host cell, or a fragment or a variant thereof (such as an RBD domain or a fragment or a variant thereof).
• the antigenic coronavirus peptide may comprise 1, 2, or 3 or more distinct domains of a coronavirus spike protein connected together in sequence, and in such embodiments, a linker may optionally separate the distinct domains.
• the fusion protein comprises a variant that comprises about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of a coronavirus spike protein (e.g., SEQ ID NO: 18), so long as the fragment is able to elicit an immune response (i.e., it is an antigenic fragment).
• a coronavirus spike protein e.g., SEQ ID NO: 18
• the spike protein of SARS- CoV-2 attaches to human angiotensin converting enzyme (ACE)-2 cell surface receptors facilitating human infection.
• ACE angiotensin converting enzyme
• the SARS-CoV-2 spike protein (NCBI Reference Sequence: YP 009724390.1) comprises 1273 amino acids and consists of a signal peptide (amino acids 1-13) located at the N-terminus, the SI subunit (14-685 residues), and the S2 subunit (686-1273 residues); the last two regions are responsible for receptor binding and membrane fusion, respectively.
• the amino acid sequence is shown below.
• NTD N-terminal domain
• the antigenic coronavirus peptide may comprise a variant of an RBD (e.g., SEQ ID NO: 19) with one or more specific modifications made to reduce “sticky” hydrophobic regions, which may increase expression and/or the ability to form nanoparticles, for example, one of more of the following modifications.
• RBD e.g., SEQ ID NO: 19
• an antigenic coronavirus peptide of the present disclosure may be or comprise an NTD from a coronavirus other than SARS-CoV-2.
• the NTD domain may be derived from MERS or SARS-CoV-1.
• Exemplary NTD sequences can be found in the full length constructs provided in attached Table 6 and Table 7.
• a particle may comprise multiple NTDs from the same or different coronaviruses.
• a particle may comprise a combination of one or more RBD(s) and one or more NTD(s), and the RBD(s) and NTD(s) may be derived from the same or different coronaviruses or strains.
• an antigenic coronavirus peptide of the present disclosure may comprise an SI protein sequence.
• An SI protein sequence may comprise a SARS-CoV-2 SI protein amino acid sequence
• the antigenic coronavirus peptide may comprise a variant of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23 that may comprise an amino acid sequence that shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to 100% sequence identity or homology with SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.
• the antigenic coronavirus peptide may comprise a fragment of SI that may be a fragment of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23 that comprises about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23, so long as the fragment is able to elicit an immune response (i.e., it is an antigenic fragment).
• SI may be a fragment of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23 that comprises about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,
• an antigenic coronavirus peptide of the present disclosure may comprise an S-2P sequence or a fragment or variant thereof.
• An S-2P sequence is a stabilized version of the spike ectodomain that includes two proline substitutions and stabilizes the prefusion conformation.
• the S-2P domain includes a transmembrane domain.
• S-2P comprises proline modifications K986P and V987P, as well as the removal of the Furin cleavage site (RRAS to GSAS).
• the antigenic coronavirus peptide may comprise a variant of the S-2P sequence that may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations in the S-2P sequence.
• the antigenic coronavirus peptide may comprise a variant of the S-2P sequence that may comprise an amino acid sequence that shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to 100% sequence identity or homology with a stabilized S-2P.
• the antigenic coronavirus peptide may comprise a fragment of S-2P that comprises about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the stabilized S-2P, so long as the fragment is able to elicit an immune response (i.e., it is an antigenic fragment).
• an antigenic coronavirus peptide of the present disclosure may comprise a spike S domain or an extracellular spike S domain (e.g., a stabilized spike S domain or stabilized extracellular spike S domain) or a fragment or variant thereof.
• a stabilized extracellular spike S domain may comprise one or more modifications to stabilize the refusion conformation of the domain or extracellular domain.
• the antigenic coronavirus peptide may comprise a stabilized extracellular spike S domain that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations in an extracellular spike S domain.
• the antigenic coronavirus peptide may comprise a stabilized extracellular spike S domain that comprises an amino acid sequence that shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to 100% sequence identity or homology with an extracellular spike S domain.
