Patent Application
20240277831
A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on Feb. 22, 2024, having the file name “20-1008-WO—US_amended.txt” and is 1,118,061 bytes in size.
The recent emergence of a previously unknown virus in Wuhan, China has resulted in the ongoing COVID-19 pandemic that has caused more than 18,700,000 infections and 700,000 fatalities as of Aug. 6, 2020 (WHO). Rapid viral isolation and sequencing revealed by January 2020 that the newly emerged zoonotic pathogen was a coronavirus closely related to SARS-CoV and was therefore named SARS-CoV-2. SARS-CoV-2 is believed to have originated in bats based on the isolation of the closely related RaTG13 virus from Rhinolophus affinis and the identification of the RmYN02 genome sequence in metagenomics analyses of Rhinolophus malayanus, both from Yunnan, China.
In one aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 1-84, 138-146, and 167-184, wherein X1 is absent or is an amino acid linker, and wherein residues in parentheses are optional and may be present or some or all of the optional residues may be absent. In various specific embodiments, the polypeptides comprise the amino acid sequence selected from the group consisting of SEQ ID NOS:1-12 and 142-151, comprise the amino acid sequence selected from the group consisting of SEQ ID NOS: 1-8, or comprise the amino acid sequence selected from the group consisting of SEQ ID NOS: 1 or 5. In another embodiment, the disclosure provides nanoparticles comprising a plurality of such polypeptides.
In another aspect, the disclosure provides nanoparticles, comprising:
In various other aspects, the disclosure provides compositions comprising a plurality of nanoparticles disclosed herein, nucleic acid molecules, such as mRNA, encoding the polypeptide disclosed herein, expression vectors comprising the nucleic acid molecules disclosed herein operatively linked to a suitable control sequence, cells comprising the polypeptide, the nanoparticle, the composition, the nucleic acid, and/or the expression vector disclosed herein, and pharmaceutical compositions, kits, and vaccines comprising the polypeptide, the nanoparticle, the composition, the nucleic acid, the expression vector, and/or the cell disclosed herein.
In another aspect, the disclosure provides methods to treat or limit development of a SARS-CoV-2 infection, comprising administering to a subject in need thereof an amount effective to treat or limit development of the infection the polypeptide, nanoparticle, composition, nucleic acid, pharmaceutical composition, or vaccine disclosed herein.
All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), “Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX).
As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, “about” means+/−5% of the recited parameter.
As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.
Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.
In a first aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 1-84, 138-146, and 167-184, wherein X1 is absent or is an amino acid linker, and wherein residues in parentheses are optional and may be present or some or all of the optional residues may be absent.
As shown in the examples that follow, the polypeptides of this aspect can be used to generated self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2. The nanoparticle vaccines induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease.
The amino acid sequence of exemplary polypeptides of this aspect of the disclosure are provided below.
GGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFT
GGSGSEKAAKAEEAARKMEELFKEHKIVAVLRANSVEEAKKKALAVFLGGVDLIEITFT
In various embodiments, the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOS:1-12 and 142-151. In various other embodiments, the polypeptides comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 1-8, or the group consisting of SEQ ID NOS: 1-4, SEQ ID NOS: 5-8, or the group consisting of SEQ ID NOS: 1 and 5, provided as exemplary embodiments in the examples that follow.
As used throughout the present application, the term “polypeptide” is used in its broadest sense to refer to a sequence of subunit D- or L-amino acids, including canonical and non-canonical amino acids. The polypeptides described herein may be chemically synthesized or recombinantly expressed. The polypeptides may be linked to other compounds to promote an increased half-life in vivo, such as by PEGylation, HESylation, PASylation, glycosylation, or may be produced as an Fc-fusion or in deimmunized variants. Such linkage can be covalent or non-covalent as is understood by those of skill in the art.
In a second aspect, the disclosure provides nanoparticles comprising a plurality of polypeptides according to any embodiment or combination of embodiments of the first aspect of the disclosure. In this aspect, a plurality (2, 3, 4, 5, 10. 20, 25, 50, 60, 100, or more) polypeptides of the first aspect of the disclosure are present in any suitable nanoparticle. Nanoparticles of any embodiment or aspect of this disclosure can be of any suitable size for an intended use, including but not limited to about 10 nm to about 100 nm in diameter.
In a third aspect, the disclosure provides nanoparticles, comprising:
In this aspect, the nanoparticle forms a three-dimensional structure formed by the non-covalent interaction of the first and second assemblies. A plurality (2, 3, 4, 5, 6, or more) of first polypeptides self-assemble to form a first assembly, and a plurality (2, 3, 4, 5, 6, or more) of second polypeptides self-assemble to form a second assembly. Non-covalent interaction of the individual self-assembling proteins results in self-assembly of the first protein into first assemblies, and self-assembly of the second proteins into second assemblies. A plurality of these first and second assemblies then self-assemble non-covalently via interfaces to produce the nanoparticles. The number of first polypeptides in the first assemblies may be the same or different than the number of second polypeptides in the second assemblies. Nanoparticles of this disclosure can have any shape and/or symmetry suitable for an intended use, including, but not limited to, tetrahedral, octahedral, icosahedral, dodecahedral, and truncated forms thereof. In one exemplary embodiment, each first assembly is pentameric and each second assembly is trimeric.
Assembly of the first and second assemblies into nanoparticles is not random, but is dictated by non-covalent interactions (e.g., hydrogen bonds, electrostatic, Van der Waals, hydrophobic, etc.) between the various assemblies (i.e., the cumulative effect of interactions between first assemblies, interactions between second assemblies, and interactions between first and second assemblies). Consequently, nanoparticles of this disclosure comprise symmetrically repeated, non-natural, non-covalent, protein-protein interfaces that orient the first and second assemblies into a nanoparticle having a highly ordered structure. While the formation of nanoparticles is due to non-covalent interactions of the first and second assemblies, in some embodiments, once formed, nanoparticles may be stabilized by covalent linking between proteins in the first assemblies and the second assemblies. Any suitable covalent linkage may be used, including but not limited to disulfide bonds and isopeptide linkages.
First proteins and second proteins suitable for producing assemblies of this disclosure may be of any suitable length for a given nanoparticle. First proteins and second proteins may be between 30 and 250 amino acids in length.
In one embodiment, the second proteins comprise an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS:85-124 or 185-193 (Table 2), wherein X1 for at least one second protein comprises an immunogenic portion of a SARS-CoV-2 antigen or a variant or homolog thereof, X2 is absent or an amino acid linker, and residues in parentheses are optional. The optional residues may be present, or some (i.e.: 1, 2, 3, 4, 5, 6, or more) or all of the optional residues may be absent.
GGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGV
(X2)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTV
GGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGV
(X2)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTV
GSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVE
(X2)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTV
GSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVE
(X2)KMEELFKKHKIVAVLRANSVEEAIEKAVAVGAGGVHLIEITFTVPDADTV
gsgrenlyfqggggsgyipeaprdgqayvrkdgewvllstflgGSGSGGSGGSG
SEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITF
(X2)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTV
gsgrenlyfqggggsgyipeaprdgqayvrkdgewvllstflgGSGSGGSGGSG
SEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITE
(X2)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTV
GSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVE
(X2)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTV
GSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVE
(X2)RKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADT
GGSGGSGSEKAAKAEEAARMEELFKEHKIVAVLRANSVEEAKKKALAVFLGGVD
(X2)MEELFKEHKIVAVLRANSVEEAKKKALAVFLGGVDLIEITFTVPDADTVI
GGSGGSGSEKAAKAEEAARMEELFKEHKIVAVLRANSVEEAKKKALAVELGGVD
(X2)MEELFKEHKIVAVLRANSVEEAKKKALAVFLGGVDLIEITFTVPDADTVI
GSGSGGSGGSGSEKAAKAEEAARMEELFKEHKIVAVLRANSVEEAKKKALAVEL
(X2)MEELFKEHKIVAVLRANSVEEAKKKALAVFLGGVDLIEITFTVPDADTVI
GSGSGGSGGSGSEKAAKAEEAARMEELFKEHKIVAVLRANSVEEAKKKALAVEL
(X2)MEELFKEHKIVAVLRANSVEEAKKKALAVELGGVDLIEITFTVPDADTVI
GGSGGSGSGGSGGSGSEKAAKAEEAARMEELFKEHKIVAVLRANSVEEAKKKAL
(X2)MEELFKEHKIVAVLRANSVEEAKKKALAVFLGGVDLIEITFTVPDADTVI
GGSGGSGSGGSGGSGSEKAAKAEEAARMEELFKEHKIVAVLRANSVEEAKKKAL
(X2)MEELFKEHKIVAVLRANSVEEAKKKALAVFLGGVDLIEITFTVPDADTVI
gsgrenlyfqggggsgyipeaprdgqayvrkdgewvllstflgGSGSGGSGGSG
SEKAAKAEEAARMEELFKEHKIVAVLRANSVEEAKKKALAVELGGVDLIEITFT
(X2)MEELFKEHKIVAVLRANSVEEAKKKALAVFLGGVDLIEITFTVPDADTVI
gsgrenlyfqggggsgyipeaprdgqayvrkdgewvllstflgGSGSGGSGGSG
SEKAAKAEEAARMEELFKEHKIVAVLRANSVEEAKKKALAVELGGVDLIEITFT
(X2)MEELFKEHKIVAVLRANSVEEAKKKALAVFLGGVDLIEITFTVPDADTVI
GSGSGGSGGSGSEKAAKAEEAARMEELFKEHKIVAVLRANSVEEAKKKALAVEL
(X2)MEELFKEHKIVAVLRANSVEEAKKKALAVELGGVDLIEITFTVPDADTVI
GSGSGGSGGSGSEKAAKAEEAARMEELFKEHKIVAVLRANSVEEAKKKALAVEL
(X2)MEELFKEHKIVAVLRANSVEEAKKKALAVELGGVDLIEITFTVPDADTVI
In various embodiments of this third aspect, the second proteins comprise an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS:85-88. In various other embodiments, the polypeptides comprise the amino acid sequence selected from the group consisting of SEQ ID NOS: 85-88, or the group consisting of SEQ ID NOS:85-86, or SEQ ID NOS: 85, provided as exemplary embodiments in the examples that follow.
