WO2021237124A1 - Sars-cov-2 spike protein pseudotyped vsv-delta g particles and uses thereof - Google Patents

Sars-cov-2 spike protein pseudotyped vsv-delta g particles and uses thereof Download PDF

Info

Publication number
WO2021237124A1
WO2021237124A1 PCT/US2021/033713 US2021033713W WO2021237124A1 WO 2021237124 A1 WO2021237124 A1 WO 2021237124A1 US 2021033713 W US2021033713 W US 2021033713W WO 2021237124 A1 WO2021237124 A1 WO 2021237124A1
Authority
WO
WIPO (PCT)
Prior art keywords
vsv
cov
sars
protein
pseudotyped
Prior art date
Application number
PCT/US2021/033713
Other languages
French (fr)
Inventor
Benhur Lee
Kasopefoluwa OGUNTUYO
Satoshi IKEGAME
Christian Stevens
Original Assignee
Icahn School Of Medicine At Mount Sinai
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Icahn School Of Medicine At Mount Sinai filed Critical Icahn School Of Medicine At Mount Sinai
Publication of WO2021237124A1 publication Critical patent/WO2021237124A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20241—Use of virus, viral particle or viral elements as a vector
    • C12N2760/20243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • VNA virus neutralization assay
  • a SARS-CoV-2 spike protein pseudotyped vesicular stomatitis virus (VSV) particle comprising an encapsidated negative sense, single-stranded RNA genome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, and a nucleotide sequence encoding luciferase, wherein genome does not express VSV glycoprotein (G).
  • VSV VSV glycoprotein
  • the luciferase is renilla luciferase or nanoluciferase.
  • the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the SARS-CoV-2 spike protein is encoded by the nucleotide sequence set forth in SEQ ID NO:l.
  • the genome does not comprise a nucleotide sequence sequence encoding VSV glycoprotein (G). In some embodiments, the genome further comprises a nucleotide sequence encoding a fluorescent protein. In some embodiments, the fluorescent protein is red fluorescent protein or enhanced green fluorescent protein.
  • the SARS-CoV-2 spike protein pseudotyped VSV particle has been treated with trypsin. In some embodiments, the SARS-CoV-2 spike protein pseudotyped VSV particle has been treated with trypsin and soybean inhibitor.
  • composition comprising a SARS-CoV-2 spike protein pseudotyped VSV particle described herein and a carrier.
  • the carrier is serum free media.
  • the carrier is phosphate buffered saline.
  • composition comprising a SARS-CoV-2 spike protein pseudotyped VSV particle disclosed herein and trypsin. Also provided is a composition comprising a SARS-CoV-2 spike protein pseudotyped VSV particle disclosed herein and trypsin and soybean inhibitor.
  • a method for generating SARS-CoV-2 spike protein pseudotyped VSV particles comprising:
  • N nucleotide sequence encoding for VSV nucleoprotein
  • M nucleotide sequence encoding for VSV matrix
  • L nucleotide
  • the cells are cultured in optiMEM containing anti-VSV-G antibody.
  • the SARS-CoV-2 spike protein pseudotyped VSV particles are purified from the supernatant by low speed centrifugation.
  • the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:2.
  • the SARS-CoV-2 spike protein is encoded by the nucleotide sequence set forth in SEQ ID NO:l.
  • the luciferase is renilla luciferase.
  • step (a) farther comprises contacting the particle with soybean inhibitor.
  • the certain period of time is 15 minutes.
  • a method for detecting sera that neutralizes SARS-CoV- 2 comprising:
  • step (c) measuring the luciferase activity after a third period of time, wherein a lower level of luciferase activity is detected if the sera neutralizes SARS-CoV-2 spike protein pseudotyped VSV particle than if steps (b) to (c) are performed without performing step (a), and the lower level of luciferase activity indicates that the sera neutralizes SARS-CoV-2.
  • the first period of time is about 30 minutes. In some embodiments, the second period of time is about 1 hour. In some embodiments, the third period of time is about 18 to 22 hours.
  • the subject is a human subject.
  • the sera is heat inactivated.
  • the sera is diluted in plain DMEM or DMEM and 10% heat inactivated fetal bovine serum.
  • the method for detecting sera that neutralizes SARS-CoV-2 comprises concurrently repeating steps (a) to (c) with a positive control antibody or sera that does neutralize SARS-CoV-2. In some embodiments, the method further comprises concurrently repeating steps (a) to (c) with a negative control antibody or sera that does not neutralize SARS-CoV-2.
  • a method for assessing the ability of an antibody to neutralize SARS-CoV-2 comprising:
  • the first period of time is about 30 minutes.
  • the second period of time is about 1 hour.
  • the third period of time is about 18 to 22 hours.
  • the cells overexpress human ACE-2, TMPRSS2, or both.
  • kits comprising the SARS-CoV-2 spike protein pseudotyped VSV particle disclosed herein, and optionally instructions for performing a neutralization assay using the SARS-CoV-2 spike protein pseudotyped VSV particle.
  • a nucleic acid sequence comprising a VSV antigenome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding for luciferase, and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G).
  • the nucleic acid further comprises a T7 promoter, autocatalytic hammerhead ribozyme sequences, and a T7 terminator, optionally wherein the hammerhead ribozyme sequences is immediately upstream of the 3 ’ leader sequence.
  • the luciferase is renilla luciferase or nanoluciferase.
  • the fluorescent protein is red fluorescent protein or enhanced green fluorescent protein.
  • a recombinant VSV particle pseudotyped with VSV glycoprotein comprising an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding for luciferase, and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G).
  • the luciferase is renilla luciferase or nanoluciferase.
  • the fluorescent protein is red fluorescent protein or enhanced green fluorescent protein.
  • a method for generating the recombinant VSV particle pseudotyped with VSV glycoprotein disclosed herein comprising:
  • nucleic acid sequence comprises a VSV antigenome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding for luciferase, and a nucleotide sequence encoding a fluorescent protein, wherein the nucleic acid sequence comprises a VSV antigenome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding for luciferase,
  • the method farther comprising transfecting the cells with a sixth vector comprising a codon-optimized sequence encoding a T7 polymerase.
  • a method for generating pseudotyped VSV particle comprising:
  • the cells are 293T-ACE2 clone 5-7 or 293T-ACE2- TMPRSS2 clone F8-2.
  • FIGs. 1A, IB, 1C, ID, and IE illustrate the production of VSV ⁇ G-rLuc bearing SARS-CoV-2 spike glycoprotein.
  • FIG. 1A Overview of VSV ⁇ G-rLuc pseudotyped particles bearing CoV-2 spike (top panel) with annotated spike glycoprotein domains and cleavage sites (bottom panel). SARS-CoV is referred to as SARS-CoV-1 for greater clarity.
  • FIG. IB Overview of VSV ⁇ G-rLuc pseudotyped particles bearing CoV-2 spike (top panel) with annotated spike glycoprotein domains and cleavage sites.
  • SARS-CoV is referred to as SARS-CoV-1 for greater clarity.
  • FIG. IB Overview of VSV ⁇ G-rLuc pseudotyped particles bearing CoV-2 spike (top panel) with annotated spike glycoprotein domains and cleavage sites.
  • FIG. IB is referred to as SARS-CoV-1 for greater clarity.
  • pseudotyped particles bearing the Nipah virus receptor binding protein alone (NiV- RBPpp), SARS-CoV-2-S (CoV2pp), or VSV-G (VSV-Gpp) were titered on Vero-CCL81 cells using a 10-fold serial dilution. Symbols represent the mean +/- SEM (error bars) of each titration performed in technical triplicates.
  • FIG. 1C Genome copy number and particle to infectivity ratio. Genome copy number was assessed using primers against the VSV-L protein as previously described (Pryce R, Azarm K et al Life Sci Alliance 2020 Jan; 3(1): e201900578).
  • FIG. ID Expression of the indicated viral glycoproteins on producer cells and their incorporation into VSVpp. Western blots performed using anti-Sl or anti-S2 specific antibodies.
  • FIG. IE CoV2pp entry is inhibited by soluble receptor binding domain (sRBD) derived from SARS-CoV-2-S. CoV2pp and VSV-Gpp infection of Vero-CCL81 cells was performed as in FIG. IB in the presence of the indicated amounts of sRBD. Neutralization curves were generated by fitting data points using a variable slope, 4- parameter logistics regression curve (robust fitting method). The last point (no sRBD) was fixed to represent 100% maximal infection.
  • FIGs. 2A, 2B, 2C, 2D, and 2E illustrate that CoV2pp entry is enhanced by trypsin treatment.
  • FIG. 2A Optimizing trypsin treatment conditions. Supernatant containing CoV2pp were trypsin-treated at the indicated concentrations for 15 min. at room temperature prior to the addition of 625 pg/mL of soybean trypsin inhibitor (SBTI). These particles were then titered on Vero-CCL81 cells in technical triplicates. Data shown as mean +/- SEM.
  • FIG. 2B Optimizing trypsin treatment conditions. Supernatant containing CoV2pp were trypsin-treated at the indicated concentrations for 15 min. at room temperature prior to the addition of 625 pg/mL of soybean trypsin inhibitor (SBTI). These particles were then titered on Vero-CCL81 cells in technical triplicates. Data shown as mean +/- SEM.
  • FIG. 2B The calculated IC50 for sRBD neutralization
  • FIG. 2C Dilution in serum free media (SEM, DMEM only) provides the highest signalmoise ratio for trypsin-treated CoV2pp entry. Particles were diluted 1:10 in Opti-MEM, SFM, or DMEM + 10%FBS prior to infection of Vero-CCL81 cells and spinoculation as described in Fig. IE. Cells infected without spinoculation show approximately 3x less signalmoise ratios.
  • FIG. 2C Dilution of CoV2pp in the absence of serum free media produces the highest signalmoise for trypsin treated CoV2pp. Presented are the results from an experiment in technical triplicate and error bars show the SEM.
  • FIG. 2D Dilution in serum free media
  • FIG. 2E Effect of different trypsin concentration on CoV2pp activation. Supernatant containing CoV2pp were treated with different concentrations of trypsin for 15 minutes, then used to infect Vero-CCL81 cells.
  • FIG. 3 illustrates that trypsin- treated CoV2pp depend on ACE2 and TMPRSS2 for entry. Parental and TMPRSS2 or ACE2 transduced VeroCCL81 cells were infected with the indicated pseudotyped viruses.
  • FIGs. 4A, 4B, and 4C illustrate sera neutralization in the absence of 10% FBS and optimization of neutralizations.
  • FIG. 4A Negative sera potently inhibit trypsin treated CoV2pp. CoV2pp were diluted in serum free media (SFM), then pooled negative sera and a positive serum were used to neutralize entry. An aliquot was heat inactivated (HI) for lhr in a 56°C water bath prior to use. Data are presented on a linear (top panel) and log scale (bottom panel). Each replicate from one experiment in technical duplicates are shown and neutralization curves were generated as done in Fig. ID. FIG. 4B.
  • SFM serum free media
  • Sera neutralizations were performed with untreated CoV2pp (top panel) or CoV2pp treated with trypsin (middle panel). Both particles were diluted in DMEM + 10% FBS and neutralization curves are presented as described above. VSV-G was not neutralized by the negative or positive sera (bottom panel).
  • FIG. 4C sRBD neutralizes CoV2pp equivalently across all conditions tested. Data presented in Fig. IE (i.e. the untreated CoV2pp) is duplicated here.
  • FIG. 5 shows CoV2pp viral neutralization assay and absIC50/80 versus Spike binding of patient sera.