• the antigenic coronavirus peptide may comprise a fragment of the extracellular spike S domain (e.g., a fragment of a stabilized extracellular spike S domain) that comprises about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the extracellular spike S domain (e.g., a stabilized extracellular spike S domain), so long as the fragment is able to elicit an immune response (i.e., it is an antigenic fragment).
• a fragment of the extracellular spike S domain e.g., a fragment of a stabilized extracellular spike S domain
• the antigenic coronavirus peptide may comprise a stabilized spike S domain that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations in a spike S domain.
• the antigenic coronavirus peptide may comprise a stabilized spike S domain that comprises an amino acid sequence that shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to 100% sequence identity or homology with a spike S domain.
• the antigenic coronavirus peptide may comprise a fragment of the spike S domain (e.g., a fragment of a stabilized spike S domain) that comprises about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the spike S domain (e.g., a stabilized spike S domain), so long as the fragment is able to elicit an immune response (i.e., it is an antigenic fragment).
• a fragment of the spike S domain e.g., a fragment of a stabilized spike S domain
• the antigenic coronavirus peptide may comprise a stabilized extracellular spike S trimer that comprises an amino acid sequence that shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to 100% sequence identity or homology with an extracellular spike S trimer.
• All of the proteins disclosed in Table 6 and some of the proteins disclosed in Table 7 are exemplary nanoparticle-forming proteins that can form RBD-Ferritin or Spike-Ferritin nanoparticles. These sequences contain a set of alternate sequences to improve the stability and immunogenicity of the RBD-Ferritin or Spike-Ferritin constructs.
• a glycan at N146 or N77 in the Ferritin sequence can improve and stabilize the Ferritin molecule.
• multiple RBD, NTD, or a combination thereof “beads” comprised of different antigenic sequences can be provided together on a single “string” (i.e., in a single construct) to elicit broad immune responses against coronaviruses.
• a “string” of antigens such as SARS-CoV-2-RBD-SARS-CoV-l- RBD-Khosta-2-RBD-B ANAL-20-247-RBD or SARS-CoV-2-RBD-S ARS-CoV- 1 -RBD-HKU- 1 - RBD-MERS-CoV-RBD-229E-RBD-NL63-RBD could be used with a “string” of antigens such as SARS-CoV-2-Omicron-BQ.l.l-RBD-S ARS-CoV- 1 -RBD or SARS-CoV-2-RBD- pangolinSARS-CoV-l-RBD-OC43-RBD-camelMERS-CoV-RBD-229E-RBD-NL63-RBD to increase or focus the immune response to a specific pan-reactive or pan-protective immunity.
• the “beads on a string” may also be added onto a SpFN or mos-SpFN molecule comprising an additional 1-10 RBDs, NTD, or both in series, or, in other words, may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RBDs linked to the SpFN or mos-SpFN molecule.
• a linker sequence including but not limited to a linker selected from the linker sequences disclosed in Table 1, may link one or more or each of the RBD and/or NTD sequences in series.
• the “domain fusion” format of fusion protein can be used to create a nanoparticle with antigenic components from multiple coronaviruses such as SARS-CoV-2 and its variants and subvariants, including those of concern (e.g., Alpha, Beta, Delta, Omicron), SARS-CoV-1, Khosta-2, BANAL-20-247, HKU-1, MERS-CoV, 229E, NL63, OC43, or related coronaviruses including those identified from bats, camels, or pangolins.
• coronaviruses such as SARS-CoV-2 and its variants and subvariants, including those of concern (e.g., Alpha, Beta, Delta, Omicron), SARS-CoV-1, Khosta-2, BANAL-20-247, HKU-1, MERS-CoV, 229E, NL63, OC43, or related coronaviruses including those identified from bats, camels, or pangolins.
• one or more RBDs are attached or inserted into the loop domain of an NTD via a linker (e.g., a linker from Table 1).
• a linker e.g., a linker from Table 1.
• at least two RBDs will be inserted, either in the same loop or different loops of the NTD.
• the RBDs may be from different strains or variants of coronavirus.
• the NTD may be from the same strain or variant as one or both of the RBDs, or the NTD may also be from a different strain or variant of coronavirus relative to one or both of the RBDs.