The nanoparticles of this third aspect display on their surface an immunogenic portion of a SARS-CoV-2 antigen or a variant or homolog thereof, present in the at least one second protein. In one embodiment, the immunogenic portion of a SARS-CoV-2 antigen or a variant or homolog thereof is present as fusion protein with at least one second protein; it can be present on a single second protein in the nanoparticle (present in a single copy on the nanoparticle), or present in a plurality of second proteins present in the nanoparticle. In various embodiments, the SARS-CoV-2 antigen or a variant or homolog thereof is present in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the second proteins in the nanoparticle.
In these fusion proteins, the second protein may be joined directly to the SARS-CoV-2 antigen or a variant or homolog thereof, or the second protein and the SARS-CoV-2 antigen or a variant or homolog thereof may be joined using a linker. As used throughout this disclosure, a linker is a short (e.g., 2-30) amino acid sequence used to covalently join two polypeptides. Any suitable linker sequence may be used, including but not limited to those disclosed herein.
Any suitable SARS-CoV-2 antigen or a variant or homolog thereof may be used. In one embodiment of this third aspect, X1 in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the second proteins comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a Spike (S) protein extracellular domain (ECD) amino acid sequence, an S1 subunit amino acid sequence, an S2 subunit amino acid sequence, an Si receptor binding domain (RBD) amino acid sequence, and/or an N-terminal domain (NTD) amino acid sequence, from SARS-CoV-2, or a variant or homolog thereof.
In various further embodiments, X1 in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the second proteins comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NO:125-137.
In one specific embodiment, X1 in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the second proteins comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:125, the SARS-CoV-2 RBD provided as exemplary embodiments in the examples that follow. In various embodiments, X1 in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the second proteins comprise mutations at 1, 2, 3, 4, 5, 6, 7, or all 8 positions relative to SEQ ID NO:125 selected from the group consisting of K90N, K90T, G119S, Y126F, T151I, E157K, E157A, S167P, N174Y, and L 125R, including but not limited to mutations comprising one of the following naturally occurring mutations or combinations of mutations:
The amino acid residue numbering of these naturally occurring variants is based on their position within SEQ ID NO:125, while they are generally described based on their residue number in the Spike protein (i.e.: K417 in spike=K90 in RBD; G446 in spike=G119 in RBD; L452 in spike=L125 in RBD; Y453 in spike=Y126 in RBD; T478 in spike =T151 in RBD; E484 in spike=E157 in RBD; S494 in spike=S167 in RBD; N501 in spike=N174 in RBD).
In various further embodiments, X1 in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the second proteins comprise mutations at 1, 2, 3, 4, 5, 6, 7, or all 8 positions relative to SEQ ID NO:130 selected from the group consisting ofL18F, T20N, P26S, deletion of residues 69-70, D80A, D138Y, R190S, D215G, K417N, K417T, G446S, L452R, Y453F, T4781, E484K, S494P, N501Y, A570D, D614G, H655Y, P681H, A701V, T716L including but not limited to mutations comprising one of the following naturally occurring mutations or combinations of mutations:
As will be understood by those of skill in the art, when X1 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:125 (or any other disclosed antigen), it may include additional amino acids at the amino- or carboxy-terminus. Thus, for example, when X1 comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:125, X1 may comprise the amino acid sequence of SEQ ID NO:126, which includes additional amino acids at its N-terminus relative to SEQ ID NO:125.
In a further embodiment, X1 in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the second proteins comprise 1, 2, 3, or all 4 mutations relative to SEQ ID NO:125 selected from the group consisting of K90N, K90T, E157K, and N174Y.
The plurality of second assemblies may in total comprise a single SARS-CoV-2 antigen, or may comprise 2 or more different SARS-CoV-2 antigen. In one embodiment, the plurality of second assemblies in total comprises 2, 3, 4, 5, 6, 7, 8, or more different SARS-CoV-2 antigens. In one exemplary such embodiment, the plurality of second assemblies in total comprise 2, 3, 4, 5, 6, 7, 8, or more polypeptides comprising the amino acid sequence of any one of SEQ ID NOS: 1-84.
In one embodiment, X1 in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the second proteins comprises the amino acid sequence of SEQ ID NO:125. In another embodiment, X1 in 100% of the second proteins comprises the amino acid sequence of SEQ ID NO:125, and all second proteins are identical.
In a further embodiment, all second assemblies comprise at least one second protein comprising the amino acid sequence of any one of SEQ ID NOS: 1-84. In another embodiment, all second proteins comprise the amino acid sequence of any one of SEQ ID NOS: 1-84.
The nanoparticles comprise a plurality of identical first proteins. In one embodiment, the first protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected the group consisting of SEQ ID NOS:152-159, wherein residues in parentheses are optional and may be present or some (i.e.: 1, 2, 3, 4, 5, 6, or more) or all of the optional residues may be absent. In a specific embodiment, the first protein comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:155.
(MGSSHHHHHHSSGLVPRGS
EQKLISEEDLGS)NQHSQKDQETVRIAVVRARWHAFIVDACV
In an exemplary embodiment, the first protein comprises the amino acid sequence of SEQ ID NO:155. In various further such embodiments, the at least one or a plurality (20,%, 33%, 40%, 50%, 75%, etc.) of the second assemblies comprises at least one second protein comprising the amino acid sequence selected from the group consisting of SEQ ID NO:85-88, or all second assemblies comprise at least one second protein comprising the amino acid sequence selected from the group consisting of SEQ ID NO:85-88.
In one specific embodiment,
In another specific embodiment,
In a further specific embodiment:
In one specific embodiment:
The disclosure further provides compositions, comprising a plurality of nanoparticles of any embodiment or combination of embodiments of the disclosure. In one embodiment, the compositions comprise a plurality of nanoparticles of the specific embodiments disclosed above.
In a fourth aspect, the disclosure provides nucleic acids encoding a polypeptide or fusion protein of the disclosure. The nucleic acid sequence may comprise RNA (such as mRNA) or DNA. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the proteins of the invention.
In a fifth aspect, disclosure provides expression vectors comprising the isolated nucleic acid of any embodiment or combination of embodiments of the disclosure operatively linked to a suitable control sequence. “Expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including but not limited to plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive).
In a sixth aspect, the present disclosure provides cells comprising the polypeptide, the nanoparticle, the composition, the nucleic acid, and/or the expression vector of any embodiment or combination of embodiments of the disclosure, wherein the cells can be either prokaryotic or eukaryotic, such as mammalian cells. In one embodiment the cells may be transiently or stably transfected with the nucleic acids or expression vectors of the disclosure. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art. A method of producing a polypeptide according to the invention is an additional part of the invention. The method comprises the steps of (a) culturing a host according to this aspect of the invention under conditions conducive to the expression of the polypeptide, and (b) optionally, recovering the expressed polypeptide.
In a seventh aspect, the disclosure provides pharmaceutical compositions/vaccines comprising
As shown in the examples that follow, the nanoparticle immunogens elicit potent and protective antibody responses against SARS-CoV-2. The nanoparticle vaccines of the disclosure induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease.
The compositions/vaccines may further comprise (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the composition includes a bulking agent, like glycine. In yet other embodiments, the composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the composition additionally includes a stabilizer, e.g., a molecule which substantially prevents or reduces chemical and/or physical instability of the nanostructure, in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.