  • CoV2pp were used to infect Vero-CCF81 cells in the presence of a 4-fold serial dilution of patient sera. Samples in light grey do not neutralize CoV2pp.
  • Neutralization curves were fit using a variable slope, 4-parameter logistics regression curve with a robust fitting method.
  • FIG. 6 shows a comparison of CoV2pp Absolute IC values across all 4 groups. Shown are the CoV2pp absolute IC50 (top panel), IC80 (middle panel) and IC90 (bottom panel) from all four groups with error bars showing the median and interquartile range. The dotted line presents the median from the aggregated positive neutralization samples as reported in Table 1. The dashed line indicates neat serum and the shaded gray region highlights samples that fall below this value. An ordinary one-way ANOVA with Dunnett’s correction for multiple comparisons was performed for statistical analysis. This analysis revealed no statistically significant difference between the Absolute IC values obtained across the 4 groups. There were notable outliers in this data set, including individuals that show poor neutralization (i.e.
  • FIGs. 7A, 7B, 7C, and 7D illustrate that 293T stably transduced with ACE2 and TMPRSS2 (293T-ACE2+TMPRSS2) are ultra-permissive for SARS-CoV-2pp infection.
  • FIG. 7A Infection of 293T cells lines transduced to stably express, TMPRSS2, ACE2, or both. A single dilution of particles was used to infect cells prior to spinoculation. Infections were done in technical triplicates. Presented are the aggregated results from two independent replicates and error bars show SEM.
  • FIG. 7B Normalized CoV2pp entry into single cell clones. Entry was normalized to the wild type parental cell line and further normalized to VSV-G entry. Presented are the average of one experiment in technical triplicates. Error bars show the median and interquartile range.
  • FIG. 7C CoV2pp were titered on Vero-CCL81 cells, 293T-ACE2 clone 5-7, and 293T-ACE2-TMPRSS2 clone F8-2. Titrations were performed with untreated CoV2pp and without spinoculation. Presented are the results from technical triplicates and bars show the SEM.
  • FIG. 7D Entry inhibition of CoV2pp by Nafamostat mesylate, a serine protease inhibitor.
  • Nafamostat was mixed with CoV2pp (top panel) or VSV-Gpp (bottom panel) prior addition to cells. Shown are the results from one experiment in technical triplicates. Error bars show SEM.
  • FIGs. 8A and 8B illustrate that ultra-permissive 293T-ACE2+TMPRSS2 cell clones retain the same phenotypic sensitivity to convalescent COVID-19 sera.
  • FIG. 8A Selection of pooled sera samples. Presented are the subset of samples that were pooled for use in viral neutralization assays (VNAs).
  • FIG. 8B Vero CCL81 and transduced 293T cells were used for VNAs. Sera previously shown to be negative, weakly positive, or strongly positive for CoV2pp neutralizations were selected to be pooled in equal volumes. These were subsequently used for VNAs. Notably, these VNAs were performed in the absence of exogenous trypsin or spinoculation.
  • FIGs. 9A and 9B illustrate the robust and efficient generation of an EGFP-reporter replication-competent VSV bearing SARS-CoV-2 spike (rcVSV-CoV2-S).
  • FIG. 9A Schematic of the rcVSV-CoV2-S genomic coding construct and the virus rescue procedure.
  • the maximal T7 promoter (T7prom) followed by a hammer-head ribozyme (HhRbz) and the HDV ribozyme (HDVRbz) plus T7 terminator (T7term) are positioned at the 3’ and 5’ ends of the viral cDNA, respectively.
  • An EGFP(E) transcriptional unit is placed at the 3’ terminus to allow for high level transcription.
  • SARS-CoV-2-S is cloned in place of VSV-G using the indicated restriction sites designed to facilitate easy exchange of spike variant or mutants.
  • FIG. 9B For virus rescue, highly permissive 293T cells stably expressing human ACE2 and TMPRSS2 (293T- [ACE2+TMPRSS2], F8-2 clone) cells were transfected with the genome coding plasmid, helper plasmids encoding CMV-driven N, P, M, and L genes, and pCAGS encoding codon-optimized T7-RNA polymerase(T7opt). 48-72 hpi, transfected cells turn EGFP+ and start forming syncytia. Supernatant containing rcVSV-CoV2-S are then amplified in Vero-TMPRSS2 cells at the scale shown. The blue arrowsat the bottom indicate the timeline for production of each sequence verified stock.
  • FIGs. 10A and 10B illustrate the generation of replication-competent VSV bearing SARS-CoV-2 spike (rcVSV- CoV2-S).
  • FIG. 10A Representative images of de novo generation of rcVSV-CoV2-S, carrying an EGFP reporter, in transfected 293T- ACE2+TMPRSS2 (F8-2) cells as described in FIG. 9. Single GFP+ cells detected at 2-3 days post-transfection (dpt) form a foci of syncytia by 4 dpt. Images are taken by Celigo imaging cytometer (Nexcelom) and are computational composites from the identical number of fields in each well. White bar is equal to 1 millimeter.
  • FIG. 10B illustrates the generation of replication-competent VSV bearing SARS-CoV-2 spike
  • FIGS. 11A and 11B illustrate the results of a neutralization activity of antibody responses elicited by the Sputnik V vaccine.
  • FIG. 11B For each serum sample, the fold-change in IC50 (reciprocal inhibitory dilution factor) against the indicated variant and mutant spike proteins relative to its IC50 against wild- type (WT) spike (set at 1) is plotted. Adjusted p values were calculated as in FIG. 11 A. Medians are represented by the bars and whi skersdem arcate the 95 % Cl. Neutralization dose- response curves were performed in triplicates, and the mean values from each triplicate experiment are shown as the single data points for each sera sample.
  • VNA Standardized virus neutralization assay
  • VNT virus neutralization titers
  • SARS-CoV-2 spike glycoprotein is embedded in the viral envelope and facilitates bothreceptor recognition and membrane fusion.
  • SARS-CoV-2-S is 1273 amino acids in length and, like other coronaviruses, is a trimeric class I fusion protein.
  • the S glycoprotein contains two subunits, the N-terminal, SI subunit and the C-terminal, S2 subunit.
  • the SI subunit contains the receptor-binding domain (RBD), which is responsible for host receptor binding.
  • RBD receptor-binding domain
  • the S2 subunit contains the transmembrane domain, cytoplasmic tails, and machinery necessary for fusion, notably the fusion peptide and heptad repeats.
  • Angiotensin-converting enzyme 2 (ACE2), a cell surface enzyme found in a variety of tissues, facilitates binding and entry of SARS-CoV-2.
  • ACE2 angiotensin-converting enzyme 2
  • ACE2 alone is not sufficient for efficient entry into cells.
  • entry depends on the SI subunit binding ACE2, entry is further enhanced by proteolytic cleavage between the S1/S2 and S2’ subunits.
  • proteolytic cleavage between the S1/S2 and S2’ subunits For both SARS-CoV-1 and SARS-CoV-2, this cleavage-mediated activation of S-mediated entry is supported by the expression of cell-associated proteases, like cathepsins or transmembrane serine protease 2 (TMPRSS2), or the addition of exogenous proteases that mimic the various trypsin-like proteases present in the extracellular lung milieu.
  • TMPRSS2 transmembrane serine protease 2
  • VNA viral neutralization assay
  • Antibody titers appear to be durable at greater than 40 days post infection, but in the case of SARS-CoV-1, reductions in IgG positive titers begin around 4-5 months post infection and show a significant drop by 36 months. Although there are reports of SARS- CoV-2 infected individuals testing positive by RT-PCR weeks after being confirmed as recovered by two consecutive negative tests, these are more likely the result of false negatives than of reinfection. A better understanding of the durability and efficacy of the neutralizing antibody response in patients previously infected with SARS-CoV-2 is of paramount importance.
  • HCVs human coronaviruses
  • Humoral immune responses to the SARS-CoV-2 S protein are typically evaluated by enzyme-linked immunosorbent assays (ELISAs) and its many variants (CLIA, LFA, etc.). These serological binding assays rightfully play a central role in determining patient antibody responses and can complement diagnostics and sero-epidemiological studies, especially when combined with antibody subclass determination (IgM, IgA and IgG). Nonetheless, as many antibodies generated to the spike protein bind but do not block virus entry, ELISA-based assays that detect titers of spike-binding antibodies cannot always correlate perfectly with neutralizing antibody titers as measured by plaque reduction neutralization or microneutralization tests.
  • ELISAs enzyme-linked immunosorbent assays
  • compositions and methods utilizing a SARS-CoV-2 pseudotyped viral particle by using vesicular stomatitis virus bearing e fluorescent report egene (e.g., the Renilla luciferase gene) in place of its G glycoprotein (VSV ⁇ G-rLuc).
  • egene e.g., the Renilla luciferase gene
  • G glycoprotein VSV ⁇ G-rLuc
  • This assay can provide robust metrics (absIC50, absIC80, absIC90) for meaningful comparisons between labs.
  • ultra-permissive 293T cell clones that stably express either ACE2 alone or ACE2+TMPRSS2 and methods of using these clones. These isogenic cell lines support either the late (293 T-ACE2) or early (293 T-ACE2/TMPRSS2) entry pathways that SARS-CoV-2 uses. These ultra-permissive 293T clones allow for the use of unpurified virus supernatant from the standard vims production batch, which can now provide for -150,000 infections per week (96-well format) with no further scale-up.
  • VSV vesicular stomatitis virus
  • SARS-CoV-2 spike protein pseudotyped vesicular stomatitis virus (VSV) particles comprising an encapsidated negative sense, single- stranded RNA genome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, and a nucleotide sequence encoding a reporter protein, wherein genome does not express VSV glycoprotein (G).
  • N VSV nucleoprotein
  • M VSV matrix
  • L nucleotide sequence encoding for VSV large (L) protein
  • P VSV phosphoprotein
  • G nucleotide sequence encoding a reporter protein
  • the reporter protein is luciferase.
  • SARS-CoV-2 spike protein refers to a SARS-CoV-2 spike protein known to those of skill in the art.
  • the spike protein comprises the amino acid or nucleic acid sequence found at GenBank Accession No. MN908947.3, MT291835.2, MT358639.1, MT079851, MT079848.1, or MT079845.1.
  • a typical spike protein comprises domains known to those of skill in the art including an S 1 domain, a receptor binding domain, an S2 domain, a transmembrane domain and a cytoplasmic domain.
  • the spike protein may be characterized has having a signal peptide (e.g., a signal peptide of 1-14 amino acid residues of the amino acid sequence of GenBank Accession No. MN908947.3), a receptor binding domain (e.g., a receptor binding domain of 319-541 amino acid residues of GenBank Accession No. MN908947.3), an ectodomain (e.g., an ectodomain of 15-1213 amino acid residues of GenBank Accession No.
  • a signal peptide e.g., a signal peptide of 1-14 amino acid residues of the amino acid sequence of GenBank Accession No. MN908947.3
  • a receptor binding domain e.g., a receptor binding domain of 319-541 amino acid residues of GenBank Accession No. MN908947.3
  • an ectodomain e.g., an ectodomain of 15-1213 amino acid residues of GenBank Accession No.
  • the spike protein may also be characterized as having an SI subunit and S2 subunit.
  • the SARS-CoV-2 spike protein is full length.
  • a SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:2. Due to the degeneracy of the code, any nucleotide sequence that encodes a SARS-CoV-2 spike protein (e.g., any nucleotide sequence encoding SEQ ID NO:2) may be used as described herein. In another specific embodiment, a nucleotide sequence encoding the SARS-CoV-2 spike protein is codon optimized for humans. In another specific embodiment, a SARS-CoV-2 spike protein is encoded by the nucleotide sequence set forth in SEQ ID NO:l.