• the “domain insertion” or “mosaic” format of fusion protein can be used to create a nanoparticle with antigenic components from multiple coronaviruses such as SARS-CoV-2 and its variants and subvariants, including those of concern (e.g., Alpha, Beta, Delta, Omicron), SARS- CoV-1, Khosta-2, BANAL-20-247, HKU-1, MERS-CoV, 229E, NL63, OC43, or other coronaviruses including those identified from bats, camels, or pangolins.
• coronaviruses such as SARS-CoV-2 and its variants and subvariants, including those of concern (e.g., Alpha, Beta, Delta, Omicron), SARS- CoV-1, Khosta-2, BANAL-20-247, HKU-1, MERS-CoV, 229E, NL63, OC43, or other coronaviruses including those identified from bats, camels, or pangolins.
• coronaviruses examples include, but are not limited to sarbecoviruses (e.g., ZXC21, BANAL-20-247, Rf4092, Shaanxi2011, HeB2013, Rp3, Rs_672, HKU3-1, Rs4081, RmYN02, Rfl, Yunl 1, BM48- 31, BB9904, Khosta-1, Khosta-2, RhGBOl, BtKY72, RsYN04, RatG15 (Ra7909), SHC014, WIV1, LyRa3, Rs4084, Rs4231, BANAL-20-103, RaTG13, BANAL-20-52, Pangl7 (GX-P5L), or RshSTTl 82/200), merbecoviruses (e.g., MER1 (EnnaceusCoV/2012-174/GER/2012), MER2 (Neoromicia/5038), MER3 (HKU4 SM3A), MER4 (Bat)
• pan-P-coronavirus vaccine or pan-coronavirus vaccine.
• a spike protein or segment thereof is attached to a ferritin peptide, and one or more heterologous domains (e.g., RBD, NTD, or any combination thereof) are substituted in place of the native domain or added as an additional domain.
• heterologous domains e.g., RBD, NTD, or any combination thereof
• a hetero Igous RBD of one strain may be substituted for the native RBD of a given spike protein to form a “mosaic.”
• the RBD of a heterologous species or strain may be substituted in place of the native NTD of the spike protein to form a “mosaic.”
• one or more RBDs of a heterologous strain may be added to one end (i.e., C-terminus or N-terminus) of a native spike protein to form a mosaic.
• Multiple constructs can be combined together in a single nanoparticle by co-expression to produce a stable protein nanoparticles wherein the Spike trimer on the surface of the nanoparticle can be a heterologous mixture e.g.
• heterologous nanoparticles can also be encoded as mRNA constructs where mRNA molecules encoding different Spike-ferritin molecules can be encapsulated in a single lipid nanoparticle to facilitate heterologous nanoparticle formation within a vaccinated person.
• the heterologous nanoparticles could also be encoded within a single construct where exemplary cleavage sites are encoded between a given construct such as F2A (see, e.g., ncbi. nlm. nih. gov/pmc/articles/PMC4622431 /).
• an immunogenic composition as described herein comprises antigenic coronavirus peptides from two or more coronavirus strains independently selected from Clade la, Clade lb, Clade 2, Clade 3, and Middle East respiratory syndrome-related coronavirus (MERS-CoV) (or mRNA molecule(s) encoding them).
• MERS-CoV Middle East respiratory syndrome-related coronavirus
• an immunogenic composition as described herein comprises antigenic coronavirus peptides from different coronavirus strains independently selected from WA-1, Beta, Omicron BQ.1.1, and XBB.1.5; a strain of SARS-CoV-1, BANAL20-247, Khosta2, and MERS-CoV (or mRNA molecule(s) encoding them).
• an immunogenic composition as described herein comprises antigenic coronavirus peptides selected from the following combinations of strains (or mRNA molecule(s) encoding them): (i) two or more selected from WA-1, Beta, and Omicron BQ 1.1 or XBB.1.5; (ii) one or more selected from WA-1, Beta, Omicron XBB.1.5, and Omicron BQ 1.1 , and strains of SARS-CoV-1; (iii) one or more selected from WA-1, Beta, Omicron XBB.1.5, and Omicron BQ1.1, and one or more selected from strains of SARS-CoV-1, BANAL20-247, and Khosta-2; (iv) one or more selected from WA-1, Beta, Omicron XBB.1.5, and Omicron BQ1.1, and one or more selected from strains of SARS-CoV-1, BANAL20-247, and Khosta-2, and MERS-CoV; and (v) two or more antigenic coron
• fusion proteins used in a single immunogenic composition may have different configurations of the antigenic coronavirus peptides (e.g., A-B and B-A) to provide antigens presented peripherally and laterally on the nanoparticle.