The nanoparticles may be the sole active agent in the composition, or the composition may further comprise one or more other agents suitable for an intended use, including but not limited to adjuvants to stimulate the immune system generally and improve immune responses overall. Any suitable adjuvant can be used. The term “adjuvant” refers to a compound or mixture that enhances the immune response to an antigen. Exemplary adjuvants include, but are not limited to, Adju-Phos™, Adjumer™, albumin-heparin microparticles, Algal Glucan, Algammulin, Alum, Antigen Formulation, AS-2 adjuvant, autologous dendritic cells, autologous PBMC, Avridine™, B7-2, BAK, BAY R1005, Bupivacaine, Bupivacaine-HCl, BWZL, Calcitriol, Calcium Phosphate Gel, CCR5 peptides, CFA, Cholera holotoxin (CT) and Cholera toxin B subunit (CTB), Cholera toxin A1-subunit-Protein A D-fragment fusion protein, CpG, CRL 1005, Cytokine-containing Liposomes, D-Murapalmitine, DDA, DHEA, Diphtheria toxoid, DL-PGL, DMPC, DMPG, DOC/Alum Complex, Fowlpox, Freund's Complete Adjuvant, Gamma Inulin, Gerbu Adjuvant, GM-CSF, GMDP, hGM-CSF, hIL-12 (N222L), hTNF-alpha, IFA, IFN-gamma in pcDNA3, IL-12 DNA, IL-12 plasmid, IL-12/GMCSF plasmid (Sykes), IL-2 in pcDNA3, IL-2/Ig plasmid, IL-2/Ig protein, IL-4, IL-4 in pcDNA3, Imiquimod™, ImmTher™, Immunoliposomes Containing Antibodies to Costimulatory Molecules, Interferon-gamma, Interleukin-1 beta, Interleukin-12, Interleukin-2, Interleukin-7, ISCOM(s)™, Iscoprep 7.0.3™, Keyhole Limpet Hemocyanin, Lipid-based Adjuvant, Liposomes, Loxoribine, LT(R192G), LT-OA or LT Oral Adjuvant, LT-R192G, LTK63, LTK72, MF59, MONTANIDE ISA 51, MONTANIDE ISA 720, MPL.TM., MPL-SE, MTP-PE, MTP-PE Liposomes, Murametide, Murapalmitine, NAGO, nCT native Cholera Toxin, Non-Ionic Surfactant Vesicles, non-toxic mutant E112K of Cholera Toxin mCT-E112K, p-Hydroxybenzoique acid methyl ester, pCIL-10, pCIL12, pCMVmCAT1, pCMVN, Peptomer-NP, Pleuran, PLG, PLGA, PGA, and PLA, Pluronic L121, PMMA, PODDS1M, Poly rA: Poly rU, Polysorbate 80, Protein Cochleates, QS-21, Quadri A saponin, Quil-A, Rehydragel HPA, Rehydragel LV, RIBI, Ribilike adjuvant system (MPL, TMD, CWS), S-28463, SAF-1, Sclavo peptide, Sendai Proteoliposomes, Sendai-containing Lipid Matrices, Span 85, Specol, Squalane 1, Squalene 2, Stearyl Tyrosine, Tetanus toxoid (TT), Theramide™, Threonyl muramyl dipeptide (TMDP), Ty Particles, and Walter Reed Liposomes. Selection of an adjuvant depends on the subject to be treated. Preferably, a pharmaceutically acceptable adjuvant is used.
In an eighth aspect, the disclosure provides methods to treat or limit development of a SARS-CoV-2 infection, comprising administering to a subject in need thereof an amount effective to treat or limit development of the infection of the polypeptide, nanoparticle, composition, nucleic acid, pharmaceutical composition, or vaccine of any embodiment herein (referred to as the “immunogenic composition”). The subject may be any suitable mammalian subject, including but not limited to a human subject.
When the method comprises limiting a SARS-CoV-2 infection, the immunogenic composition is administered prophylactically to a subject that is not known to be infected, but may be at risk of exposure to SARS-CoV-2. As used herein, “limiting development” includes, but is not limited to accomplishing one or more of the following: (a) generating an immune response (antibody and/or cell-based) to of SARS-CoV-2 in the subject; (b) generating neutralizing antibodies against SARS-CoV-2 in the subject (b) limiting build-up of SARS-CoV-2 titer in the subject after exposure to SARS-CoV-2; and/or (c) limiting or preventing development of SARS-CoV-2 symptoms after infection. Exemplary symptoms of SARS-CoV-2 infection include, but are not limited to, fever, fatigue, cough, shortness of breath, chest pressure and/or pain, loss or diminution of the sense of smell, loss or diminution of the sense of taste, and respiratory issues including but not limited to pneumonia, bronchitis, severe acute respiratory syndrome (SARS), and upper and lower respiratory tract infections.
In one embodiment, the methods generate an immune response in a subject in the subject not known to be infected with SARS-CoV-2, wherein the immune response serves to limit development of infection and symptoms of a SARS-CoV-2 infection. In one embodiment, the immune response comprises generation of neutralizing antibodies against SARS-CoV-2. In an exemplary such embodiment, the immune response comprises generation of SARS-CoV-2 spike protein antibody-specific responses with a mean geometric titer of at least 1×105. In a further embodiment, the immune response comprises generation of antibodies against multiple antigenic epitopes.
As used herein, an “effective amount” refers to an amount of the immunogenic composition that is effective for treating and/or limiting SARS-CoV-2 infection. The polypeptide, nanoparticle, composition, nucleic acid, pharmaceutical composition, or vaccine of any embodiment herein are typically formulated as a pharmaceutical composition, such as those disclosed above, and can be administered via any suitable route, including orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, subcutaneous, intravenous, intra-arterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally. Polypeptide compositions may also be administered via microspheres, liposomes, immune-stimulating complexes (ISCOMs), or other microparticulate delivery systems or sustained release formulations introduced into suitable tissues (such as blood). Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). A suitable dosage range may, for instance, be 0.1 μg/kg-100 mg/kg body weight of the polypeptide or nanoparticle thereof. The composition can be delivered in a single bolus, or may be administered more than once (e.g., 2, 3, 4, 5, or more times) as determined by attending medical personnel.
In one embodiment, the administering comprises administering a first dose and a second dose of the immunogenic composition, wherein the second dose is administered about 2 weeks to about 12 weeks, or about 4 weeks to about 12 weeks after the first does is administered. In various further embodiments, the second dose is administered about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the first dose. In another embodiment, three doses may be administered, with a second dose administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the first dose, and the third dose administered about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 weeks after the second dose.
In various other embodiments of prime-boost dosing, the administering comprises
In an alternative embodiment, the administering comprises
In either of these embodiments, any suitable DNA, mRNA, or adenoviral vector vaccine may be used in conjunction with the immunogenic compositions of the present disclosure, including but not limited to vaccines to be developed as well as those available from Moderna, Pfizer/BioNTech, Johnson & Johnson, etc.
In another embodiment of the methods, the subject is infected with a severe acute respiratory (SARS) virus, including but not limited to SARS-CoV-2, wherein the administering elicits an immune response against the SARS virus in the subject that treats a SARS virus infection in the subject. When the method comprises treating a SARS-CoV-2 infection, the immunogenic compositions are administered to a subject that has already been infected with SARS-CoV-2, and/or who is suffering from symptoms (as described above) indicating that the subject is likely to have been infected with SARS-CoV-2.
As used herein, “treat” or “treating” includes, but is not limited to accomplishing one or more of the following: (a) reducing SARS-CoV-2 titer in the subject; (b) limiting any increase of SARS-CoV-2 titer in the subject; (c) reducing the severity of SARS-CoV-2 symptoms; (d) limiting or preventing development of SARS-CoV-2 symptoms after infection; (e) inhibiting worsening of SARS-CoV-2 symptoms; (f) limiting or preventing recurrence of SARS-CoV-2 symptoms in subjects that were previously symptomatic for SARS-CoV-2 infection; and/or (e) survival.
The disclosure further provides kits, which may be used to prepare the nanoparticles and compositions of the disclosure. In one embodiment, the kits comprise:
In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 5, and the first protein comprises the amino acid sequence of SEQ ID NO:155.
In another embodiment, the kits comprise:
In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 5, and the first protein comprises the amino acid sequence of SEQ ID NO:155.
In a further embodiment, the kits comprise:
In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 5, and the first protein comprises the amino acid sequence of SEQ ID NO:155.
In another embodiment, the kits comprise:
In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 5, and the first protein comprises the amino acid sequence of SEQ ID NO:155.
A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, we describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, indicate that manufacture of the nanoparticle vaccines will be highly scalable.
To design vaccine candidates that induce potent neutralizing Ab responses, we focused on the RBD of the SARS-CoV-2 S glycoprotein (
Size-exclusion chromatography (SEC) of the SARS-CoV-2 RBD-I53-50 nanoparticles revealed predominant peaks corresponding to the target icosahedral assemblies and smaller peaks comprising residual unassembled RBD-I53-50A components (
Each experiment was performed at least twice, and the values and fitting errors presented are derived from a representative experiment. The corresponding binding curves and fits are presented in
We used recombinant human ACE2 ectodomain and two S-specific mAbs (CR3022 and S309) to characterize the antigenicity of the RBD when fused to I53-50A as well as the accessibility of multiple RBD epitopes in the context of the assembled nanoparticle immunogens. CR3022 and S309 were both isolated from individuals infected with SARS-CoV and cross-react with the SARS-CoV-2 RBD. CR3022 is a weakly neutralizing Ab that binds to a conserved, cryptic epitope in the RBD that becomes accessible upon RBD opening but is distinct from the receptor binding motif (RBM), the surface of the RBD that interacts with ACE2 (Huo et al., 2020; ter Meulen et al., 2006; Yuan et al., 2020). S309 neutralizes both SARS CoV and SARS-CoV-2 by binding to a glycan-containing epitope that is conserved amongst sarbecoviruses and accessible in both the open and closed prefusion S conformational states (Pinto et al., 2020).