  • VSV strains and genomic sequences are known in the art.
  • the ATCC offers VSV (ATCC VR-1238).
  • nucleotide sequences for VSV and the proteins encoded by VSV may be found on, e.g., GenBank. See, e.g., GenBank Accession Nos. NC_001560.1 (GI: 9627229) and J02428.1 (GI: 335873).
  • the genome of the SARS-CoV-2 spike protein pseudotyped VSV particle further comprises a nucleotide sequence encoding a fluorescent protein.
  • the nucleotide sequence encoding the reporter protein and the nucleotide sequence encoding fluorescent protein are separated by a nucleotide sequence encoding a self-cleaving peptide (e.g., a 2 A self-cleaving peptide) such that a single polypeptide comprising the reporter protein, the self-cleaving peptide, and fluorescent protein is generated and may be cleaved to produce the reporter protein and the fluorescent protein.
  • a self-cleaving peptide e.g., a 2 A self-cleaving peptide
  • the fluorescent protein is a red fluorescent protein or green fluorescent protein.
  • Additional examples of fluorescent proteins compatible with the compositions and methods disclosed herein include, include, but are not limited to, (3-F)Tyr- EGFP, A44-KR, aacuGFPl, aacuGFP2, aceGFP, aceGFP-G222E-Y220L, aceGFP-h, AcGFPl, AdRed, AdRed-C148S, aeurGFP, afraGFP, alajGFPl, alajGFP2, alajGFP3, amCyanl, amFP486, amFP495, amFP506, amFP515, amilFP484, amilFP490, amilFP497, amilFP504, amilFP512, amilFP513, amilFP593, amilFP597, anmlGFPl, anmlGFP2, anm2CP, anobCFPl, anobCFP2, ano
  • the reporter protein is luciferase.
  • the luciferase is renilla luciferase, firefly luciferase or nano luciferase. See, e.g., England et al., 2016, Bioconjug. Chem 27(5): 1175-1187 for examples of luciferases, including nanoluciferase.
  • the genome of the SARS-CoV-2 spike protein pseudotyped VSV particles does not comprise a nucleotide sequence encoding VSV glycoprotein (G). In some embodiments, the genome of the SARS-CoV-2 spike protein pseudotyped VSV particles only comprises a fragment of the nucleotide sequence sequence encoding VSV glycoprotein (G) (e.g., 10, 15, 20, 25, 30 or so nucleotides of the sequence that would encode VSV glycoprotein). In another embodiment, the SARS-CoV-2 spike protein is one described in the Examples. In another specific embodiment, provided herein are SARS-CoV-2 spike protein pseudotyped VSV particles such as described in in the Examples.
  • the SARS-CoV-2 spike protein pseudotyped VSV particles are treated with trypsin. In some embodiments, the SARS-CoV-2 spike protein pseudotyped VSV particles are treated with trypsin and soybean inhibitor.
  • the SARS-CoV-2 spike protein pseudotyped VSV particles are unable to undergo multiple rounds of replication. In a particular embodiment, the SARS- CoV-2 spike protein pseudotyped VSV particles are only able to undergo a single round of replication. [0062] Compositions
  • compositions comprising a SARS-CoV-2 spike protein pseudotyped VSV particle disclosed herein.
  • a composition comprising a SARS-CoV-2 spike protein pseudotyped VSV particle described herein and a carrier.
  • the carrier is phosphate buffered saline or another buffered saline solution.
  • the carrier is media (e.g., serum free media).
  • compositions comprising a SARS-CoV-2 spike protein pseudotyped VSV particle described herein and trypsin, soybean inhibitor or both.
  • a composition comprising supernatant containing SARS-CoV-2 spike protein pseudotyped VSV particles described herein and trypsin, soybean inhibitor or both.
  • composition comprising (1) SARS- CoV-2 spike protein pseudotyped VSV particles described herein (2) trypsin and (3) dextran (e.g., DEAE-dextran).
  • composition comprising (1) supernatant containing SARS-CoV-2 spike protein pseudotyped VSV particles described herein, (2) trypsin and (3) dextran (e.g., DEAE-dextran).
  • the trypsin concentration is between 5 and 1000, between 2 and 20, between 5 and 15, or between 6.25 and 12.5 pg/ml. In some embodiments the trypsin concentration is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 pg/ml.
  • the soybean inhibitor concentration is about 5, 10, 50,
  • soybean inhibitor concentration is about 625 pg/ml.
  • kits for generating SARS-CoV-2 spike protein pseudotyped VSV particles are, for example, described in the Examples.
  • a method for generating SARS-CoV- 2 spike protein pseudotyped VSV particle comprising: (a) infecting cells (e.g., HEK293T cells) overexpressing SARS-CoV-2 spike protein with a recombinant VSV particle, wherein the recombinant VSV particle comprises an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding for a reporter protein (e.g., luciferase), wherein the genome does not express VSV glycoprotein (G), wherein the VSV particle is pseudotyped with VSV glycoprotein; and (b) purifying SARS-
  • the genome of the recombinant VSV particles further comprise a nucleotide sequence encoding a fluorescent protein.
  • the nucleotide sequence encoding the reporter protein (e.g., luciferase) and the nucleotide sequence encoding fluorescent protein are separated by a nucleotide sequence encoding a self-cleaving peptide (e.g., a 2 A self-cleaving peptide) such that a single polypeptide comprising the reporter protein (e.g., luciferase), the self-cleaving peptide, and fluorescent protein is generated and may be cleaved to produce comprising the reporter protein (e.g., luciferase) and the fluorescent protein.
  • a self-cleaving peptide e.g., a 2 A self-cleaving peptide
  • the cells are cultured in optiMEM containing anti-VSV-G antibody.
  • anti-VSV G neutralizing antibody may minimize the background sometimes seen with “bald” VSV pseudotypes.
  • optiMEM media for production of SARS-CoV-2 spike protein pseudotyped VSV particles may be preferred over DMEM + 10% FBS because an increase of CoV-2 spike cleavage relative to DMEM + 10%FBS.
  • chemically defined serum free media is used to culture the cells.
  • Cells may be transiently or stably transfected with vector (e.g., plasmid) comprising a nucleotide sequence encoding SARS-CoV-2 spike protein.
  • vector e.g., plasmid
  • the cells do not express ACE-2 and do not support SARS-CoV-2 entry.
  • the SARS-CoV-2 spike protein pseudotyped VSV particles are purified from the supernatant by low speed centrifugation. In some embodiments, the SARS-CoV-2 spike protein pseudotyped VSV particles are purified from the supernatant by low speed centrifugation to remove cell debris and concentrated via ultracentrifugation through a sucrose cushion, and/or Amicon and PEG concentration. [0076] Methods for infecting cells with SARS-CoV-2 spike protein pseudotyped VSV particles
  • kits for infecting cells with SARS-CoV- 2 spike protein pseudotyped VSV particles described herein are described, for example, in the Examples.
  • a method for infecting cells with SARS- CoV-2 spike protein pseudotyped VSV particles described herein comprising: (a) contacting the SARS-CoV-2 spike protein pseudotyped VSV particles with trypsin for a certain period of time; and (b) infecting cells with the SARS-CoV-2 spike protein pseudotyped VSV particles.
  • a method for infecting cells with s SARS- CoV-2 spike protein pseudotyped VSV particle comprising: (a) contacting the SARS-CoV-2 spike protein pseudotyped VSV particles with trypsin and soybean inhibitor for a certain period of time; and (b) infecting cells with the SARS-CoV-2 spike protein pseudotyped VSV particles.
  • step (a) further comprises contacting the particles with soybean inhibitor.
  • step (a) further comprises contacting the particles with dextran (e.g., DEAE-dextran).
  • dextran e.g., DEAE-dextran
  • Trypsin can be used to induce entry enhancement of SARS-CoV-2 spike protein pseudotyped VSV particles.
  • the trypsin concentration is between 5 and 1000, between 2 and 20, between 5 and 15, or between 6.25 and 12.5 pg/ml. In some embodiments the trypsin concentration is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 pg/ml.
  • the soybean inhibitor concentration is about 5, 10, 50,
  • soybean inhibitor concentration is about 625 pg/ml.
  • the certain period of time is 15 minutes.
  • low concentrations of trypsin and soybean inhibitor can be used to achieve maximal SAS-CoV-2 spike protein pseudotyped VSV particles entry with limited toxicity.
  • methods for detecting blood, sera or plasma that neutralizes SARS-CoV-2 are provided, for example, in the Examples.
  • the SARS-CoV-2 spike protein pseudotyped VSV particles provide a surrogate for SARS-CoV-2 and allow neutralization assays to be conducted without the need for a biosafety level higher than level 2.
  • a method for detecting sera that neutralizes SARS-CoV-2 comprising: (a) incubating with the SARS-CoV-2 spike protein pseudotyped VSV particles described herein with sera from a subject for a first period of time; (b) spinoculating cells expressing human ACE-2, TMPRSS2 or both with the sera-treated SARS-CoV-2 spike protein pseudotyped VSV particle for a second period of time; and (c) measuring the reporter protein (e.g., luciferase) activity after a third period of time, wherein a lower level of reporter protein (e.g., luciferase) activity is detected if the sera neutralizes SARS-CoV-2 spike protein pseudotyped VSV particle than if steps (b) to (c) are performed without performing step (a) or a negative control (e.g., an antibody that is known not neutralize SARS-CoV-2) is used in step (a) when performing steps (a) to (c), and
  • a negative control
  • reporter protein e.g., luciferase
  • SARS-CoV-2 spike protein pseudotyped VSV particles further express fluorescent protein, it may also be detected using techniques known in the art (e.g., cytometry).
  • the first period of time is about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour or more. In certain embodiments, the first period of time is 10 to 45 minutes, 15 to 45 minutes, 15 to 60 minutes, or 30 to 60 minutes.
  • the second period of time is about 30 minutes, 45 minutes,
  • the second period of time is 30 minutes to 1 hour, 45 minutes to 1.5 hours, or 1 hour to 2 hours.
  • the third period of time is about 16 to 24 hours, about 18 to 20 hours, about 18 to 22 hours, or about 18 to 24 hours.
  • the subject is a human subject.
  • the serum is heat inactivated (e.g., the serum is incubated at 56° C for 30-60 minutes).
  • the serum is diluted in plain DMEM.
  • the serum is diluted in DMEM containing heat inactivated 10% heat inactivated FBS.
  • the method further comprises concurrently repeating steps (a) to (c) with a positive control antibody or sera that does not neutralize SARS-CoV-2. In some embodiments, the method comprises concurrently repeating steps (a) to (c) with a negative control antibody or sera that does not neutralize SARS-CoV-2.
  • the cells overexpress human angiotensin-converting enzyme 2 (ACE-2), Transmembrane protease serine 2 (TMPRSS2), or both. Cells may be engineered to overexpress human ACE-2, TMPRSS2, or both using sequences and techniques known to one of skill in the art.
  • the cells may be transiently or stably transfected with vectors (e.g., plasmids) comprising nucleotide sequences encoding human ACE-2, TMPRSS2, or both.
  • vectors e.g., plasmids
  • the neutralization assay is carried out in a high-throughput manner (e.g., using a 96 well microtiter plate).
  • the sera is serially diluted. In some embodiments, between 1:4 and 1:10 dilution of the SARS-CoV-2 spike protein pseudotyped VSV particles (e.g., the dilution may be done in plain DMEM or DMEM containing 10% heat inactivated FBS) is used in the method.