• peripherally refers to epitopes on adjacent fusion proteins on the nanoparticle
• laterally refers to epitopes within the same fusion protein.
• multi- domained fusion proteins e.g., RBD-RBD or RBD-RBD-Spike, etc.
• fusion proteins may be prepared to “pattern” the nanoparticle surface by changing the order of the component strains’ antigens (e.g., RBDs), such that epitope combinations are adjacent both laterally and peripherally.
• RBDs antigens
• “mosaic” formats can be designed, where the spike protein is from one strain and the RBD domain of the spike protein or an additional RBD domain is from a different coronavirus (mosSp, RmosSp).
• fusion protein where the series of antigenic peptides are RBD-RBD-Spike (wherein Spike includes an RBD domain) taken from different strains of sarbecoviruses (A, B, and C, respectively, with Spike of strain C); additional fusion proteins could be RBD-RBD-mosSpike where the RBDs are (B, C, A, respectively, with Spike of strain C), and (C, A, B, respectively, with Spike of strain C), to allow the antigenic domains of each strain to be presented in each possible position of the fusion protein.
• RBD-RBD-Spike wherein Spike includes an RBD domain
• additional fusion proteins could be RBD-RBD-mosSpike where the RBDs are (B, C, A, respectively, with Spike of strain C), and (C, A, B, respectively, with Spike of strain C), to allow the antigenic domains of each strain to be presented in each possible position of the fusion protein.
• fusion proteins were designed according to the following antigen conjugation frameworks (“antigen presentations”) across various antigenic distance sets to provide multivalent nanoparticles displaying antigens of the selected antigenic distances to permit common and conserved epitopes to be optimally recognized and crosslinked by B cell receptors.
• antigen presentations antigen conjugation frameworks
• antigen presentation format RFN, SpFN, RRFN, RR-SpFN
• an immunogenic composition as described herein comprising several different mRNA molecules (e.g., encoding different fusion proteins), either co-encapsulated in the same LNP or administered in the same composition after separate encapsulation in separate LNPs, result in co-expression of each encoded fusion protein (e.g., comprising ferritin-conjugated antigens).
• Ferritin-conjugated antigens produced in the same cell auto-assemble into a ferritin nanoparticle displaying the expressed fusion proteins on the same nanoparticle, resulting in what is referred to herein as a “mosaic nanoparticle” or “multivalent nanoparticle.”
• strains with different levels of antigenic (i.e., sequence) distance that span the antigenic space of sarbeco viruses were selected.
• the strains include three antigenically distinct SARS-CoV-2 (Clade lb) strains: Parental WA-1, Beta, Omicron XBB.1.5, and Omicron BQ.1.1; a SARS-CoV-1 (Clade la) strain (Frankfurt); two increasingly distant bat zoonotic coronaviruses (Clades 2 and 3): BANAL20-247 and Khosta2, respectively; and a Merbecovirus (MERS-CoV) strain, representing a non-ACE2 binding strain as an outlier (FIG. 16, Panel A).
• mRNA molecules were constructed encoding antigenic coronavirus peptides (e.g., spike and/or RBD antigens) from strains of these antigenic distance sets (with or without ferritin moieties), to obtain immunogenic compositions that provide multivalent presentation of antigens reflecting various antigenic distance paradigms.