We used bio-layer interferometry (BLI) to confirm the binding affinities of the monomeric human ACE2 (hACE2) ectodomain and the CR3022 Fab for the monomeric RBD. Equilibrium dissociation constants (KD) of these reagents for immobilized RBD-I53-50A fusion proteins closely matched those obtained for the monomeric RBD (Table 4 and
To evaluate the possibility that the magnitude and quality of nanoparticle immunogen-elicited Ab responses can be modulated by the accessibility of specific epitopes in the context of a dense, multivalent antigen array, we measured the binding of the nanoparticle immunogens to immobilized dimeric macaque ACE2 (mACE2-Fc) and the CR3022 and S309 mAbs, the latter of which roughly mimics the B cell receptor (BCR)-antigen interaction that is central to B cell activation. This approach does not allow the calculation of KD values due to the multivalent nature of the interactions, but does enable qualitative comparisons of epitope accessibility in different nanoparticle constructs. We compared the full-valency nanoparticles displaying 60 RBDs to a less dense antigen array by leveraging the versatility of in vitro assembly to prepare nanoparticle immunogens displaying the RBD antigen at 50% valency (˜30 RBDs per nanoparticle) (
We first used chemical denaturation in guanidine hydrochloride (GdnHCl) to compare the stability of the RBD-I53-50A fusion proteins and RBD-12GS-I53-50 nanoparticle immunogen to recombinant monomeric RBD and the S-2P ectodomain trimer (
We compared the immunogenicity of the three RBD-I53-50 nanoparticles, each displaying the RBD at either 50% or 100% valency, to the S-2P ectodomain trimer and the monomeric RBD in BALB/c mice. Groups of ten mice were immunized intramuscularly at weeks 0 and 3 with AddaVax™-adjuvanted formulations containing either 0.9 or 5 μg of SARS-CoV-2 antigen in either soluble or particulate form. Three weeks post-prime, all RBD nanoparticles elicited robust S-specific Ab responses with geometric mean reciprocal half-maximal effective concentrations ranging between 8×102 and 1×104 (
We prototyped potential human antibody responses to the RBD nanoparticle immunogens using the Kymab proprietary IntelliSelect™ Transgenic mouse platform (known as ‘Darwin’) that is transgenic for the non-rearranged human antibody variable and constant region germline repertoire. In contrast to previous mice with chimeric antibody loci that have been described (Lee et al., 2014), the mice in the present study differed in that they were engineered to express fully human kappa light chain Abs. Groups of five Darwin mice were immunized intramuscularly with S-2P trimer, 100% RBD-12GS-, or 100% RBD-16GS-I53-50 nanoparticles at antigen doses of 0.9 μg (nanoparticles only) or 5 μg (
We then evaluated the neutralizing activity elicited by each immunogen using both pseudovirus and live virus neutralization assays. In BALB/c mice, all RBD nanoparticle immunogens elicited serum neutralizing Abs after a single immunization, with reciprocal half-maximal inhibition dilutions (IC50) ranging from 1×102 to 5×102 (GMT) in pseudovirus and 3×103 to 7×103 in live virus neutralization assays (
In both mouse models, a second immunization with the RBD-I53-50 nanoparticles led to a large increase in neutralizing Ab titers. In BALB/c mice, pseudovirus neutralization GMT reached 2×103 to 3×104, exceeding that of the HCS by 1-2 orders of magnitude, and live virus neutralization titers reached 2×104 to 3×104 (
Several conclusions can be drawn from these data. First, the RBD nanoparticles elicit potent neutralizing Ab responses in two mouse models that exceed those elicited by the prefusion-stabilized S-2P trimer and, after two doses, by infection in humans. Second, linker length and antigen valency did not substantially impact the overall immunogenicity of the RBD nanoparticles, although there is a trend suggesting that RBD-16GS-I53-50 may be more immunogenic than the nanoparticles with shorter linkers. These observations are consistent with the antigenicity and accessibility data presented in Table 4 and
Eight mice immunized with AddaVax™ only, monomeric RBD, S-2P trimer, or RBD-8GS- or RBD-12GS-I53-50 nanoparticles were challenged seven weeks post-boost with a mouse-adapted SARS-CoV-2 virus (SARS-CoV-2 MA) to determine whether these immunogens confer protection from viral replication. The RBD-8GS- and RBD-12GS-I53-50 nanoparticles provided complete protection from detectable SARS-CoV-2 MA replication in mouse lung and nasal turbinates (
Germinal center (GC) responses are a key process in the formation of durable B cell memory, resulting in the formation of affinity-matured, class-switched memory B cells and long-lived plasma cells. We therefore evaluated the antigen-specific GC B cell responses in mice immunized with the monomeric RBD, S-2P trimer, and RBD-8GS-, RBD-12GS-, or RBD-16GS-I53-50 nanoparticles. The quantity and phenotype of RBD-specific B cells were assessed 11 days after immunization to determine levels of GC precursors and B cells (B220+CD3−CD138−CD38−GL7+) (
We compared the ratio of binding to neutralizing antibodies elicited by the S-2P and the RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50 nanoparticles and HCS as a measure of the quality of the Ab responses elicited by the nanoparticle immunogens. In Kymab Darwin™ mice, the nanoparticle vaccines had lower (better) ratios than S-2P-immunized mice, but higher than HCS (
We set out to identify the epitopes recognized by Abs elicited upon immunization with the nanoparticle immunogens in a nonhuman primate model that more closely resembles humans in their immune response to vaccination. We immunized a pigtail macaque with 250 μg of RBD-12GS-I53-50 (88 μg of RBD antigen) at weeks 0 and 4 and found that serum collected at week 8 had high levels of S-specific Abs (EC50˜1×106). Polyclonal Fabs were generated and purified for use in competition BLI with hACE2, CR3022, and S309, which recognize three distinct sites targeted by neutralizing Abs on the SARS-CoV-2 RBD (
Here we showed that two-component self-assembling SARS-CoV-2 RBD nanoparticle vaccine candidates elicit potent neutralizing Ab responses targeting multiple distinct RBD epitopes. The greater neutralizing Ab responses elicited by the RBD nanoparticles compared to the prefusion-stabilized ectodomain trimer are very promising. Our data indicate that RBD-12GS-I53-50 and RBD-16GS-I53-50 elicit nearly ten-fold higher levels of S-specific Abs and, more importantly, roughly ten-fold higher levels of neutralizing activity compared to the S-2P ectodomain trimer. This enhancement in potency is maintained at a more than five-fold lower antigen dose by mass, suggesting that presentation on the nanoparticle also has a dose-sparing effect. Both enhanced potency and dose-sparing could be critical for addressing the need to manufacture an unprecedented number of doses of vaccine to respond to the SARS-CoV-2 pandemic.
Although the RBD is poorly immunogenic as a monomer, our data establish that it can form the basis of a highly immunogenic vaccine when presented multivalently in our designs. The exceptionally low binding:neutralizing ratio elicited upon immunization with the RBD nanoparticles suggests that presentation of the RBD on I53-50 focuses the humoral response on epitopes recognized by neutralizing Abs. This metric is a potentially important indicator of vaccine safety, as high levels of binding yet non-neutralizing or weakly neutralizing Abs may contribute to vaccine-associated enhancement of respiratory disease. Our data further show that RBD-12GS-I53-50 elicited Ab responses targeting several of the non-overlapping epitopes recognized by neutralizing Abs that have been identified in the RBD. Such polyclonal responses targeting multiple distinct epitopes might explain the magnitude of neutralization observed and should minimize the risk of selection or emergence of escape mutations. Finally, the high production yield of RBD-I53-50A components and the robust stability of the antigen-bearing RBD nanoparticles makes them amenable to large-scale manufacturing.
HEK293F is a female human embryonic kidney cell line transformed and adapted to grow in suspension (Life Technologies). HEK293F cells were grown in 293FreeStyle™ expression medium (Life Technologies), cultured at 37° C. with 8% C02 and shaking at 130 rpm. Expi293F™ cells are derived from the HEK293F cell line (Life Technologies). Expi293F™ cells were grown in Expi293™ Expression Medium (Life Technologies), cultured at 36.5° C. with 8% CO2 and shaking at 150 rpm. VeroE6 is a female kidney epithelial cell from African green monkey. HEK293T/17 is a female human embryonic kidney cell line (ATCC). The HEK-ACE2 adherent cell line was obtained through BEI Resources, NIAID, NIH: Human Embryonic Kidney Cells (HEK-293T) Expressing Human Angiotensin-Converting Enzyme 2, HEK-293T-hACE2 Cell Line, NR-52511. All adherent cells were cultured at 37° C. with 8% C02 in flasks with DMEM+10% FBS (Hyclone)+1% penicillin-streptomycin. Cell lines other than Expi293F were not tested for mycoplasma contamination nor authenticated.
Female BALB/c mice four weeks old were obtained from Jackson Laboratory, Bar Harbor, Maine. Animal procedures were performed under the approvals of the Institutional Animal Care and Use Committee of University of Washington, Seattle, WA, and University of North Carolina, Chapel Hill, NC. Kymab's proprietary IntelliSelect™ Transgenic mouse platform, known as Darwin™, has complete human antibody loci with a non-rearranged human antibody variable and constant germline repertoire. Consequently, the antibodies produced by these mice are fully human.
Two adult male Pigtail macaques (Macaca nemestrina) were immunized in this study. All animals were housed at the Washington National Primate Research Center (WaNPRC), an American Association for the Accreditation of Laboratory Animal Care International (AAALAC)-accredited institution, as previously described (Erasmus et al., 2020). All procedures performed on the animals were with the approval of the University of Washington's Institutional Animal Care and Use Committee (IACUC).