  • the cells are Vero-CCL81 cells, 293T cells, or human ACE2, primary HAECs.
  • Methods for assessing the ability of an antibody to neutralize SARS-CoV-2 [0092] In another aspect, provided herein are methods for assessing the ability of an antibody to neutralize SARS-CoV-2. Methods for assessing the ability of an antibody to neutralize SARS-CoV-2 are provided, for example, in the Examples.
  • a method for assessing the ability of an antibody to neutralize SARS-CoV-2 comprising: (a) incubating with the SARS-CoV-2 spike protein pseudotyped VSV particles described herein with an antibody of interest for a first period of time; (b) spinoculating cells expressing human ACE-2, TMPRSS2 or both with the antibody-treated SARS-CoV-2 spike protein pseudotyped VSV particle for a second period of time; and (c) measuring the reporter protein (e.g., luciferase) activity after a third period of time, wherein a lower level of reporter protein (e.g., luciferase) activity is detected if the antibody neutralizes SARS-CoV-2 spike protein pseudotyped VSV particle than if steps (b) to (c) are performed without performing step (a) or a negative control (e.g., an antibody that is known not neutralize SARS-CoV-2) is used in step (a) when performing steps (a)
  • the cells are spinoculated in the presence of serum free media (e.g., DMEM only).
  • serum free media e.g., DMEM only.
  • reporter protein e.g., luciferase
  • the SARS-CoV-2 spike protein pseudotyped VSV particles further express fluorescent protein, it may also be detected using techniques known in the art (e.g., cytometry).
  • the first period of time is about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour or more. In certain embodiments, the first period of time is 10 to 45 minutes, 15 to 45 minutes, 15 to 60 minutes, or 30 to 60 minutes.
  • the second period of time is about 30 minutes, 45 minutes,
  • the second period of time is 30 minutes to 1 hour, 45 minutes to 1.5 hours, or 1 hour to 2 hours.
  • the third period of time is about 16 to 24 hours, about 18 to 20 hours, about 18 to 22 hours, or about 18 to 24 hours.
  • the method farther comprises concurrently repeating steps (a) to (c) with a positive control antibody or sera that does neutralize SARS-CoV-2. In some embodiments, the method comprises concurrently repeating steps (a) to (c) with a negative control antibody or sera that does not neutralize SARS-CoV-2.
  • the cells overexpress human angiotensin-converting enzyme 2 (ACE-2), Transmembrane protease serine 2 (TMPRSS2), or both. Cells may be engineered to overexpress human ACE-2, TMPRSS2, or both using sequences and techniques known to one of skill in the art.
  • the cells may be transiently or stably transfected with vectors (e.g., plasmids) comprising nucleotide sequences encoding human ACE-2, TMPRSS2, or both.
  • vectors e.g., plasmids
  • plasmids comprising nucleotide sequences encoding human ACE-2, TMPRSS2, or both.
  • the neutralization assay is carried out in a high-throughput manner (e.g., using a 96 well microtiter plate).
  • the antibody is serially diluted. In some embodiments, between 1:4 and 1:10 dilution of the SARS-CoV-2 spike protein pseudotyped VSV particles (e.g., the dilution may be done in plain DMEM or DMEM ontaining 10% heat inactivated FBS) is used in the method.
  • the cells are Vero-CCL81 cells, 293T cells, or human ACE2, primary HAECs. In certain embodiments, RLUs >10 5 are achieved with a 1:10 dilution in SFM and spinoculation.
  • the particlednfectivity ratio may be calculated as A:B, where A is genome copies/ml and B is TCID50/ml.
  • the SARS-CoV-2 SI incorporation may be determined by SI subunit of SARS-CoV-2 spike protein/VS V matrix ratio on Western blot.
  • the percent cleavage of incorporated SI may be determined by SI subunit of SARS-CoV-2 spike protein/SARS-CoV-2 spike protein ratio on Western blot.
  • cells e.g., 293T cells
  • the cells may be used in a neutralization assay described herein, including cell lines Vero-CCL81 TMPRSS2, HEK 293T-hACE2 (clone 5-7), and 293T-hACE2-TMPRSS2 (clone F8-2).
  • kits comprising SARS-CoV-2 spike protein pseudotyped VSV particles described herein in a container.
  • a kit comprising SARS-CoV-2 spike protein pseudotyped VSV particles in a container, and optionally instructions for performing a neutralization assay using the SARS- CoV-2 spike protein pseudotyped VSV particle.
  • the kit may further comprise a positive control antibody (e.g., an antibody known to neutralize SARS- CoV-2), a negative control antibody (e.g., an antibody known not to neutralize SARS-CoV- 2), or both.
  • the kit may further comprise one or more reagents need to detect reporter protein (e.g., lucif erase) activity.
  • a nucleic acid sequence comprising a VSV antigenome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding a reporter protein (e.g., luciferase), and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G).
  • the nucleic acid sequence further comprises a T7 promoter, autocatalytic hammerhead ribozyme sequences, and a T7 terminator ⁇ See, e.g., Beaty et a , 2017, mSphere 2(2):e00376-16 for a description of the structure of the nucleic acid sequence (in particular, see, e.g., FIG. 4A of Beaty et ak).
  • the nucleic acid sequence comprises an optimized T7 promoter and hammerhead ribozyme (HhRbz) just before the 5 ’ end of the viral genome.
  • the optimized T7 promoter comprises the sequence TAATACGACTCACTATAGGGAGA (SEQ ID NO:9).
  • the HhRbz sequence comprises the sequence
  • the use of a codon-optimized T7 polymerase may alleviate the use of a vaccinia-driven T7 polymerase, resulting in higher rescue efficiency.
  • a nucleic acid comprising the sequence of the codon-optimized T7 RNA polymerase comprises SEQ ID NO: 11.
  • the T7 RNA polymerase encoding sequence is provided by a helper plasmid. The sequence of a codon optimized T7 RNA polymerase has been deposited to Addgene (Cat no. 65974).
  • nucleotide sequence encoding the reporter protein e.g., luciferase
  • nucleotide sequence encoding the fluorescent protein are separated by a nucleotide sequence encoding a self-cleaving peptide (e.g., a P2A self-cleaving peptide) so that a single polypeptide is produced that is cleaved into the reporter protein (e.g., luciferase) and fluorescent protein.
  • a self-cleaving peptide e.g., a P2A self-cleaving peptide
  • the nucleotide sequence encoding the fluorescent protein and the nucleotide sequence encoding the reporter protein are separated by a nucleotide sequence encoding a self-cleaving peptide (e.g., a P2A self-cleaving peptide) so that a single polypeptide is produced that is cleaved into fluorescent protein and the reporter protein (e.g., luciferase).
  • the nucleic acid sequence is in a plasmid.
  • the reporter protein is a luciferase such as a renilla luciferase, firefly luciferase or nano luciferase.
  • the fluorescent protein is a fluorescent protein disclosed herein.
  • vectors comprising the nucleic acids disclosed herein.
  • kits comprising the nucleic acid sequence in a container and optionally instructions for generating pseudotyped VSV particles.
  • the kit further comprises a one, two, three or all of the following: (1) a first vector (e.g., a plasmid) comprising a nucleotide sequence encoding VSV M protein in a container, (2) a second vector (e.g., a plasmid) comprising a nucleotide sequence encoding VSV L protein in a container, (3) a third vector (e.g., a plasmid) encoding VSV N protein in a container, (4) a fourth vector (e.g., a plasmid) comprising a nucleotide sequence encoding VSV G protein in a container; (5) a fifth vector (e.g.
  • kits comprising a nucleotide sequence encoding VSV P protein; (6) a sixth vector (e.g., a plasmid comprising a codon- optimized gene encoding T7 RNA polymerase.
  • the kit may further comprise one or more ingredients to transfect cells with a plasmid.
  • VSV particles pseudotyped with VSV glycoprotein comprising an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding a reporter protein (e.g., luciferase), and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G).
  • the reporter protein is a luciferase such as renilla luciferase, firefly luciferase or nano luciferase.
  • the fluorescent protein fluorescent protein disclosed herein in one embodiment, the fluorescent protein fluorescent protein disclosed herein.
  • a method for generating pseudotyped VSV particles comprising:
  • the viral surface protein is the SARS-CoV-2 spike protein.
  • a method for generating recombinant VSV particles pseudotyped with VSV glycoprotein comprising an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding a reporter protein (e.g., luciferase), and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G), the method comprising techniques similar to those described in Beaty et al.
  • a method for generating recombinant VSV particles pseudotyped with VSV glycoprotein comprising an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding a reporter protein (e.g., luciferase), and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G), the method comprising: (a) transfecting cells with a nucleic acid sequence described herein that comprises a VSV antigenome, a first vector (e.g., a plasmid) comprising a nucleotide sequence encoding VSV M
  • the method further comprises transfecting the cells with a sixth vector comprising a codon-optimized sequence encoding a T7 polymerase.
  • the cells are 293T-ACE2 clone 5-7 or 293T-ACE2-TMPRSS2 clone F8-2.
  • SARS-CoV-2 Spike nucleotide sequence [0108] ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGA
  • Example 1 Material and Methods for Examples 3-10 [0113] Plasmids
  • SARS-CoV-2 spike is in a pCAGG backbone and expresses the codon optimized Wuhan-Hu-1 isolate (NCBI ref. seq. NC_045512.2).
  • SARS-CoV-2 sRBD (NCBI GenBank MT380724.1 from Krammer lab) is in a pCAGG backbone and expresses the codon optimized sequence from the Wuhan-Hu-1 isolate. sRBD-His used for neutralization studies was generated from this construct.
  • VSV-G is in a pCAGG backbone and expresses wild type Indiana strain VSV-G (Genbank: ACK77583.1).
  • ACE2 packaging construct (GeneCopoeia, cat no EX-U1285-Lvl05) uses a CMV promoter to express TMPRSS2 and bears a puromycin selection marker in the integrating cassette.
  • TMPRSS2 packaging construct (GeneCopoeia, cat no EX-Z7591-Lvl97) uses a CMV promoter to express TMPRSS2 and bears a blasticidin selection marker in the integrating cassette.
  • psPAX22nd generation lentiviral packaging plasmid (Addgene #12259) expresses HIV-1 Gag, Pol, and Pro proteins.
  • NiV-RBP is in a pCAGG backbone and expresses the HA-tagged codon optimized NiV receptor binding protein.
  • Vero-CCL81 and 293T cells were cultured in DMEM with 10% heat inactivated FBS at 37 °C with 5% C02.
  • VSV-G pseudotyped lentiviruses packaging ACE2 or TMPRSS2 expression constructs were generated by using Bio-T (Bioland; BOl-01) to transfect 293T cells with the second-generation lentiviral packaging plasmid (Addgene; 12259), pCAGG- VSV-G, and the desired expression construct (i.e. ACE2 or TMPRSS2). The media was changed the next morning.
  • Vero-CCL81 and 293T cells were transduced in a 6-well plate with the prepared lentiviral constructs. Two days after transduction, these cells were expanded into a 10cm plate and placed under selection with puromycin (for ACE2 transduced cells) or blasticidin (for TMPRSS2 transduced cells). 293T and Vero-CCL81 cells were selected with 2 or 10pg/mL of puromycin, respectively.
  • 293T were selected with 5pg/mL and Vero- CCL81 cells were selected with 15pg/ml.
  • 293T-ACE2 cells were transduced with the VSV-G with 5pg/mL blasticidin.