• antigenic coronavirus peptides e.g., spike and/or RBD antigens
• strains of these antigenic distance sets with or without ferritin moieties
• an immunogenic composition as described herein comprises antigenic coronavirus peptides (or mRNA molecule(s) encoding them) selected from the following combinations of strains: (i) WA-1, Beta, and Omicron BQ.1.1 (or XBB.1.5), wherein the antigenic coronavirus peptides are RBD antigens comprised in RFN fusion proteins R(WA-1)FN, R(Beta)FN, and R(BQ1.1)FN, or are Spike antigens comprised in S-2P (e.g., S(WA-1)-2P, S(Beta)-2P, and S(BQ1.1)-2P) or SpFN fusion proteins Sp(WA-l)FN, Sp(Beta)FN, and Sp(BQl.l)FN, or are mosaic antigens comprised in RmosSpFN or RRmosSpFN fusion proteins, or any combination thereof; (ii) WA-1, Omicron BQ.1.1 (or XBB1.5),
• any of the fusion proteins, nanoparticles, mRNA molecules and immunogenic compositions (e.g., vaccines) disclosed herein can be used for treating or preventing a coronavirus infection.
• Optimal doses and routes of administration may vary depending on the nature of the immunogenic composition (e.g., mRNA vs. nanoparticle), virus(es) being targeted, and subject being treated.
• the DNA may be incorporated into a plasmid, which may comprise the necessary components (e.g., promoter) to express the DNA in vivo after administration to a subject, and the plasmid can be operably organized for expression in a mammal, such as a human.
• a plasmid which may comprise the necessary components (e.g., promoter) to express the DNA in vivo after administration to a subject, and the plasmid can be operably organized for expression in a mammal, such as a human.
• a subject may receive an initial dose followed by one or more subsequent doses of an equal or lesser concentration at a set time after this initial dose, such as 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, or 20 or more weeks, such as 24 weeks, 52 weeks, 104 weeks, 260 weeks, or 520 weeks.
• dosing may be based on the number of nanoparticles administered to a subject.
• a dose of the disclosed vaccines may comprise 1.0 x 10 8 to 1 ,0 x 10 12 nanoparticles.
• a single dose may comprise 1.0 x 10 8 , 1.5 x 10 8 , 2.0 x 10 8 , 2.5 x 10 8 , 3.0 x 10 8 , 3.5 x 10 8 , 4.0 x 10 8 , 4.5 x 10 8 , 5.0 x 10 8 , 5.5 x 10 8 , 6.0 x 10 8 , 6.5 x 10 8 , 7.0 x 10 8 , 7.5 x 10 8 , 8.0 x 10 8 , 8.5 x 10 8 , 9.0 x 10 8 , 9.5 x 10 8 , 1.0 x 10 9 , 1.5 x 10 9 , 2.0 x 10 9 , 2.5 x 10 9 , 3.0 x IO 9 , 3.5 x IO 9 , 4.0 x IO 9 , 4.5 x IO 9 , 5.0 x IO 9 , 5.5 x IO 9 , 6.0 x IO 9 , 6.5 x IO 9 , 7.0 x
• the dose may be about 9.5 x 10 8 , about 9.75 x 10 8 , about 9.85 x 10 8 , about 9.95 x 10 8 , about 1.0 x 10 9 , about 1.1 x 10 9 , about 1.15 x 10 9 , about 1.2 x 10 9 , about 1.25 x 10 9 , about 1.3 x 10 9 , about 1.35 x 10 9 , about 1.4 x 10 9 , about 1.45 x 10 9 , or about 1.5 x 10 9 nanoparticles
• the subject is a mammal. In some embodiments, the subject is a human. In embodiments in which the subject is a human, the subject may be at least 18 years old, 40 years old, at least 45 years old, at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old or older. In some embodiments, the subject is a pediatric subject (i.e., less than 18 years old).
• a recombinant IgGl immunoglobulin structure can be “switched” to the corresponding regions of immunoglobulin structures from other immunoglobulin classes, such as recombinant secretory IgAl or recombinant secretory IgA2, such as may be useful for topical application onto mucosal surfaces.
• immunoglobulin IgA structures are known to have applications in protective immune surveillance directed against invasion of infectious diseases, which makes such structures suitable for methods of using the disclosed binding proteins in such contexts, e.g., treating or preventing coronavirus infection (e.g., COVID-19 or SARS-CoV-1 infection) or the spread of coronavirus from one individual to another.