Samples collected between 1-60 days post infection from 31 individuals who tested positive for SARS-CoV-2 by PCR were profiled for anti-SARS-CoV-2 S antibody responses and the 29 with anti-S Ab responses were maintained in the cohort (
The SARS-CoV-2 RBD (BEI NR-52422) construct was synthesized by GenScript into pcDNA3.1-with an N-terminal mu-phosphatase signal peptide and a C-terminal octa-histidine tag (GHHHHHHHH) (SEQ ID NO:164). The boundaries of the construct are N-32sRFPN331 and 528KKST531-C(Walls et al., 2020). The SARS-CoV-2 S-2P ectodomain trimer (GenBank: YP_009724390.1, BEI NR-52420) was synthesized by GenScript into pCMV with an N-terminal mu-phosphatase signal peptide and a C-terminal TEV cleavage site (GSGRENLYFQG) (SEQ ID NO: 165), T4 fibritin foldon (GGGSGYIPEAPRDGQAYVRKDGEWVLLSTFL) (SEQ ID NO:166), and octa-histidine tag (GHHHHHHHH) (SEQ ID NO:164) (Walls et al., 2020). The construct contains the 2P mutations (proline substitutions at residues 986 and 987; (Pallesen et al., 2017)) and an 682SGAG685 substitution at the furin cleavage site. The SARS-CoV-2 RBD was genetically fused to the N terminus of the trimeric I53-50A nanoparticle component using linkers of 8, 12, or 16 glycine and serine residues. RBD-8GS- and RBD-12GS-I53-50A fusions were synthesized and cloned by Genscript into pCMV. The RBD-16GS-I53-50A fusion was cloned into pCMV/R using the XbaI and AvrII restriction sites and Gibson assembly (Gibson et al., 2009). All RBD-bearing components contained an N-terminal mu-phosphatase signal peptide and a C-terminal octa-histidine tag. The macaque or human ACE2 ectodomain was genetically fused to a sequence encoding a thrombin cleavage site and a human Fc fragment at the C-terminal end. hACE2-Fc was synthesized and cloned by GenScript with a BM40 signal peptide. Plasmids were transformed into the NEB 5-alpha strain of E. coli (New England Biolabs) for subsequent DNA extraction from bacterial culture (NucleoBond Xtra Midi™ kit) to obtain plasmid for transient transfection into Expi293F cells. The amino acid sequences of all novel proteins used in this study can be found in Table 3.
SARS-CoV-2 S and ACE2-Fc proteins were produced in Expi293F cells grown in suspension using Expi293F expression medium (Life Technologies) at 33° C., 70% humidity, 8% CO2 rotating at 150 rpm. The cultures were transfected using PEI-MAX™ (Polyscience) with cells grown to a density of 3.0 million cells per mL and cultivated for 3 days. Supernatants were clarified by centrifugation (5 minutes at 4000 ref), addition of PDADMAC solution to a final concentration of 0.0375% (Sigma Aldrich, #409014), and a second spin (5 minutes at 4000 ref).
Genes encoding CR3022 heavy and light chains were ordered from GenScript and cloned into pCMV/R. Antibodies were expressed by transient co-transfection of both heavy and light chain plasmids in Expi293F cells using PEI MAX™ (Polyscience) transfection reagent. Cell supernatants were harvested and clarified after 3 or 6 days as described above.
Proteins containing His tags were purified from clarified supernatants via a batch bind method where each clarified supernatant was supplemented with 1 M Tris-HCl pH 8.0 to a final concentration of 45 mM and 5 M NaCl to a final concentration of ˜310 mM. Talon cobalt affinity resin (Takara) was added to the treated supernatants and allowed to incubate for 15 minutes with gentle shaking. Resin was collected using vacuum filtration with a 0.2 μm filter and transferred to a gravity column. The resin was washed with 20 mM Tris pH 8.0, 300 mM NaCl, and the protein was eluted with 3 column volumes of 20 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole. The batch bind process was then repeated and the first and second elutions combined. SDS-PAGE was used to assess purity. RBD-I53-50A fusion protein IMAC elutions were concentrated to >1 mg/mL and subjected to three rounds of dialysis into 50 mM Tris pH 7, 185 mM NaCl, 100 mM Arginine, 4.5% glycerol, and 0.75% w/v 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in a hydrated 10K molecular weight cutoff dialysis cassette (Thermo Scientific). S-2P IMAC elution fractions were concentrated to −1 mg/mL and dialyzed three times into 50 mM Tris pH 8, 150 mM NaCl, 0.25% L-Histidine in a hydrated 10K molecular weight cutoff dialysis cassette (Thermo Scientific). Due to inherent instability, the S-2P trimer was immediately flash frozen and stored at −80° C.
Clarified supernatants of cells expressing monoclonal antibodies and human or macaque ACE2-Fc were purified using a MabSelect PrismA™ 2.6×5 cm column (Cytiva) on an AKTA Avant150 FPLC (Cytiva). Bound antibodies were washed with five column volumes of 20 mM NaPO4, 150 mM NaCl pH 7.2, then five column volumes of 20 mM NaPO4, 1 M NaCl pH 7.4 and eluted with three column volumes of 100 mM glycine at pH 3.0. The eluate was neutralized with 2 M Trizma base to 50 mM final concentration. SDS-PAGE was used to assess purity.
Recombinant S309 was expressed as a Fab in expiCHO cells transiently co-transfected with plasmids expressing the heavy and light chain, as described above (see Transient transfection) (Stettler et al., 2016). The protein was affinity-purified using a HiTrap™ Protein A Mab select Xtra™ column (Cytiva) followed by desalting against 20 mM NaPO4, 150 mM NaCl pH 7.2 using a HiTrap™ Fast desalting column (Cytiva). The protein was sterilized with a 0.22 μm filter and stored at 4° C. until use.
The I53-50A and I53-50B.4.PT1 proteins were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) grown in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf). Cells were grown at 37° C. to an OD600 ˜0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18° C. and the cells shaken for ˜16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6×10 cm Ni Sepharose™ 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM to 500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex™ 200 Increase 10/300, or HiLoad™ S200 μg GL SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53-50A elutes at ˜0.6 column volume (CV). I53-50B.4PT1 elutes at ˜0.45 CV. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.
Total protein concentration of purified individual nanoparticle components was determined by measuring absorbance at 280 nm using a UV/vis spectrophotometer (Agilent Cary 8454) and calculated extinction coefficients (Gasteiger et al., 2005). The assembly steps were performed at room temperature with addition in the following order: RBD-I53-50A trimeric fusion protein, followed by additional buffer as needed to achieve desired final concentration, and finally I53-50B.4PT1 pentameric component (in 50 mM Tris pH 8, 500 mM NaCl, 0.75% w/v CHAPS), with a molar ratio of RBD-I53-50A:I53-B.4PT1 of 1.1:1. In order to produce partial valency RBD-I53-50 nanoparticles (50% RBD-I53-50), both RBD-I53-50A and unmodified I53-50A trimers (in 50 mM Tris pH 8, 500 mM NaCl, 0.75% w/v CHAPS) were added in a slight molar excess (1.1×) to I53-50B.4PT1. All RBD-I53-50 in vitro assemblies were incubated at 2-8° C. with gentle rocking for at least 30 minutes before subsequent purification by SEC in order to remove residual unassembled component. Different columns were utilized depending on purpose: Superose™ 6 Increase 10/300 GL column was used analytically for nanoparticle size estimation, a Superdex™ 200 Increase 10/300 GL column used for small-scale pilot assemblies, and a HiLoad™ 26/600 Superdex™ 200 pg column used for nanoparticle production. Assembled particles elute at ˜11 mL on the Superose™ 6 column and in the void volume of Superdex™ 200 columns. Assembled nanoparticles were sterile filtered (0.22 μm) immediately prior to column application and following pooling of fractions.
hACE2-Fc and CR3022 Digestion
hACE2-Fc was digested with thrombin protease (Sigma Aldrich) in the presence of 2.5 mM CaCl2 at a 1:300 w/w thrombin:protein ratio. The reaction was incubated at ambient temperature for 16-18 hours with gentle rocking. Following incubation, the reaction mixture was concentrated using Ultracel™ 10K centrifugal filters (Millipore Amicon Ultra) and sterile filtered (0.22 μM). Cleaved hACE2 monomer was separated from uncleaved hACE2-Fc and the cleaved Fc regions using Protein A purification (see Protein purification above) on a HiScreen MabSelect SuRe™ column (Cytiva) using an AKTA avant 25 FPLC (Cytiva). Cleaved hACE2 monomer was collected in the flow through, sterile filtered (0.22 μm), and quantified by UV/vis.
LysC (New England BioLabs) was diluted to 10 ng/μL in 10 mM Tris pH 8 and added to CR3022 IgG at 1:2000 w/w LysC:IgG and subsequently incubated for 18 hours at 37° C. with orbital shaking at 230 rpm. The cleavage reaction was concentrated using Ultracel™ 10K centrifugal filters (Millipore Amicon Ultra) and sterile filtered (0.22 μM). Cleaved CR3022 mAb was separated from uncleaved CR3022 IgG and the Fc portion of cleaved IgG, using Protein A purification as described above. Cleaved CR3022 was collected in the flow through, sterile filtered (0.22 μm), and quantified by UV/vis.