  • Low passage stock of each cell line generated were immediately frozen down using BamBanker (Fisher Scientific; NC9582225).
  • Single cell, isogenic clones were isolated via serial dilution in a 96 well plate. Wells with only a single cell were grown up and eventually expanded while under selection.
  • 293T producer cells were transfected to overexpress SARS-CoV-2 or VS V-G glycoproteins.
  • pCAGG empty vector was transfected into 293T cells.
  • cells were infected with the VSV ⁇ G-rLuc reporter virus for 2 hours, then washed with DPBS.
  • supernatants were collected and clarified by centrifugation at 1250 rpm for 5 mins.
  • VSVAG-rLuc particles bearing the CoV2pp were then treated with TPCK-treated trypsin (Sigma- Aldrich; T1426-1G ) at room temperature for 15 minutes prior to inhibition with soybean trypsin inhibitor (SBTI) (Fisher Scientific; 17075029). Particles were aliquoted prior to storage in - 80 °C to avoid multiple freeze-thaws.
  • SBTI soybean trypsin inhibitor
  • VSV ⁇ G reporter backbone is from the Indiana lab-adapted strain which is cleared for use at BSL-2 (reviewed in PMID: 20709108). Bona fide recombination amongst negative sense RNA viruses (as opposed to positive sense RNA viruses) is exceedingly rare if not absent (reviewed in PMID: 21994784). Finally, the VSV-G provided in trans lacks the VSV-G gene start and gene stop signals present in the VSV ⁇ G backbone, making even the possibility of a productive homologous recombination event vanishingly small.
  • the media was (optionally) replaced with 5 mL DMEM + 10% FBSi.
  • the purpose of this media exchange is to reduce the volume that the transfection reagents are added to i.e. 5 mLs instead of 10 mLs.
  • 24 pg total DNA (glycoprotein expression plasmid) were transfect per plate.
  • 120 uL PEI reagent were diluted in 500 uL PBS.
  • a total of 24 ug of DNA was diluted in a separate tube of 500 uL PBS.
  • the PEI mix was added to the DNA mix and incubated at room temperature for 30 mins. The mixture was added drop wise to cells.
  • VSV-AG-G infection can be varied depending on the cell surface expression kinetics of the specific envelope protein. For example, 6, 8, 12, and 24 hpt can be used. optiMEM may be used for CoV2pp production, as this leads to an increase of CoV-2 spike cleavage relative to DMEM + 10% FBS.
  • ⁇ 8 hpt see comment above, cells were infected with a VSV[Rluc]- AG-G* stock ( ⁇ 1 X 10 8 TCID50 units) in an inoculum volume of 5 mL per plate. The inoculum was incubated for 1-2 hours at 37 °C to permit infection. The inoculum was removed. The plates were washed 2x with dPBS to remove excess VSV-G particles that did not infect.
  • the incubation medium opti-MEM (modification of Eagle's Minimum Essential Medium, buffered with HEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, and growth factors) containing a 1:20,000 dilution of anti- VSV-G antibody (8G5F11 from Kerafast), was prepared.
  • This potently neutralizing antibody minimizes the background sometimes seen with “bald” VSV pseudotypes (293T cells transfected with 24pg empty vector, then infected as normal).
  • 10 mL incubation medium was added to each plate of washed 293T cells. At 48 hpi the supernatant into a 50 mL conical tube was collected — roughly 10 mL per plate.
  • CoV2pp were clarified, but not concentrated through a sucrose cushion. If desired, other concentration methods such as Amicon and Peg concentration may be used.
  • the pseudotype-containing supernatant of cell debris was clarified by centrifugation in a standard benchtop centrifuge at 1,250 rpm (approximately. 450 x g) for 5 min.
  • the clarified supernatant was transferred to Seton Open-Top Polyclear Centrifuge Tubes for SW28 rotor. 15 mL serum- free media was added.
  • a pipette containing 10.5 mL 20% sucrose was inserted to the bottom of the tube. 10 mL 20% sucrose were getnly added to the bottom of the tube to create cushion. The pipette was removed without disrupting the sucrose-media interface.
  • DMEM + 10% FBS for Vero or 293T cells were prepared such that one was able to transfer a final volume of 100 uL inoculum per well. This dilution series was performed in 6 replicates in order to generate a TCID50 value.
  • Promega Renilla or Passive Lysis buffer was prepared by diluting the stock 1/5 in ddH20. The culture media/inoculum was removed. Each well was washed with 100 pL of DPBS.
  • the cells were lysed by adding 25 pL prepared lysis buffer to each well. From this point on, all samples can be handled safely outside the biosafety cabinet, as all living and infectious material has been inactivated. It is recommended that, in addition to using the passive lysis buffer provided in the kit, one freeze-thaw cycle is performed to release the rLuc. Alternatively, incubation on an orbital shaker for 15 mins at 500 rpm can be performed. [0152] After lysis the same plate was assayed for Renilla luciferase production on a plate reader using the Promega rLuc kit.
  • the assay buffer 1:1 was diluted with DPBS and a 1:200 dilution of assay substrate was used.
  • the following procedure was used: (1) delay of 5 seconds between each well, (2) dispense 40pL of assay reagent, (3) shake for 2 seconds, (4) delay for 2 seconds, (5) read luminescence, (6) quench the reaction by dispensing 50pL of 70% ethanol, (7), shake for 5 seconds and (8) proceed to the next well.
  • the limit of detection for the Cytation3 is -300 RLUs.
  • the Spearman & Karber algorithm was used. Positive wells are those with >2x the average background signal i.e. for the Cytation3, this would be >600 RLUs. For other instruments, uninfected wells were assayed to determine the background signal.
  • trypsin For two separate batches of trypsin, the following conditions were used: 1st batch: 625 pg/mL of trypsin with 625 pg/mL of soybean inhibitor; 2nd batch: 475 pg/mL of trypsin with 600 pg/mL of soybean inhibitor.
  • 1st batch 625 pg/mL of trypsin with 625 pg/mL of soybean inhibitor
  • 2nd batch 475 pg/mL of trypsin with 600 pg/mL of soybean inhibitor.
  • certain trypsin might be particularly useful, such as concentrations between 6.25 pg/mL and 12.5 pg/mL, to induce this entry enhancement.
  • drastically lower concentrations of trypsin and soybean inhibitor can be used to achieve maximal CoV2pp entry with limited toxicity.
  • Day -1 pre-infection: PLL coating of 10 cm dishes, seeding of 293T cells.
  • Day 0 Transfection of 293T cells; infection of transfected cells with parental VSV[Rluc]- AG-G* stock.
  • Day 1 post-infection Seeding of susceptible cells in 96-well plates for titration (optional).
  • Day 2 post-infection Collection of supernatant from transfected/infected cells; clarification, and, if needed, concentration (i.e ultracentrifugation, Amicon filter, or PEG) of supernatant; if needed, trypsin-treatment of CoV2pp, aliquotting and freezing; tittering of new pseudotyped virus stocks on susceptible cells.
  • concentration i.e ultracentrifugation, Amicon filter, or PEG
  • Vero-CCL81 cells were seeded in a 96 well plate 20-24 hrs prior to infection.
  • a single aliquot of BALDpp, CoV2pp, and VSV-Gpp were used for infections and titrations were performed in technical triplicates.
  • the infected cells were washed with DPBS, lysed with passive lysis buffer, and processed for detection of Renilla luciferase.
  • the Cytation3 BioTek was used to read luminescence.
  • Vero CCL81 cells or isogenic cells (293T-ACE2 clone 5-7 or 293T- ACE2+TMPRSS2 clone F8), all maintained in DMEM + 10% FBS, DMEM with 10% FBS; Promega Renilla luciferase assay system (100 assays-E2810; 1000 assays-E2820); CoV2pp: VSV ⁇ G-Rluc bearing SARS-CoV-2 Spike glycoprotein; VSV-Gpp: VSV ⁇ G-Rluc bearing VSV-G entry glycoprotein; BALDpp: VSV-G-Rluc bearing no protein (produced in parallel with samples above)
  • the CoV2pp are a VSV pseudotyped particle (pp) system that do not encode any viral glycoprotein in the VSV genome and can be worked with under Bio-Safety Level 2 (BSL2) conditions.
  • the desired serial dilution of virus was prepared in Serum Free Media (SFM; DMEM only) such that one was able to transfer a final volume of 100 pL/well.
  • the media was removed from the Vero cells. Starting from the lowest dilution, 100 pL from the titration plate were carefully transferred to the cells, which were then incubated at 37 °C.
  • the Promega Renilla lysis buffer was prepared by diluting the stock 1 :5 in ddH20. The culture media/inoculum was removed. Each well was washed by adding 100 pL of DPBS, then removing this volume with a multichannel pipette.
  • the cells were lysed by adding 25 pL prepared lysis buffer to each well. It is recommended that, in addition to using the passive lysis buffer provided in the kit, one freeze-thaw cycle is performed to release the rLuc. Alternatively, incubation on an orbital shaker for 15 mins at 500 rpm can be performed.
  • the same plate was assayed for Renilla luciferase production on a plate reader using the Promega rLuc kit.
  • the assay buffer 1:1 was diluted with DPBS and a 1:200 dilution of assay substrate was used.
  • the following procedure was used: (1) delay of 5 seconds between each well, (2) dispense 40pL of assay reagent, (3) shake for 2 seconds, (4) delay for 2 seconds, (5) read luminescence, (6) quench the reaction by dispensing 50pL of 70% ethanol, (7), shake for 5 seconds and (8) proceed to the next well.
  • the limit of detection for the Cytation3 is -300 RLUs.
  • the Spearman & Karber algorithm was used for calculating TCID50. Positive wells are those with >3x the average background signal of uninfected wells *For the Cytation3, the limit of detection is 300 RLUs.
  • the limit of detection is 300 RLUs.
  • positive wells as those with >1000 RLUs were considered. For other instruments, multiple uninfected wells can be assayed to determine the background signal.
  • a dilution of the monoclonal antibody or sera in DMEM + 10%FBS in a V bottom 96 well plate was prepared as follows: If using a 4-fold serial dilution, 28.75 pL media were added to all wells except the top well. To the top well, 38.34 pL of the entry inhibitor were added to the well containing the starting dilution. Then 9.58 pL were transferred for a 4-fold serial dilution. This ensured that there were 28.7 5pL of the entry inhibitor (e.g. sera, monoclonal antibody or small molecule) in each well, which will be further diluted 1:4 after the addition of virus.
  • the entry inhibitor e.g. sera, monoclonal antibody or small molecule
  • the virus stock was diluted in DMEM + 10%FBS and, using a multichannel and a sterile basin, the desired amount of virus was transferred to each well of a V bottom 96 well plate. For example, between a 1:4 and 1:50 dilution of the CoV2pp can be used.
  • RLUs -105 > 100 x signalmoise
  • the same RLUs can be achieved with a 1:20-1:50 dilution.
  • All CoV2pp batches were tittered first prior to use for viral neutralization assays or entry inhibition assays. Each point of the neutralization curve was performed in triplicate.
  • Cell lysates were collected from producer cells with lOmM EDTA in DPBS. Cells were subsequently lysed with RIPA buffer (Thermo Scientific, 89900) containing protease inhibitor (Thermo Scientific, 87785) for 30 minutes on ice. Lysates were centrifuged at 25,000 x g for 30 minutes at 4°C, and the supernatants were collected and stored at -80°C. Total protein concentrations were determined by the Bradford assay.