• coronavirus infection e.g., COVID-19 or SARS-CoV-1 infection
• coronavirus infection e.g., COVID-19 or SARS-CoV-1 infection
• any of the coronavirus-specific binding proteins or antibodies obtained from a subject inoculated with a disclosed immunogenic composition or screened/selected using the disclosed fusion proteins can be used for treating and/or preventing a coronavirus infection, such as COVID- 19 or SARS-CoV-1 infection, for example.
• a coronavirus infection such as COVID- 19 or SARS-CoV-1 infection
• Optimal doses and routes of administration may vary, such as based on the route of administration and dosage form, the age and weight of the subject, and/or the subject’s condition, including the type and severity of the coronavirus infection, and can be determined by the skilled practitioner.
• the binding proteins can be formulated in a pharmaceutical composition suitable for administration to a subject by any intended route of administration.
• Embodiment 1 A nanoparticle comprising a fusion protein comprising a nanoparticleforming peptide and at least two antigenic coronavirus peptides selected from: a receptor-binding domain (RBD) of a coronavirus, or a fragment or variant thereof, an N-terminal domain (NTD) of a coronavirus, or a fragment or variant thereof, an SI domain of a coronavirus, or a fragment or variant thereof, a stabilized extracellular spike S-2P domain of a coronavirus, or a fragment or variant thereof, a stabilized extracellular spike S domain of a coronavirus, or a fragment or variant thereof, a stabilized extracellular spike S-trimer of a coronavirus, or a fragment or variant thereof, and a mosaic coronavirus spike protein, wherein at least one domain of the mosaic coronavirus spike protein is substituted or added from a heterologous.
• RBD receptor-binding domain
• NTD N-terminal domain
• SI
• Embodiment 2 A nanoparticle of embodiment 1 , wherein the nanoparticle-forming peptide comprises or is a ferritin protein or a fragment or variant thereof.
• Embodiment 3 A nanoparticle of embodiment 1 or 2, wherein the nanoparticle-forming peptide comprises or is Helicobacter pylori ferritin (Hpf) or a fragment or variant thereof.
• Hpf Helicobacter pylori ferritin
• Embodiment 4 A nanoparticle of any one of embodiments 1-3, wherein the nanoparticleforming peptide comprises an amino acid sequence selected from: a. ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFL FDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHE QHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGN ENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 1) or a fragment or variant thereof, b.
• Embodiment 5 A nanoparticle of any one of embodiments 1-4, wherein the at least two antigenic coronavirus peptides are connected via a linker.
• Embodiment 6 A nanoparticle of any one of embodiments 1-5, wherein the at least two antigenic coronavirus peptides are connected to the nanoparticle-forming peptide via a linker.
• Embodiment 7 A nanoparticle of embodiment 5 or 6, wherein the linker comprises an amino acid sequence selected from: GSGGGG (SEQ ID NO: 11), GGGG (SEQ ID NO: 15), GSGG (SEQ ID NO: 5), GGG (SEQ ID NO: 16), and SGG (SEQ ID NO: 17).
• Embodiment 8 A nanoparticle of any one of embodiments 1-7, wherein the fusion protein comprises 3-10 antigenic coronavirus peptides connected in series, optionally wherein the antigenic coronavirus peptides are connected via peptide linkers.
• Embodiment 9 A nanoparticle of any one of embodiments 1-8, wherein the at least two antigenic coronavirus peptides are isolated or derived from one or more coronaviruses selected from SARS-CoV-2, human coronavirus OC43 (hCoV-OC43), Middle East respiratory syndrome- related coronavirus (MERS-CoV), severe acute respiratory syndrome-related coronavirus (SARS- CoV-1), HKU-1, 229E, or NL63.
• SARS-CoV-2 human coronavirus OC43
• MERS-CoV Middle East respiratory syndrome- related coronavirus
• SARS- CoV-1 severe acute respiratory syndrome-related coronavirus
• HKU-1 HKU-1
• 229E or NL63.
• Embodiment 10 A nanoparticle of any one of embodiments 1 -9, wherein the fusion protein comprises a format selected from beads on a string, domain fusion, loop insertion, or domain insertion.
• Embodiment 11 A nanoparticle of any one of embodiments 1-10, wherein the fusion protein comprises a format shown in FIG. 2.
• Embodiment 12 A nanoparticle of any one of embodiments 1-11, wherein the fusion protein comprises an amino acid sequence disclosed in Table 6.