Antigenicity assays were performed and analyzed using BLI on an Octet™ Red 96 System (Pall Forté Bio/Sartorius) at ambient temperature with shaking at 1000 rpm. RBD-I53-50A trimeric components and monomeric RBD were diluted to 40 μg/mL in Kinetics buffer (1× HEPES-EP+(Pall Fortd Bio), 0.05% nonfat milk, and 0.02% sodium azide). Monomeric hACE2 and CR3022 Fab were diluted to 750 nM in Kinetics buffer and serially diluted three-fold for a final concentration of 3.1 nM. Reagents were applied to a black 96-well Greiner Bio-one microplate at 200 μL per well as described below. RBD-I53-50A components or monomeric RBD were immobilized onto Anti-Penta-HIS (HIS1K) biosensors per manufacturer instructions (Fortd Bio) except using the following sensor incubation times. HIS1K biosensors were hydrated in water for 10 minutes, and were then equilibrated in Kinetics buffer for 60 seconds. The HIS1K tips were loaded with diluted trimeric RBD-I53-50A component or monomeric RBD for 150 seconds and washed with Kinetics buffer for 300 seconds. The association step was performed by dipping the HIS1K biosensors with immobilized immunogen into diluted hACE2 monomer or CR3022 Fab for 600 seconds, then dissociation was measured by inserting the biosensors back into Kinetics buffer for 600 seconds. The data were baseline subtracted and the plots fitted using the Pall™ FortéBio/Sartorius analysis software (version 12.0). Plots in
Binding of mACE2-Fc, CR3022 IgG, and S309 IgG to monomeric RBD, RBD-I53-50A trimers, and RBD-I53-50 nanoparticles was analyzed for accessibility experiments and real-time stability studies using an Octet™ Red 96 System (Pall™ FortéBio/Sartorius) at ambient temperature with shaking at 1000 rpm. Protein samples were diluted to 100 nM in Kinetics buffer. Buffer, immunogen, and analyte were then applied to a black 96-well Greiner Bio-one microplate at 200 μL per well. Protein A biosensors (FortéBio/Sartorius) were first hydrated for 10 minutes in Kinetics buffer, then dipped into either mACE2-Fc, CR3022, or S309 IgG diluted to 10 μg/mL in Kinetics buffer in the immobilization step. After 500 seconds, the tips were transferred to Kinetics buffer for 60 seconds to reach a baseline. The association step was performed by dipping the loaded biosensors into the immunogens for 300 seconds, and subsequent dissociation was performed by dipping the biosensors back into Kinetics buffer for an additional 300 seconds. The data were baseline subtracted prior to plotting using the FortéBio analysis software (version 12.0). Plots in
RBD-I53-50 nanoparticles were first diluted to 75 μg/mL in 50 mM Tris pH 7, 185 mM NaCl, 100 mM Arginine, 4.5% v/v Glycerol, 0.75% w/v CHAPS, and S-2P protein was diluted to 0.03 mg/mL in 50 mM Tris pH 8, 150 mM NaCl, 0.25% L-Histidine prior to application of 3 μL of sample onto freshly glow-discharged 300-mesh copper grids. Sample was incubated on the grid for 1 minute before the grid was dipped in a 50 μL droplet of water and excess liquid blotted away with filter paper (Whatman). The grids were then dipped into 6 μL of 0.75% w/v uranyl formate stain. Stain was blotted off with filter paper, then the grids were dipped into another 6 μL of stain and incubated for ˜70 seconds. Finally, the stain was blotted away and the grids were allowed to dry for 1 minute. Prepared grids were imaged in a Talos model L120C electron microscope at 45,000× (nanoparticles) or 92,000× magnification (S-2P).
Dynamic Light Scattering (DLS) was used to measure hydrodynamic diameter (Dh) and % Polydispersity (% Pd) of RBD-I53-50 nanoparticle samples on an UNcle Nano-DSF (UNchained Laboratories). Sample was applied to a 8.8 μL quartz capillary cassette (UNi, UNchained Laboratories) and measured with 10 acquisitions of 5 seconds each, using auto-attenuation of the laser. Increased viscosity due to 4.5% v/v glycerol in the RBD nanoparticle buffer was accounted for by the UNcle™ Client software in Dh measurements.
Monomeric RBD, RBD-I53-50A fusion proteins, and RBD-I53-50 nanoparticle immunogens were diluted to 2.5 μM in 50 mM Tris pH 7.0, 185 mM NaCl, 100 mM Arginine, 4.5% v/v glycerol, 0.75% w/v CHAPS, and guanidine chloride [GdnHCl] ranging from 0 M to 6.5 M, increasing in 0.25 M increments, and prepared in triplicate. S-2P trimer was also diluted to 2.5 μM using 50 mM Tris pH 8, 150 mM NaCl, 0.25% L-Histidine, and the same GuHCl concentration range. Dilutions were mixed 10× by pipetting. The samples were then incubated 18-19 hours at ambient temperature. Using a Nano-DSF (UNcle™, UNchained Laboratories) and an 8.8 μL quartz capillary cassette (UNi™, UNchained Laboratories), fluorescence spectra were collected in triplicate, exciting at 266 nm and measuring emission from 200 nm to 750 nm at 25° C.
Endotoxin levels in protein samples were measured using the EndoSafem Nexgen-MCS System (Charles River). Samples were diluted 1:50 or 1:100 in Endotoxin-free LAL reagent water, and applied into wells of an EndoSafe™ LAL reagent cartridge. Charles River EndoScan™-V software was used to analyze endotoxin content, automatically back-calculating for the dilution factor. Endotoxin values were reported as EU/mL which were then converted to EU/mg based on UV/vis measurements. Our threshold for samples suitable for immunization was <50 EU/mg.
Ultraviolet-visible spectrophotometry (UV/vis) was measured using an Agilent Technologies Cary™ 8454. Samples were applied to a 10 mm, 50 μL quartz cell (Starna Cells, Inc.) and absorbance was measured from 180 to 1000 nm. Net absorbance at 280 nm, obtained from measurement and single reference wavelength baseline subtraction, was used with calculated extinction coefficients and molecular weights to obtain protein concentration. The ratio of absorbance at 320/280 nm was used to determine relative aggregation levels in real-time stability study samples. Samples were diluted with respective purification/instrument blanking buffers to obtain an absorbance between 0.1 and 1.0. All data produced from the UV/vis instrument was processed in the 845× UV/visible System software.
To identify site-specific glycosylation profiles, including glycoform distribution and occupancy determination, a bottom up mass spectrometry (MS) approach was utilized. Aliquots of 1 mg/mL monomeric, 8GS, 12GS and 16GS RBD protein were prepared to evaluate the glycosylation profiles at N331 and N343 of the four RBD variants. Comprehensive glycoprofiling on the stabilized Spike ectodomain (S-2P) was performed in parallel using 1.5 mg/mL SARS-CoV-2 S-2P protein. All the samples were denatured in a solution containing 25 mM Tris (pH 8.0), 7 M guanidinium chloride (GdnHCl) and 50 mM dithiothreitol (DTT) at 90° C. for 30 minutes. Reduced cysteines were alkylated by adding fresh iodoacetamide (IAA) to 100 mM and incubating at room temperature for 1 hour in the dark. 50 mM excess DTT was then added to quench the remaining IAA. The GndHCl concentration was reduced to 0.6 M by diluting the samples 11-fold with a 10 mM Tris (pH 8.0), 2 mM calcium chloride solution. Each sample was then split in half. One half (275 μL) was mixed with 10 units of recombinant Peptide N-glycanase F (GST-PNGase F) (Krenkova et al., 2013) and incubated at 37° C. for 1 hour in order to convert glycosylated Asn into deglycosylated Asp.
Protease digestions were performed in the following manner: all RBD samples and one S-2P sample were digested with Lys-C at a ratio of 1:40 (w/w) for RBD and 1:30 (w/w) for S-2P for 4 hours at 37° C., followed by Glu-C digestion overnight at the same ratios and conditions. The other three S-2P samples were digested with trypsin, chymotrypsin and alpha lytic protease, respectively, at a ratio of 1:30 (w/w) overnight at 37° C. All the digestion proteases used were MS grade (Promega). The next day, the digestion reactions were quenched by 0.02% formic acid (FA, Optima™, Fisher).
The glycoform determination of four S-2P samples was performed by nano LC-MS using an Orbitrap Fusion™ mass spectrometer (Thermo Fisher). The digested samples were desalted by Sep-Pak C18 cartridges (Waters) following the manufacturer's suggested protocol. A 2 cm trapping column and a 35 cm analytical column were freshly prepared in fused silica (100 μm ID) with 5 μM ReproSil-Pur™ C18 AQ beads (Dr. Maisch). 8 μL sample was injected and run by a 60-minute linear gradient from 2% to 30% acetonitrile in 0.1% FA, followed by 10 minutes of 80% acetonitrile. An EThcD method was optimized as followed: ion source: 2.1 kV for positive mode; ion transfer tube temperature: 350° C.; resolution: MS1=120000, MS2=30000; AGC target: MS1=2e5, MS2=1e5; and injection time: MS1=50 ms, MS2=60 ms.
Glycopeptide data were visualized and processed by Byonic™ and Byologic™ (Version 3.8, Protein Metrics Inc.) using a 6 ppm precursor and 10 ppm fragment mass tolerance. Glycopeptides were searched using the N-glycan 309 mammalian database in Protein Metrics PMI-Suite and scored based on the assignment of correct c- and z-fragment ions. The true-positive entities were further validated by the presence of glycan oxonium ions m/z at 204 (HexNAc ions) and 366 (HexNAcHex ions) and the absence in its corresponding spectrum in the deglycosylated sample. The relative abundance of each glycoform was determined by the peak area analyzed in Byologic™. Glycoforms were categorized in Oligo (Oligomannose), Hybrid, and Complex as well as subtypes in Complex, described in the previous study (Watanabe et al., 2020). HexNAc(2)Hex(9-5) is M(annose)9 to M5; HexNAc(3)Hex(5-6) is classified as Hybrid; HexNAc(3)Hex(3-4)X is A1 subtype; HexNAc(4)X is A2/A1B; HexNAc(5)X is A3/A2B and HexNAc(6)X is A4/A3B subtype. Hybrid and Complex forms with fucosylation are separately listed as FHybrid and FComplex (eg. FA1), respectively.
