Polypeptides, compositions, and their use to treat or limit development of an infection

#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.

#FIG. 1 (A-H). Design, In Vitro Assembly, and Characterization of SARS-COV-2 RBD Nanoparticle Immunogens (A) Molecular surface representation of the SARS-COV-2 S-2P trimer in the prefusion conformation (PDB 6VYB). The glycan at position N343 was modeled based on PDB 6WPS and the receptor-binding motif (RBM) was modeled from PDB 6M0J). The single open RBD is boxed. (B) Molecular surface representation of the SARS-COV-2 S RBD, including the N-linked glycans at positions 331 and 343. The ACE2 receptor-binding site or RBM is indicated with a black outline. (C) Structural models of the trimeric RBD-I53-50A and pentameric I53-50B components. Upon mixing in vitro, 20 trimeric and 12 pentameric components assemble to form nanoparticle immunogens with icosahedral symmetry. Each nanoparticle displays 60 copies of the RBD. (D) Structural model of the RBD-12GS-I53-50 nanoparticle immunogen. Although a single orientation of the displayed RBD antigen and 12-residue linker are shown for simplicity, these regions are expected to be flexible relative to the I53-50 nanoparticle scaffold. (E) Dynamic light scattering (DLS) of the RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50 nanoparticles compared to unmodified I53-50 nanoparticles. (F) Representative electron micrographs of negatively stained RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50 nanoparticles. The samples were imaged after one freeze/thaw cycle. Scale bars, 100 nm. (G) Hydrogen/Deuterium-exchange mass spectrometry of monomeric RBD versus trimeric RBD-8GS-I53-50A component, represented here as a butterfly plot, confirms preservation of the RBD conformation, including at epitopes recognized by known neutralizing Abs. In the plot, each point along the horizontal sequence axis represents a peptide where deuterium uptake was monitored from 3 seconds to 20 hours. Error bars shown on the butterfly plot indicate standard deviations from two experimental replicates. The difference plot below demonstrates that monomeric RBD and RBD-8GS-I53-50A are virtually identical in local structural ordering across the RBD. (H) Pie charts summarizing the glycan populations present at the N-linked glycosylation sites N331 and N343 in five protein samples: monomeric RBD, S-2P trimer, and RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50A trimeric components. The majority of the complex glycans at both sites were fucosylated; minor populations of afucosylated glycans are set off by dashed lines. Oligo, oligomannose.

#FIG. 2 (A-B). Antigenic Characterization of SARS-COV-2 RBD-I53-50 Nanoparticle Immunogens (A) Bio-layer interferometry of immobilized mACE2-Fc, CR3022 mAb, and S309 mAb binding to RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50 nanoparticles displaying the RBD antigen at 50% or 100% valency. The monomeric SARS-CoV-2 RBD was included in each experiment as a reference. (B) The binding signal at 880 s, near the end of the association phase, is plotted for each experiment in panel (A) to enable comparison of the binding signal obtained from each nanoparticle.

#FIG. 3 (A-E). Physical and Antigenic Stability of RBD Nanoparticle Immunogens and S-2P Trimer (A) Chemical denaturation by guanidine hydrochloride. The ratio of intrinsic tryptophan fluorescence emission at 350/320 nm was used to monitor protein tertiary structure. Major transitions are indicated by shaded regions. Representative data from one of three independent experiments are shown. (B) Summary of SDS-PAGE and nsEM stability data over four weeks. SDS-PAGE showed no detectable degradation in any sample. nsEM revealed substantial unfolding of the S-2P trimer at 2-8° C. after three days incubation, and at 22-27° C. after four weeks. N/A, not assessed. (C) Summary of antigenicity data over four weeks. The antigens were analyzed for mACE2-Fc (solid lines) and CR3022 mAb (dashed lines) binding by bio-layer interferometry after storage at the various temperatures. The plotted value represents the amplitude of the signal near the end of the association phase normalized to the corresponding <−70° C. sample at each time point. (D) Summary of UV/vis stability data over four weeks. The ratio of absorbance at 320/280 nm is plotted as a measure of particulate scattering. Only the S-2P trimer and the RBD-12GS-I53-50 nanoparticle showed any increase in scattering, and only at ambient temperature. (E) DLS of the RBD-12GS-I53-50 nanoparticle indicated a monodisperse species with no detectable aggregate at all temperatures and time points. The data in panels B-E is from a four-week real-time stability study that was performed once.

#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.