• Embodiment 13 A vaccine comprising a nanoparticle of any one of embodiments 1-12.
• Embodiment 14 A vaccine of embodiment 13, wherein the vaccine further comprises one or more adjuvants selected from ALFQ, alhydrogel, and combinations thereof.
• Embodiment 15 A messenger RNA (mRNA) encoding a nanoparticle according to any one of embodiments 1-12.
• mRNA messenger RNA
• Embodiment 16 A method of treating or preventing a coronavirus infection in a subject in need thereof, comprising administering to a subject in need thereof the nanoparticle according to any one of embodiments 1-12, the vaccine according to any one of embodiments 13-14 or the mRNA according to embodiment 15.
• Embodiment 17 A method of embodiment 16, wherein the subject is at risk of contracting a coronavirus infection.
• Embodiment 18 A method of embodiment 16, wherein the subject has already contracted a coronavirus infection.
• Embodiment 19 A nanoparticle according to any one of embodiments 1-12, a vaccine according to any one of embodiments 13-14, or a mRNA according to embodiment 15, for use in treating or preventing a coronavirus infection in a subject in need thereof.
• Embodiment 20 A nanoparticle, vaccine or mRNA for use of embodiment 19, wherein the subject is at risk of contracting a coronavirus infection.
• Embodiment 21 A nanoparticle, vaccine, or mRNA for use of embodiment 19, wherein the subject has already contracted a coronavirus infection.
• Embodiment 22 Use of a nanoparticle according to any one of embodiments 1-12, a vaccine according to any of embodiments 13-14, or the mRNA according to embodiment 16 in the preparation of a medicament for treating or preventing a coronavirus infection in a subject in need thereof.
• Embodiment 23 A DNA molecule, comprising a sequence encoding a nanoparticle according to any one of embodiments 1-12.
• Embodiment 24 A plasmid comprising the DNA molecule of embodiment 23.
• Embodiment 25 A plasmid of embodiment 24, wherein the plasmid can express the DNA molecule in vivo.
• Binding studies of MERS-CoV RBD-ferritin nanoparticle immunogens were performed, assessing MERS-CoV RBD-ferritin constructs for binding to MERS-CoV-neutralizing human monoclonal antibody CDC-C2 in two formats. Briefly, biosensors are hydrated in PBS prior to use. Assay steps are performed at 30°C with agitation set at l,000crpm in the Octet RED96 instrument (ForteBio). Biosensors are equilibrated in assay buffer (PBS) for 15 seconds before loading of IgG antibodies (30 pg/ml diluted in PBS).
• PBS assay buffer
• MERS-CoV neutralizing antibodies that target the spike RBD used include D12, Fl l, CDC2-C2, and JC57-11, while SARS-CoV-2 neutralizing antibodies include WRAIR-2125, WRAIR-5001, and ShAbO2.
• MERS-CoV and SARS-CoV-2 antibodies were immobilized onto AHC biosensors (ForteBio) for 100 seconds, followed by a brief baseline in assay buffer for 15 sec. Immobilized antibodies were then dipped in various antigens for 180 seconds. Response values were measured at the end of the association step. Results for constructs M.1-M3.6 are shown in FIG. 10.
• Values are response (nm) after 180 seconds.
• Values are response (nm) after 180 seconds.
• RR-SpFN constructs were designed and tested for expression, yield, and nanoparticle formation.
• Construct pCoV323 (RR-SpFN MR14-SARSl-SpFN) was expressed inExpi293F cells for 5 days at 37°C and purified by Galanthus nivalis lectin (GN A) affinity chromatography. This construct showed reasonable expression levels of 0.4 mg/L medium supernatant. Purified protein was assessed by SDS-PAGE and size-exclusion chromatography to assess expression and to evaluate size. The results are shown in FIG. 14, with the RR-SpFN construct showing appropriate size by SDS-PAGE and nanoparticle formation by size-exclusion chromatography.
• mRNA constructs encoding spike antigens were provided and expressed in HeLa cells. Additionally, expressed fusion proteins were assessed using a set of neutralizing antibodies by octet biolayer interferometry.