  • ACE2 (66699-1-Ig from Proteintech and Rb abl08252 from abeam), VSV-G (A00199 from Genescript), VSV-M (EB0011 from Kerafast), anti-HA (NB600-363 from Novus), and CoX IV (926-42214 from LI-COR) were used.
  • membranes were washed and incubated with the appropriate Alexa Fluor 647-conjugated anti- mouse antibody or Alexa Fluor 647-conjugated anti-rabbit antibody. Alexa Fluor 647 was detected using the ChemiDoc MP imaging system (Bio-Rad).
  • Relative ACE2 or TMPRSS2 abundance was calculated by First normalizing abundance relative to GAPDH expression, then normalizing to wild type expression.
  • RNA extraction and qPCR for ACE2 and TMPRSS2 expression [0194] Total RNA was extracted from cells using Direct-zolTM RNA Miniprep kit (Zymol, R2051), and reverse transcription (RT) was performed with the TetroTM cDNA Synthesis kit (Bioline, BIO- 65043) and random hexamers. RT PCR was performed with the SensiFASTTM SYBR & Fluorescein Kit (Bioline, BIO-96005).
  • HPRT forward (5’- ATTGTAATGACCAGTCAACAGGG-3 ’ , SEQ ID NOG) and reverse (5’- GCATTGTTTTGCCAGTGTCAA- 3’, SEQ ID NO:4) primers
  • ACE2 forward (5’- GGCCGAGAAGTTCTTTGTATCT-3 ’ , SEQ ID NOG) and reverse (5’- CCCAACTATCTCTCGCTTCATC-3 ’ , SEQ ID NO:x6) primers
  • TMPRSS2 forward (5’- CCATGGATACCAACCGGAAA-3 ’ , SEQ ID NO:7) and reverse (5’- GGATGAAGTTTGGTCCGTAGAG-3’, SEQ ID NO:8) primers were utilized.
  • the protocol from Stadlbauer et all04 was modified slightly to start from a 1:300 and end at a 1:24300 dilution of sera.
  • IgG and IgM antibodies were detected with secondary antibodies conjugated to HRP (Millipore AP101P for anti- Human IgG and Invitrogen A18841 for anti-Human IgM).
  • a pre-titrated amount of pseudotyped particle dilution was mixed with the protein or compound and added to cells immediately after. Approximately 20 hours post infection, cells were processed for detection of luciferase activity as described above.
  • Raw luminometry data were obtained from labs that volunteered VNA results from at least 12 patient samples and analyzed as indicated below.
  • IC Relative inhibitory concentrations
  • the absIC50 would be the point at which the curve matches inhibition equal to exactly 50% of the 100% assay control relative to the assay minimum (0%).lll
  • sera samples that are non-neutralizing or minimally neutralizing may have lower plateaus indicating they cannot reach certain absolute inhibitory concentrations, such as an absIC90 or absIC99.
  • Example 2 Material and Methods for Example 11 [0208] Cell lines
  • Vero-CCL81 TMPRSS2, HEK 293T-hACE2 (clone 5-7), and 293T-hACE2- TMPRSS2 (clone F8-2) cells were maintained in DMEM + 10% FBS.
  • the HEK 293T- hACE2-TMPRSS2 cells were plated on collagen coated plates or dishes.
  • BSR-T7 cells 52, which stably express T7 -polymerase were maintained in DMEM with 10% FBS.
  • VSV-eGFP-CoV2 spike A21aa genomic clone and helper plasmids
  • the VSV-eGFP sequence was cloned into the pEMC vector (pEMC-VSV-eGFP), which includes an optimized T7 promoter having the sequence TAATACGACT CACTATAGGG AGA (SEQ ID NO: 9) and hammerhead ribozyme having the sequence CTGATGAGTC CGTGAGGACG AAACGGAGTC TAGACTCCGT C (SEQ ID NO: 10) just before the 5’ end of the viral genome (see FIG. 4A of Beaty et a , 2017, mSphere 2(2):e00376-16).
  • pEMC-VSV-eGFP-CoV2-S (Genbank Accession: MW816496) was generated as follows: the VSV-G open reading frame of pEMC-VSV-eGFP was replaced with the SARS- CoV-2 S, truncated to lack the final 21 amino acids 54.
  • a Pac-I restriction enzyme site was introduced just after the open reading frame of S transcriptional unit, such that the S transcriptional unit is flanked by Mlul / Pad sites.
  • SARS-CoV-2 S is from pCAGGS-CoV-2-
  • the B.1.1.7 Spike used carries the mutations found in GISAID Accession Number EPI_ISL 668152: del 69-70, del 145, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.
  • the B.1.351 Spike carries the mutations D80A, D215G, del242-244, K417N, E484K, N501Y, D614G, and A701V (from EPI_ISL_745109).
  • the Spike sequences of WT, B.l.1.7, B.1.351, and E484K are available at Genbank (Accession Numbers: MW816497, MW816498, MW816499, and MW8 16500
  • the initial objective was to produce SARS-CoV-2 PsV sufficient for 10,000 infections/week at -1:100 signaknoise ratio when performed in a 96- well format.
  • a VSV- based rather than a lentiviral PsV system was used as lentiviruses are intrinsically limited by their replication kinetics particle production rate (10 4 -10 6 /ml for lentiviruses versus 10 7 -10 9 /ml for VSV without concentration).
  • the production of the VSVAG-rLuc pseudotyped viral particles (pp) bearing the SARS-CoV-2 spike glycoprotein was optimized as diagramed in FIG. 1A.
  • SARS-CoV-2 spike protein In the first step, cells overexpressing SARS-CoV-2 spike protein are infected with VSV[Rluc] AG-G* at a low MOI and grown in the presence of anti-VSV-G monoclonal antibody.
  • SARS-CoV-2 spike protein pseudotyped particles bud out from the infected cells. Those SARS-CoV-2 pseudotyped particles (at a certain concentration, e.g., genome copies/ml) may then be used infect target cells.
  • the purified pseudotyped particles may be trypsin treated and spinoculation may be performed to enhance entry of the pseudotyped particles.
  • the particle: infectivity ratio and SARS-CoV-2 spike protein incorporation may be determined as well as the percent cleavage of incorporated spike protein.
  • this protocol involves infecting producer cells at a low multiplicity of infection (MOI) of stock VSVAG-G*, incubating producer cells with an anti-VSV-G monoclonal antibody and generating the pseudotyped particles in Opti-MEM media.
  • MOI multiplicity of infection
  • the first two measures effectively eliminated the background signal from residual VSV-G while the last measure allowed for more cleavage of SARS-CoV-2pp in producer cells.
  • CT cytoplasmic tail
  • CT truncations in many other class I viral fusion proteins including other ACE2- using coronaviruses (HCoV-NL63 and SARS-CoV-1) can affect ectodomain conformation and lunction.
  • surrogate assay was developed that reflects the biology of the full-length virus spike.
  • BALDpp, NiV-RBPpp, CoV2pp, and VSV-Gpp were produced using the VSVAG- rLuc reporter backbone and titered them on Vero-CCL81 cells FIG. IB).
  • High background problems have resulted in low signahnoise ratios when using VSV-based PsV, especially for viral envelope proteins that do not mediate efficient entry.
  • BALDpp and NiV-RBP were used, to show that the background issue was resolved.
  • BALDpp lacks any surface glycoprotein while NiV-RBPpp incorporates the NiV receptor binding protein (RBP), which binds to the broadly expressed ephrin-B2 with sub-nanomolar affinity.
  • RBP NiV receptor binding protein
  • NiV- RBPpp without NiV-L should not fuse and effectively serves as a stricter and complementary negative control.
  • BALDpp nor NiV-RBPpp gives any background even at the highest concentration of virus particles used.
  • FIG. 1C shows the genome copy number and particle to infectivity ratio for BALDpp, NiV-G only, CoV2pp, or VAV-Gpp.
  • the genome copy number was assessed using primers against the VSV-L protein as previously described (Pryce, Azarm CedV-Bl usage).
  • the particle to infectivity ratio was calculated as a fraction of number of genomes to TCID50.
  • Example 4 CoV2pp entry is enhanced by trypsin treatment and spinoculation
  • the relative signal of CoV2pp infections was enhanced to effectively increase the number of infections one can provide or perform per batch of CoV2pp.
  • the effect of typsin treatment and spinoculation on CoV2pp entry into target cells was determined.
  • CoV2pp stocks were treated with the indicated range of trypsin concentrations for 15 min at room temperature (FIG. 2A).
  • 625 pg/mL of soybean trypsin inhibitor (SBTI) were added to all samples before titrating the trypsin-treated CoV2pp onto Vero-CCL81 cells.
  • SBTI soybean trypsin inhibitor
  • CoV2pp treated with the highest concentration of trypsin (625pg/mL) resulted in ⁇ 100-fold enhancement of entry (FIG. 2A), but this trypsin-dependent enhancement was only apparent when comparing entry of undiluted trypsin-treated CoV2pp.
  • the remaining uninhibited trypsin-dependent effect which must be present at the highest trypsin concentration, might have inadvertently been neutralized by diluting the trypsin-treated CoV2pp in Dulbecco’s modified Eagle Medium (DMEM) +10% fetal bovine serum (FBS), which is the standard infection media for titrating CoV2pp.
  • DMEM Dulbecco
  • FBS fetal bovine serum
  • CoV2pp and trypsin-treated CoV2pp were diluted 1:10 in three different media conditions before infecting Vero-CCL81 cells.
  • dilution in DMEM alone serum free media, SFM
  • SFM serum free media
  • CoV2pp treated with 62 5pg/mL of TPCK-treated trypsin was chosen, then 625 pg/mL of SBTI, diluted in SFM as the standard treatment condition.
  • spinoculation at 1,250 rpm for 1 hr enhanced entry 3-5 fold (compare signalmoise in FIG. 2B to FIG. 2C).
  • FIG. 2E shows that certain trypsin can improve CoV2pp activation. Supernatant containing CoV2pp were treated with different concentrations of trypsin for 15 minutes, then used to infect Vero-CCL81 cells.
  • Example 5 Entry of CoV2pp is independently enhanced by stable expression of ACE2 and TMPRSS2 in cells already permissive for SARS-CoV-2 entry and replication
  • Vero-CCL81 cell lines were generated stably expressing human ACE2 or human TMPRSS2.
  • Vero-CCL81 cells are already highly permissive for SARS-CoV-2 entry and replication.
  • the indicated cells were infected with CoV2pp or trypsin-treated CoV2pp diluted in serum-free media (standard treatment) and enhanced entry in both stable cell lines was observed (Fig. 3).
  • the entry enhancement of trypsin-treated CoV2pp in Vero-CCL81 + TMPRSS2 overexpressing cells was subdued relative to untreated CoV2pp.
  • Example 6 Standardizing the parameters that impact CoV2pp-based virus neutralization assay
  • This CoV2pp serum neutralizing factor was somewhat reduced but not completely diminished by heat inactivation for 1 hr at 56 °C. Notably, the effect of this neutralizing factor from negative sera was preempted by diluting the trypsin treated CoV2pp in DMEM containing 10% FBS (FIG. 4B). Importantly, recombinant sRBD neutralization was not affected by the dilution of CoV2pp in Serum Free Media or DMEM + 10% FBS (FIG. 4C). Regardless, for standardizing the CoV2pp-based VNA, all subsequent patient sera were heat inactivated for at least 30 mins prior to use an serially diluted in DMEM + 10% FBS, which also served as the infection media. Despite the data from FIG.