Glycan occupancy analysis and glycoform determination of the four RBD variants were performed by LC-MS on the Synapt G2-Si™ TOF mass spectrometer coupled to an Acquity™ UPLC system (Waters). Samples were resolved over a Waters CSH C18 1×100 mm 1.7 μm column with a linear gradient from 3% to 40% B over 30 minutes (A: 98% water, 2% acetonitrile, 0.1% FA; B: 100% acetonitrile, 0.1% FA). Data dependent acquisition (DDA) method was utilized with precursor mass range 300-2000, MS/MS mass range 50-2000 and a collision energy ramped from 70 to 100 V. Chromatographic peaks for the most abundant and non-overlapped isotopic peaks were determined and integrated with MassLynx™ (Waters). All the water and organic solvents used, unless specifically stated, were MS grade (Optima™, Fisher). The peak area ratio of the non-glycosylated (Asn) to the deglycosylated (Asp) glycopeptide was used to measure the glycan occupancy at each site.
3 μg of monomeric RBD and RBD-8GS-I53-50A were incubated and H/D exchanged (HDX) in the deuteration buffer (pH* 7.6, 85% D2O, Cambridge Isotope Laboratories, Inc.) for 3, 60, 1800, and 72000 seconds, respectively, at 23° C. Samples were subsequently mixed 1:1 with ice-cold quench buffer (200 mM tris(2-chlorethyl) phosphate (TCEP), 8 M Urea, 0.2% formic acid) for a final pH 2.5 and immediately flash frozen in liquid nitrogen. Samples were in-line pepsin digested and analyzed by LC-MS-IMS on Synapt G2-Si™ TOF mass spectrometer (Waters) as previously described (Verkerke et al., 2016) with an 18 minute gradient applied. A fully deuteration control was made by collecting the pepsin digest eluate from an undeuterated sample LC-MS run, drying by speedvac, incubating in deuteration buffer for 1 hour at 85° C., and quenching the same as all other HDX samples. Internal exchange standards (Pro-Pro-Pro-Ile [PPPI] and Pro-Pro-Pro-Phe [PPPF]) were added in each sample to ensure consistent labeling conditions for all samples (Zhang et al., 2012). Pepsin digests for undeuterated samples were also analyzed by nano LC-MS using an Orbitrap Fusion™ mass spectrometer (Thermo Fisher) with the settings as described above for glycoprofiling. The data was then processed by Byonic™ to obtain the peptide reference list. Peptides were manually validated using DriftScope™ (Waters) and identified with orthogonal retention time (rt) and drift time (dt) coordinates. Deuterium uptake analysis was performed with HX-Express v2 (Guttman et al., 2013; Weis et al., 2006). Peaks were identified from the peptide spectra with binomial fitting applied. The deuterium uptake level was normalized relative to fully deuterated standards.
Female BALB/c (Stock: 000651) mice were purchased at the age of four weeks from The Jackson Laboratory, Bar Harbor, Maine, and maintained at the Comparative Medicine Facility at the University of Washington, Seattle, WA, accredited by the American Association for the Accreditation of Laboratory Animal Care International (AAALAC). At six weeks of age, 10 mice per dosing group were vaccinated with a prime immunization, and three weeks later mice were boosted with a second vaccination. Prior to inoculation, immunogen suspensions were gently mixed 1:1 vol/vol with AddaVax™ adjuvant (Invivogen, San Diego, CA) to reach a final concentration of 0.009 or 0.05 mg/mL antigen. Mice were injected intramuscularly into the gastrocnemius muscle of each hind leg using a 27-gauge needle (BD, San Diego, CA) with 50 μL per injection site (100 μL total) of immunogen under isoflurane anesthesia. To obtain sera all mice were bled two weeks after prime and boost immunizations. Blood was collected via submental venous puncture and rested in 1.5 mL plastic Eppendorf tubes at room temperature for 30 minutes to allow for coagulation. Serum was separated from hematocrit via centrifugation at 2000 g for 10 minutes. Complement factors and pathogens in isolated serum were heat-inactivated via incubating serum at 56° C. for 60 minutes. Serum was stored at 4° C. or −80° C. until use. Six weeks post-boost, mice were exported from Comparative Medicine Facility at the University of Washington, Seattle, WA to an AAALAC accredited Animal Biosafety Level 3 (ABSL3) Laboratory at the University of North Carolina, Chapel Hill. After a 7-day acclimation time, mice were anesthetized with a mixture of ketamine/xylazine and challenged intranasally with 105 plaque-forming units (pfu) of mouse-adapted SARS-CoV-2 MA strain for the evaluation of vaccine efficacy (IACUC protocol 20-114.0). After infection, body weight was monitored daily until the termination of the study two days post-infection, when lung and nasal turbinate tissues were harvested to evaluate the viral load by plaque assay. All experiments were conducted at the University of Washington, Seattle, WA, and University of North Carolina, Chapel Hill, NC according to approved Institutional Animal Care and Use Committee protocols.
Kymab Darwin™ mice (a mix of males and females, 10 weeks of age), 5 mice per dosing group, were vaccinated with a prime immunization and three weeks later boosted with a second vaccination. Prior to inoculation, immunogen suspensions were gently mixed 1:1 vol/vol with AddaVax™ adjuvant (Invivogen) to reach a final concentration of 0.009 or 0.05 mg/mL antigen. Mice were injected intramuscularly into the tibialis muscle of each hind leg using a 30-gauge needle (BD) with 20 μL per injection site (40 μL total) of immunogen under isoflurane anesthesia. A final boost was administered intravenously (50 μL) with no adjuvant at week 7. Mice were sacrificed 5 days later under UK Home Office Schedule 1 (rising concentration of CO2) and spleen, lymph nodes, and bone marrow cryopreserved. Whole blood (0.1 ml) was collected 2 weeks after each dose (weeks 0, 2, 5, and week 8 terminal bleed). Serum was separated from hematocrit via centrifugation at 2000 g for 10 minutes. Serum was stored at −20° C. and was used to monitor titers by ELISA. All mice were maintained and all procedures carried out under United Kingdom Home Office License 70/8718 and with the approval of the Wellcome Trust Sanger Institute Animal Welfare and Ethical Review Body.
ELISA
For anti-S-2P ELISA, 25 μL of 2 μg/mL S-2P was plated onto 384-well Nunc Maxisorp™ (ThermoFisher) plates in PBS and sealed overnight at 4° C. The next day plates were washed 4× in Tris Buffered Saline Tween (TBST) using a plate washer (BioTek) and blocked with 2% BSA in TBST for 1 h at 37° C. Plates were washed 4× in TBST and 1:5 serial dilutions of mouse, NHP, or human sera were made in 25 μL TBST starting at 1:25 or 1:50 and incubated at 37° C. for 1 h. Plates were washed 4× in TBST, then anti-mouse (Invitrogen) or anti-human (Invitrogen) horseradish peroxidase-conjugated antibodies were diluted 1:5,000 and 25 μL added to each well and incubated at 37° C. for 1 h. Plates were washed 4× in TBST and 25 μL of TMB (SeraCare) was added to every well for 5 min at room temperature. The reaction was quenched with the addition of 25 μL of 1N HCl. Plates were immediately read at 450 nm on a VarioSkanLux™ plate reader (ThermoFisher) and data plotted and fit in Prism™ (GraphPad) using nonlinear regression sigmoidal, 4PL, X is log(concentration) to determine EC50 values from curve fits.
MLV-based SARS-CoV-2 S, SARS-CoV S, and WIV-1 pseudotypes were prepared as previously described (Millet and Whittaker, 2016; Walls et al., 2020). Briefly, HEK293T cells were co-transfected using Lipofectamine™ 2000 (Life Technologies) with an S-encoding plasmid, an MLV Gag-Pol packaging construct, and the MLV transfer vector encoding a luciferase reporter according to the manufacturer's instructions. Cells were washed 3× with Opti-MEM and incubated for 5 h at 37° C. with transfection medium. DMEM containing 10% FBS was added for 60 h. The supernatants were harvested by a 2,500 g spin, filtered through a 0.45 μm filter, concentrated with a 100 kDa membrane for 10 min at 2,500 g and then aliquoted and placed at −80° C.
HEK-hACE2 cells were cultured in DMEM with 10% FBS (Hyclone) and 1% PenStrep with 8% CO2 in a 37° C. incubator (Thermofisher). One day prior to infection, 40 μL of poly-lysine (Sigma) was placed into 96-well plates and incubated with rotation for 5 min. Poly-lysine was removed, plates were dried for 5 min then washed 1× with DMEM prior to plating cells. The following day, cells were checked to be at 80% confluence. In a half-area 96-well plate a 1:3 serial dilution of sera was made in DMEM starting between 1:3 and 1:66 initial dilution in 22 μL final volume. 22 μL of pseudovirus was then added to the serial dilution and incubated at room temperature for 30-60 min. HEK-hACE2 plate media was removed and 40 μL of the sera/virus mixture was added to the cells and incubated for 2 h at 37° C. with 8% CO2. Following incubation, 40 μL 20% FBS and 2% PenStrep containing DMEM was added to the cells for 48 h. Following the 48-h infection, One-Glo-EX™ (Promega) was added to the cells in half culturing volume (40 μL added) and incubated in the dark for 5 min prior to reading on a Varioskan™ LUX plate reader (ThermoFisher). Measurements were done on all ten mouse sera samples from each group in at least duplicate. Relative luciferase units were plotted and normalized in Prism™ (GraphPad) using a zero value of cells alone and a 100% value of 1:2 virus alone. Nonlinear regression of log(inhibitor) vs. normalized response was used to determine IC50 values from curve fits. Mann-Whitney tests were used to compare two groups to determine whether they were statistically different.