• HeLa cells were plated in 24-well plates at 0.075 million cells/well in 0.5 mL EMEM + 10% FBS. Cells were transfected the next day with 1 pg/million cells mRNA constructs with lipofectamine 2000. The different mRNA constructs have been tested for in vitro expression and secretion in HeLa cells after transfection. 24 hours post-transfection, the cell lysates and supernatants (to assess secretion) were analyzed in dot blot by probing the Spikes or RBDs with anti-RBD monoclonal antibodies (mAbs) targeting conformational neutralizing epitopes (SA55 for example) and/or with anti-ferritin mAb.
• mAbs monoclonal antibodies
• the RFN formulations also included a construct encoding the ferritin monomer (without a conjugated antigen), termed an “empty FN.”
• an empty FN a construct encoding the ferritin monomer (without a conjugated antigen).
• composition administered was formulated and diluted to a concentration befitting a 1 pg dose per construct per 50 pL. If an mRNA encoding only empty ferritin was included, that mRNA was added at 0.3 pl per construct per 50 pL. Mice receiving control formulations listed as “co-administration” were inoculated with three separate injections, each containing one listed construct encapsulated in an LNP at a dose of 1 pg (total dose 3 pg).
• the Spike protein (S) expression plasmid sequences for SARS-CoV-2 and SARS-CoV-1 were codon optimized and modified to remove an 18 amino acid endoplasmic reticulum retention signal in the cytoplasmic tail in the case of SARS-CoV-2, and a 28 amino acid deletion in the cytoplasmic tail in the case of SARS-CoV. This allowed increased S incorporation into pseudovirions (PSV) and thereby improve infectivity. Virions pseudotyped with the vesicular stomatitis virus (VSV) G protein were used as a non-specific control.
• SSV vesicular stomatitis virus
• SARS-CoV-2 pseudovirions were produced by co-transfection of HEK293T/17 cells with a SARS-CoV-2 S plasmid (pcDNA3.4) and an HIV-1 NL4-3 luciferase reporter plasmid (The reagent was obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: Human Immunodeficiency Virus 1 (HIV-1) NL4-3 AEnv Vpr Luciferase Reporter Vector (pNL4-3.Luc.R-E-), ARP-3418, contributed by Drs. Nathaniel Landau and Aaron Diamond).
• HIV-1 NL4-3 luciferase reporter plasmid HIV-1 NL4-3 luciferase reporter plasmid
• Infectivity and neutralization titers were determined using ACE2-expressing HEK293 target cells (Integral Molecular) in a semi-automated assay format using robotic liquid handling (Biomek NXp Beckman Coulter). Test sera were diluted 1:40 in growth medium and serially diluted, then 25 pL/well was added to a white 96-well plate. An equal volume of diluted SARS- CoV-2 PSV was added to each well and plates were incubated for 1 hour at 37°C. Target cells were added to each well (40,000 cells/ well) and plates were incubated for an additional 48 hours.
• Sera generated using the antigen designs and immunization strategies disclosed here may be considered to have an enhanced breadth of immune response if they have increased neutralization titers (as measured by pseudoneutralization assay) to one or more sarbecovirus strains not included as a vaccine component.
• the enhanced effect of multivalent antigen presentation as described herein can be assessed by comparing the pseudoneutralization titers between mouse groups inoculated with multivalent formulations and their concordant monovalent formulations in the same antigenic presentation.
• Commercial S2-P XBB.1.5 SARS CoV-2 (SoC) vaccine will be included in this study as a comparator for the proposed novel booster formulations.
• a specific dosing regimen will be as follows: Priming: Day 0 and D21; Bleed: Day 35; Pre-boost bleed: about one day before 1st boost; 1st Boost: 3 months after Day 21 (DI 19); Bleed: 2 weeks after 1st boost (D133); Pre-2nd boost bleed: about one day before 2nd boost (D150); 2nd boost: one month after 1st boost (DI 51). There will be 8 mice in each group. The dosing regimen is illustrated in FIG. 31.
• multivalent compositions will comprise admixtures of LNPs separately encapsulating mRNA molecules encoding a given antigen, but another study may employ multivalent compositions that comprise multiple mRNA molecules co-encapsulated in a single LNP.

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