  • Example 7 Performance characteristics of the standardized CoV2pp virus neutralization assay
  • Example 8 Independent validation of CoV2pp VNA with geographically distinct and ethnically diverse COVID-19 patient cohorts
  • absIC80 Although absIC80 also generally follows this trend, differences in the ranked order of absIC50 and absIC80 values calculated for all sera samples were observed. This difference is more pronounced when comparing the absIC50 and absIC90 graphs further highlighting the need for a neutralization assay with a broad dynamic range. Additionally, the samples from each of the 4 groups show no statistical difference when absIC50, 80, or 90 calculations are compared (FIG. 6). Altogether, these data support the robustness of the CoV2pp VNA and suggest that absIC80 is a more stringent and meaningful measure of Nab titers.
  • Example 9 Ultra-permissive 293T-ACE2 and 293T-ACE/TMPRSS2 clones allow for use of CoV2pp in VNA at scale
  • the untreated CoV2pp was used to screen for ultrapermissive cell lines that would allow for CoV2pp VNA to be performed with dilutions of virus supernatant without any trypsin treatment, virus purification, or spinoculation.
  • TMPRSS2 can enhance ACE2 dependent virus entry in a non-linear fashion
  • BALDpp, CoV2pp, and VSV-Gpp were used to screen 19 single cell clones derived from 293T-ACE2 or 293T-ACE2+TMPRSS2 or Vero-ACE2 bulk transduced cells.
  • the latter (FIG. 3) served as an additional control in a naturally permissive cell line for SARS-CoV-2 entry and replication. All three bulk transduced cell lines resulted in significant increases in entry of CoV2pp relative to the parental 293T and Vero CCL81 cells (FIG. 7B). However, only a subset of the single cell clones performed better than bulk transduced cells.
  • TMPRSS2 was determined to be the main driver of this entry enhancement in the F8-2 cells as treatment with Nafamostat, a serine protease inhibitor, potently inhibited entry.
  • Nafamostat a serine protease inhibitor
  • this entry inhibition plateaued at 90% of maximal infection and the remaining 10% is nearly equivalent to the raw RLU values seen with bulk 293Ts stably expressing ACE2 alone (FIG 7D), suggesting a TMPRSS2-independent mechanism of entry. Entry into 293T-ACE2 cells was not inhibited by Nafamostat, once again highlighting that CoV2pp can enter by both the early and late entry pathways that have differential protease requirements.
  • Example 10 Diverse cell lines maintain similar kinetics in CoV2pp viral neutralization assays:
  • Sera samples were identified from 15 patients shown in FIG. 5 and tiered them into three groups: negative for CoV2pp neutralization (negative), weakly positive for CoV2pp neutralization (low positive), or strongly positive for CoV2pp neutralization (high positive) (FIG. 8A). Equal volumes of each set of samples were pooled and CoV2pp neutralization assays were performed on Vero-CCL81 WT, 293T-ACE2 clone 5-7, 293T- ACE2+TMPRSS2 bulk transduced, and the 293 T - ACE2+TMPRS S2 clone F8-2.
  • CoV2pp neutralization assays show consistent patterns of neutralization, exhibiting the robust nature of the assay in tandem with its sensitivity in detecting relative differences in neutralizing titer (FIG. 8B). Patterns of neutralization as well as the calculated absIC50 and absIC80 reveal a large dynamic range between low and high neutralizing patient sera across cell lines (FIG. 8B).
  • Example 11 A replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system for virus neutralization assays (VNAs)
  • VSV-CoV2-S a replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system uses rcVSV-CoV2-S, which encodes S from SARS coronavirus 2 in place of VSV-G, and coupled with a clonal HEK-293T ACE2 TMPRSS2 cell line optimized for highly efficient S-mediated infection.
  • VNAs BSL-2 compatible virus neutralization assays
  • the rcVSV-CoV2-S genomic coding construct comprises a hammerhead ribozyme immediately upstream of the 3’ leader sequence which cleaves in cis to give the exact 3’ termini (FIG. 9A).
  • the system further uses a codon-optimized T7 -polymerase which alleviates the use of vaccinia-driven T7 -polymerase, and a highly permissive and transfectable 293T-ACE2+TMPRSS2 clone (F8-2) (FIG. 9B).
  • a 6-plasmid transfection into F8-2 cells results in GFP+ cells 2-3 days post-transfection (dpt), which turn into foci of syncytia by 4-5 dpt indicating virus replication and cell-to-cell spread (Fig. 10A).
  • Transfer of F8-2 cell supernatant into interferon-defective Vero-TMPRSS2 cells allowed for rapid expansion of low-passage viral stocks that maintain only the engineered Spike mutations. Clarified viral supernatants from Vero-TMPRSS2 cells were aliquoted, sequenced verified, then titered on F8-2 cells to determine the linear range of response (Fig. 10B).
  • Example 12 Exemplary use of the replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system
  • VOC SARS-CoV-2 ‘variants of concern’
  • S Spike
  • the S genes of B.1.351 and P.l viruses each carry a number of mutations, but include three in the receptor binding domain (RBD) that are particularly notable, the S: N501 Y substitution, found in B.1.1.7, alongside polymorphisms at positions 417 and 484, K417N/T and E484K.
  • the P.2 lineage originally detected in Rio de Janeiro, carries only the E484K mutation in the RBD and has spread to other parts of South America, including Argentina laboratories to confer escape from convalescent sera and monoclonal antibodies.
  • the assay described in Example 11, may, for example be used to neutralizing activity of vaccine sera.
  • isogenic rcVSV-CoV2-S were generated expressing the B.1.1.7 (UK SARS-CoV-2 lineage), B.1.351 or E484K S to evaluate the neutralizing activity of Sputnik V vaccine sera from Argentina.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Communicable Diseases (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Pulmonology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Provided herein are SARS-CoV-2 spike protein pseudotyped vesicular stomatitis virus (VSV) particles and methods for generating such particles. Also provided herein are neutralization assays using SARS-CoV-2 spike protein pseudotyped vesicular stomatitis virus (VSV) particles.

Description

SARS-COV-2 SPIKE PROTEIN PSEUDOTYPED VSV-DELTA G PARTICLES
AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/029,217, filed May 22, 2020, and of U.S. Provisional Patent Application No. 63/063,041 , filed August 7, 2020, which are incorporated by reference herein in their entireties.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under AI07647, AI154739, AI123449 and AI1498033 awarded by National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
[0003] There is an urgent need to develop therapeutics to treat COVID-19 and diagnostics to detect severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). As of May 14, 2021, globally more than 161 million people have tested positive for SARS-CoV-2. In addition, as of May 14, 2021, globally more than 3.35 million people have died from COVID-19.
[0004] There is an urgent need for standardized virus neutralization assay (VNA) to understand and assess humoral immunity in acute and recovered COVID-19 patients, to screen for therapeutic entry inhibitors, such as small molecules, monoclonal antibodies, or convalescent sera, and to screen for vaccine induced responses, in animals as well as in humans.
SUMMARY OF THE INVENTION
[0005] In one aspect, provided is a SARS-CoV-2 spike protein pseudotyped vesicular stomatitis virus (VSV) particle comprising an encapsidated negative sense, single-stranded RNA genome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, and a nucleotide sequence encoding luciferase, wherein genome does not express VSV glycoprotein (G).
[0006] In some embodiments, the luciferase is renilla luciferase or nanoluciferase.
[0007] In some embodiments, the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the SARS-CoV-2 spike protein is encoded by the nucleotide sequence set forth in SEQ ID NO:l.
[0008] In some embodiments, the genome does not comprise a nucleotide sequence sequence encoding VSV glycoprotein (G). In some embodiments, the genome further comprises a nucleotide sequence encoding a fluorescent protein. In some embodiments, the fluorescent protein is red fluorescent protein or enhanced green fluorescent protein.
[0009] In some embodiments, the SARS-CoV-2 spike protein pseudotyped VSV particle has been treated with trypsin. In some embodiments, the SARS-CoV-2 spike protein pseudotyped VSV particle has been treated with trypsin and soybean inhibitor.
[0010] In one aspect, provided is a composition comprising a SARS-CoV-2 spike protein pseudotyped VSV particle described herein and a carrier. In some embodiments, the carrier is serum free media. In some embodiments, the carrier is phosphate buffered saline.
[0011] Provided herein is a composition comprising a SARS-CoV-2 spike protein pseudotyped VSV particle disclosed herein and trypsin. Also provided is a composition comprising a SARS-CoV-2 spike protein pseudotyped VSV particle disclosed herein and trypsin and soybean inhibitor.
[0012] In one aspect, provided is a method for generating SARS-CoV-2 spike protein pseudotyped VSV particles, comprising:
(a) infecting cells overexpressing SARS-CoV-2 spike protein with a recombinant VSV particle, wherein the recombinant VSV particle comprises an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding luciferase, wherein the genome does not express VSV glycoprotein (G), wherein the VSV particle is pseudotyped with VSV glycoprotein; and (b) purifying SARS-CoV-2 spike protein pseudotyped VSV particles from the supernatant of the cells.
[0013] In some embodiments, the cells are cultured in optiMEM containing anti-VSV-G antibody. In some embodiments, the SARS-CoV-2 spike protein pseudotyped VSV particles are purified from the supernatant by low speed centrifugation. In some embodiments, the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the SARS-CoV-2 spike protein is encoded by the nucleotide sequence set forth in SEQ ID NO:l. In some embodiments, the luciferase is renilla luciferase.
[0014] In one aspect, provided is a method for infecting cells with a SARS-CoV-2 spike protein pseudotyped VSV particle disclosed herein, comprising:
(a) contacting the SARS-CoV-2 spike protein pseudotyped VSV particle with trypsin for a certain period of time; and
(b) infecting cells with the SARS-CoV-2 spike protein pseudotyped VSV particle.
[0015] In one aspect, provided is a method for infecting cells with a SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 7, comprising:
(a) contacting the SARS-CoV-2 spike protein pseudotyped VSV particle with trypsin and soybean inhibitor for a certain period of time; and
(b) infecting cells with the SARS-CoV-2 spike protein pseudotyped VSV particle.
[0016] In one aspect, provided is a method for infecting cells with a SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 7, comprising:
(a) contacting the SARS-CoV-2 spike protein pseudotyped VSV particle with trypsin for a certain period of time; and
(b) spinoculating the cells with the SARS-CoV-2 spike protein pseudotyped VSV particle.
[0017] In some embodiments, step (a) farther comprises contacting the particle with soybean inhibitor. In some embodiments, the certain period of time is 15 minutes.
[0018] In one aspect, provided is a method for detecting sera that neutralizes SARS-CoV- 2 comprising:
(a) incubating with the SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 7 with sera from a subject for a first period of time; (b) spinoculating cells expressing human ACE-2, TMPRSS2 or both with the sera- treated SARS-CoV-2 spike protein pseudotyped VSV particle for a second period of time; and
(c) measuring the luciferase activity after a third period of time, wherein a lower level of luciferase activity is detected if the sera neutralizes SARS-CoV-2 spike protein pseudotyped VSV particle than if steps (b) to (c) are performed without performing step (a), and the lower level of luciferase activity indicates that the sera neutralizes SARS-CoV-2.
[0019] In some embodiments, the first period of time is about 30 minutes. In some embodiments, the second period of time is about 1 hour. In some embodiments, the third period of time is about 18 to 22 hours.
[0020] In some embodiments, the subject is a human subject.
[0021] In some embodiments, the sera is heat inactivated.
[0022] In some embodiments, the sera is diluted in plain DMEM or DMEM and 10% heat inactivated fetal bovine serum.