SARS-CoV-2-nanoLuc virus (WA1 strain) in which ORF7 was replaced by nanoluciferase gene (nanoLuc), and mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA) (Dinnon et al., 2020) were generated by the coronavirus reverse genetics system described previously (Hou et al., 2020). Recombinant viruses were generated in Vero E6 cells (ATCC-CRL 1586) grown in DMEM high glucose media (Gibco #11995065) supplemented with 10% Hyclone™ Fetal Clone II (GE #SH3006603HI), 1% non-essential amino acid, and 1% Pen/Strep in a 37° C. +5% CO2 incubator. To generate recombinant SARS-CoV-2, seven DNA fragments which collectively encode the full-length genome of SARS-CoV-2 flanked by a 5′ T7 promoter and a 3′ polyA tail were ligated and transcribed in vitro. The transcribed RNA was electroporated into Vero E6 cells to generate a P0 virus stock. The seed virus was amplified twice in Vero E6 cells at low moi for 48 h to create a working stock which was titered by plaque assay (Hou et al., 2020). All the live virus experiments, including the ligation and electroporation steps, were performed under biosafety level 3 (BSL-3) conditions at negative pressure, by operators in Tyvek suits wearing personal powered-air purifying respirators.
Vero E6 cells were seeded at 2×104 cells/well in a 96-well plate 24 h before the assay. One hundred pfu of SARS-CoV-2-nanoLuc virus (Hou et al., 2020) were mixed with serum at 1:1 ratio and incubated at 37° C. for 1 h. An 8-point, 3-fold dilution curve was generated for each sample with starting concentration at 1:20 (standard) or 1:2000 (high neutralizer). Virus and serum mix was added to each well and incubated at 37° C. +5% CO2 for 48 h. Luciferase activities were measured by Nano-Glom Luciferase Assay System (Promega, WI) following manufacturer protocol using SpectraMax™ M3 luminometer (Molecular Device). Percent inhibition and 50% inhibition concentration (IC50) were calculated by the following equation: [1-(RLU with sample/RLU with mock treatment)]×100%. Fifty percent inhibition titer (IC50) was calculated in GraphPad Prism™ 8.3.0 by fitting the data points using a sigmoidal dose-response (variable slope) curve.
Recombinant SARS-CoV-2 S-2P trimer was biotinylated using the EZ-Link™ Sulfo-NHS-LC Biotinylation Kit (ThermoFisher) and tetramerized with streptavidin-APC (Agilent) as previously described (Krishnamurty et al., 2016; Taylor et al., 2012). The RBD domain of SARS-CoV-2 S was biotinylated and tetramerized with streptavidin-APC (Agilent). The APC decoy reagent was generated by conjugating SA-APC to Dylight™ 755 using a DyLight 755 antibody labeling kit (ThermoFisher), washing and removing unbound DyLight 755, and incubating with excess of an irrelevant biotinylated His-tagged protein. The PE decoy was generated in the same manner, by conjugating SA-PE to Alexa Fluor 647 with an AF647 antibody labeling kit (ThermoFisher).
For phenotyping of B cells, 6-week old female BALB/c mice, three per dosing group, were immunized intramuscularly with 50 μL per injection site of vaccine formulations containing 5 μg of SARS-CoV-2 antigen (either S-2P trimer or RBD, but not including mass from the I53-50 nanoparticle) mixed 1:1 vol/vol with AddaVax™ adjuvant on day 0. All experimental mice were euthanized for harvesting of inguinal and popliteal lymph nodes on day 11. The experiment was repeated two times. Popliteal and inguinal lymph nodes were collected and pooled for individual mice. Cell suspensions were prepared by mashing lymph nodes and filtering through 100 μM Nitex™ mesh. Cells were resuspended in PBS containing 2% FBS and Fc block (2.4G2), and were incubated with 10 nM Decoy tetramers at room temperature for 20 min. RBD-PE tetramer and Spike-APC tetramer were added at a concentration of 10 nM and incubated on ice for 20 min. Cells were washed, incubated with anti-PE and anti-APC magnetic beads on ice for 30 min, then passed over magnetized LS columns (Miltenyi Biotec). Bound B cells were stained with anti-mouse B220 (BUV737), CD3 (PerCP-Cy5.5), CD138 (BV650), CD38 (Alexa Fluor™ 700), GL7 (eFluor™ 450), IgM (BV786), IgD (BUV395), CD73 (PE-Cy7), and CD80 (BV605) on ice for 20 min. Cells were run on the Cytek Aurora™ and analyzed using FlowJom software (Treestar). Cell counts were determined using Accucheck™ cell counting beads.
A Pigtail macaque was immunized with 250 μg of RBD-12GS-I53-50 nanoparticle (88 μg RBD antigen) at day 0 and day 28. Blood was collected at days 0, 10, 14, 28, 42, and 56 days post-prime. Serum and plasma were collected as previously described (Erasmus et al., 2020). Prior to vaccination or blood collection, animals were sedated with an intramuscular injection (10 mg/kg) of ketamine (Ketaset®; Henry Schein). Prior to inoculation, immunogen suspensions were gently mixed 1:1 vol/vol with AddaVax™ adjuvant (Invivogen, San Diego, CA) to reach a final concentration of 0.250 mg/mL antigen. The vaccine was delivered intramuscularly into both quadriceps muscles with 1 mL per injection site on days 0 and 28. All injection sites were shaved prior to injection and monitored post-injection for any signs of local reactogenicity. At each study timepoint, full physical exams and evaluation of general health were performed on the animals, as previously described (Erasmus et al., 2020), and no adverse events were observed.
Purification of Fabs from NHP serum was adapted from (Boyoglu-Bamum et al., 2020). Briefly, 1 mL of day 56 serum was diluted to 10 mL with PBS and incubated with 1 mL of 3×PBS washed protein A beads (GenScript) with agitation overnight at 37° C. The next day beads were thoroughly washed with PBS using a gravity flow column and bound antibodies were eluted with 0.1 M glycine pH 3.5 into 1M Tris-HCl (pH 8.0) to a final concentration of 100 mM. Serum and early washes that flowed through were re-bound to beads overnight again for a second, repeat elution. IgGs were concentrated (Amicon 30 kDa) and buffer exchanged into PBS. 2×digestion buffer (40 mM sodium phosphate pH 6.5, 20 mM EDTA, 40 mM cysteine) was added to concentrated and pooled IgGs. 500 μL of resuspended immobilized papain resin (ThermoFisher Scientific) freshly washed in 1×digestion buffer (20 mM sodium phosphate, 10 mM EDTA, 20 mM cysteine, pH 6.5) was added to purified IgGs in 2×digestion buffer and samples were agitated for 5 h at 37° C. The supernatant was separated from resin and resin washes were collected and pooled with the resin flow through. Pooled supernatants were sterile-filtered at 0.22 μm and applied 6× to PBS-washed protein A beads in a gravity flow column. The column was eluted as described above and the papain procedure repeated overnight with undigested IgGs to increase yield. The protein A flowthroughs were pooled, concentrated (Amicon 10 kDa), and buffer exchanged into PBS. Purity was checked by SDS-PAGE.
Epitope competition was performed and analyzed using BLI on an Octet™ Red 96 System (Pall™ Fortd Bio/Sartorius) at 30° C. with shaking at 1000 rpm. NTA biosensors (Pall™ Fortd Bio/Sartorius) were hydrated in water for at least 10 minutes, and were then equilibrated in 10× Kinetics buffer (KB) (Pall™ Fortd Bio/Sartorius) for 60 seconds. 10 ng/μL monomeric RBD in 10×KB was loaded for 100 seconds prior to baseline acquisition in 10×KB for 300 seconds. Tips were then dipped into diluted polyclonal Fab in 10× KB in a 1:3 serial dilution beginning with 5000 nM for 2000 seconds or maintained in 10×KB. Tips bound at varying levels depending on the polyclonal Fab concentration. Tips were then dipped into the same concentration of polyclonal Fab plus either 200 nM of hACE2, 400 nM CR3022, or 20 nM S309 and incubated for 300-2000 seconds. The data were baseline subtracted and aligned to pre-loading with polyclonal Fabs using the Pall™ Fortd Bio/Sartorius analysis software (version 12.0) and plotted in PRISM™.
This application is a U.S. national phase of International Application No. PCT/US2021/017799, filed on Feb. 12, 2021, which claims priority to U.S. Provisional Application Ser. No. 63/064,235, filed Aug. 11, 2020; 63/046,159, filed Jun. 30, 2020, and 62/977,036 filed Feb. 14, 2020, all incorporated by reference herein in their entirety.
This invention was made with government support under Grant Nos. HHSN272201700059C and R01 GM120553, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/017799 | 2/12/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63064235 | Aug 2020 | US | |
| 63046159 | Jun 2020 | US | |
| 62977036 | Feb 2020 | US |