[0023] In some embodiments, the method for detecting sera that neutralizes SARS-CoV-2 comprises concurrently repeating steps (a) to (c) with a positive control antibody or sera that does neutralize SARS-CoV-2. In some embodiments, the method further comprises concurrently repeating steps (a) to (c) with a negative control antibody or sera that does not neutralize SARS-CoV-2.
[0024] In one aspect, provided is a method for assessing the ability of an antibody to neutralize SARS-CoV-2 comprising:
(a) incubating with the SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 7 with an antibody of interest for a first period of time;
(b) spinoculating cells expressing human ACE-2, TMPRSS2 or both with the antibody- treated SARS-CoV-2 spike protein pseudotyped VSV particle for a second period of time; and
(c) measuring the luciferase activity after a third period of time, wherein a lower level of luciferase activity is detected if the antibody neutralizes SARS-CoV-2 spike protein pseudotyped VSV particle than if steps (b) to (c) are performed without performing step (a), and the lower level of luciferase activity indicates that the antibody neutralizes SARS-CoV-2. [0025] In some embodiments, the first period of time is about 30 minutes. In some embodiments, the the second period of time is about 1 hour. In some embodiments, the third period of time is about 18 to 22 hours. In some embodiments, the cells overexpress human ACE-2, TMPRSS2, or both.
[0026] In one aspect, provided is a kit comprising the SARS-CoV-2 spike protein pseudotyped VSV particle disclosed herein, and optionally instructions for performing a neutralization assay using the SARS-CoV-2 spike protein pseudotyped VSV particle.
[0027] In one aspect, provided is a nucleic acid sequence comprising a VSV antigenome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding for luciferase, and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G).
[0028] In some embodiments, the nucleic acid further comprises a T7 promoter, autocatalytic hammerhead ribozyme sequences, and a T7 terminator, optionally wherein the hammerhead ribozyme sequences is immediately upstream of the 3 ’ leader sequence. In some embodiments, the luciferase is renilla luciferase or nanoluciferase. In some embodiments, the fluorescent protein is red fluorescent protein or enhanced green fluorescent protein.
[0029] In one aspect, provided is a recombinant VSV particle pseudotyped with VSV glycoprotein comprising an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding for luciferase, and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G). In some embodiments, the luciferase is renilla luciferase or nanoluciferase. In some embodiments, the fluorescent protein is red fluorescent protein or enhanced green fluorescent protein.
[0030] In one aspect, provided is a method for generating the recombinant VSV particle pseudotyped with VSV glycoprotein disclosed herein, the method comprising:
(a) transfecting cells with a nucleic acid disclosed, a first vector comprising a nucleotide sequence encoding VSV M protein, a second vector comprising a nucleotide sequence encoding VSV L protein, a third vector encoding VSV N protein, a fourth vector comprising a nucleotide sequence encoding VSV G protein, and a fifth vector comprising a nucleotide sequence encoding VSV P protein, wherein the nucleic acid sequence comprises a VSV antigenome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding for luciferase, and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G); and (b) purifying the recombinant VSV particle pseudotyped with VSV glycoprotein.
[0031] In some embodiments, the method farther comprising transfecting the cells with a sixth vector comprising a codon-optimized sequence encoding a T7 polymerase.
[0032] In one aspect, provided is a method for generating pseudotyped VSV particle, comprising:
(a) infecting or spinoculating cells overexpressing viral surface protein of interest with the recombinant VSV particle disclosed herein; and
(b) purifying viral surface protein pseudotyped VSV particles from the supernatant of the cells.
[0033] In some embodiments the cells are 293T-ACE2 clone 5-7 or 293T-ACE2- TMPRSS2 clone F8-2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIGs. 1A, IB, 1C, ID, and IE illustrate the production of VSV \G-rLuc bearing SARS-CoV-2 spike glycoprotein. FIG. 1A. Overview of VSV \G-rLuc pseudotyped particles bearing CoV-2 spike (top panel) with annotated spike glycoprotein domains and cleavage sites (bottom panel). SARS-CoV is referred to as SARS-CoV-1 for greater clarity. FIG. IB. VSV- \G| Rluc| pseudotyped particles (VSVpp) bearing the Nipah virus receptor binding protein alone (NiV- RBPpp), SARS-CoV-2-S (CoV2pp), or VSV-G (VSV-Gpp) were titered on Vero-CCL81 cells using a 10-fold serial dilution. Symbols represent the mean +/- SEM (error bars) of each titration performed in technical triplicates. FIG. 1C. Genome copy number and particle to infectivity ratio. Genome copy number was assessed using primers against the VSV-L protein as previously described (Pryce R, Azarm K et al Life Sci Alliance 2020 Jan; 3(1): e201900578). Particle to infectivity ratio was calculated as a fraction of number of genomes to TCID50. FIG. ID. Expression of the indicated viral glycoproteins on producer cells and their incorporation into VSVpp. Western blots performed using anti-Sl or anti-S2 specific antibodies. FIG. IE. CoV2pp entry is inhibited by soluble receptor binding domain (sRBD) derived from SARS-CoV-2-S. CoV2pp and VSV-Gpp infection of Vero-CCL81 cells was performed as in FIG. IB in the presence of the indicated amounts of sRBD. Neutralization curves were generated by fitting data points using a variable slope, 4- parameter logistics regression curve (robust fitting method). The last point (no sRBD) was fixed to represent 100% maximal infection. Each replicate from an experiment performed in duplicate is shown. The calculated IC50 for sRBD neutralization of CoV2pp is 4.65 pg/mL. [0035] FIGs. 2A, 2B, 2C, 2D, and 2E illustrate that CoV2pp entry is enhanced by trypsin treatment. FIG. 2A. Optimizing trypsin treatment conditions. Supernatant containing CoV2pp were trypsin-treated at the indicated concentrations for 15 min. at room temperature prior to the addition of 625 pg/mL of soybean trypsin inhibitor (SBTI). These particles were then titered on Vero-CCL81 cells in technical triplicates. Data shown as mean +/- SEM. FIG. 2B. Dilution in serum free media (SEM, DMEM only) provides the highest signalmoise ratio for trypsin-treated CoV2pp entry. Particles were diluted 1:10 in Opti-MEM, SFM, or DMEM + 10%FBS prior to infection of Vero-CCL81 cells and spinoculation as described in Fig. IE. Cells infected without spinoculation show approximately 3x less signalmoise ratios. FIG. 2C. Dilution of CoV2pp in the absence of serum free media produces the highest signalmoise for trypsin treated CoV2pp. Presented are the results from an experiment in technical triplicate and error bars show the SEM. FIG. 2D. Addition of soybean trypsin inhibitor at the time of infection reduces trypsin treated particle entry. This was performed in technical triplicates for two independent experiments. Shown are the combined results with error bars indicating SEM and **** indicating a p-value <0.0001. FIG. 2E. Effect of different trypsin concentration on CoV2pp activation. Supernatant containing CoV2pp were treated with different concentrations of trypsin for 15 minutes, then used to infect Vero-CCL81 cells. [0036] FIG. 3 illustrates that trypsin- treated CoV2pp depend on ACE2 and TMPRSS2 for entry. Parental and TMPRSS2 or ACE2 transduced VeroCCL81 cells were infected with the indicated pseudotyped viruses. All particles were diluted in serum free media in order to be within the linear range for the assay. Normalized infectivity data is presented as fold-over Vero-CCL81-WT for the various VSVpp shown. VSV-Gpp served as an internal control for the intrinsic permissiveness of various cell lines to VSV mediated gene expression. Data is presented as meant-/- SEM from two independent experiments done in technical triplicates. *, p <0.05, **, p <0.01, and ****, p <0.0001.
[0037] FIGs. 4A, 4B, and 4C illustrate sera neutralization in the absence of 10% FBS and optimization of neutralizations. FIG. 4A. Negative sera potently inhibit trypsin treated CoV2pp. CoV2pp were diluted in serum free media (SFM), then pooled negative sera and a positive serum were used to neutralize entry. An aliquot was heat inactivated (HI) for lhr in a 56°C water bath prior to use. Data are presented on a linear (top panel) and log scale (bottom panel). Each replicate from one experiment in technical duplicates are shown and neutralization curves were generated as done in Fig. ID. FIG. 4B. Sera neutralizations were performed with untreated CoV2pp (top panel) or CoV2pp treated with trypsin (middle panel). Both particles were diluted in DMEM + 10% FBS and neutralization curves are presented as described above. VSV-G was not neutralized by the negative or positive sera (bottom panel). FIG. 4C. sRBD neutralizes CoV2pp equivalently across all conditions tested. Data presented in Fig. IE (i.e. the untreated CoV2pp) is duplicated here.
[0038] FIG. 5 shows CoV2pp viral neutralization assay and absIC50/80 versus Spike binding of patient sera. 36 patient sera screened for CoV2pp neutralization. CoV2pp were used to infect Vero-CCF81 cells in the presence of a 4-fold serial dilution of patient sera. Samples in light grey do not neutralize CoV2pp. Neutralization curves were fit using a variable slope, 4-parameter logistics regression curve with a robust fitting method.
[0039] FIG. 6 shows a comparison of CoV2pp Absolute IC values across all 4 groups. Shown are the CoV2pp absolute IC50 (top panel), IC80 (middle panel) and IC90 (bottom panel) from all four groups with error bars showing the median and interquartile range. The dotted line presents the median from the aggregated positive neutralization samples as reported in Table 1. The dashed line indicates neat serum and the shaded gray region highlights samples that fall below this value. An ordinary one-way ANOVA with Dunnett’s correction for multiple comparisons was performed for statistical analysis. This analysis revealed no statistically significant difference between the Absolute IC values obtained across the 4 groups. There were notable outliers in this data set, including individuals that show poor neutralization (i.e. the 3 samples in the IC50 plot from FSUHS) and an individual that showed exceptionally potent neutralization (i.e. the sample in all plots from ISMMS-2). [0040] FIGs. 7A, 7B, 7C, and 7D illustrate that 293T stably transduced with ACE2 and TMPRSS2 (293T-ACE2+TMPRSS2) are ultra-permissive for SARS-CoV-2pp infection. FIG. 7A. Infection of 293T cells lines transduced to stably express, TMPRSS2, ACE2, or both. A single dilution of particles was used to infect cells prior to spinoculation. Infections were done in technical triplicates. Presented are the aggregated results from two independent replicates and error bars show SEM. For statistics, ns = not significant, ** represents p-value <0.01, and **** represents p-value <0.0001. FIG. 7B. Normalized CoV2pp entry into single cell clones. Entry was normalized to the wild type parental cell line and further normalized to VSV-G entry. Presented are the average of one experiment in technical triplicates. Error bars show the median and interquartile range. FIG. 7C. CoV2pp were titered on Vero-CCL81 cells, 293T-ACE2 clone 5-7, and 293T-ACE2-TMPRSS2 clone F8-2. Titrations were performed with untreated CoV2pp and without spinoculation. Presented are the results from technical triplicates and bars show the SEM. FIG. 7D. Entry inhibition of CoV2pp by Nafamostat mesylate, a serine protease inhibitor. Nafamostat was mixed with CoV2pp (top panel) or VSV-Gpp (bottom panel) prior addition to cells. Shown are the results from one experiment in technical triplicates. Error bars show SEM.
[0041] FIGs. 8A and 8B illustrate that ultra-permissive 293T-ACE2+TMPRSS2 cell clones retain the same phenotypic sensitivity to convalescent COVID-19 sera. FIG. 8A. Selection of pooled sera samples. Presented are the subset of samples that were pooled for use in viral neutralization assays (VNAs). FIG. 8B. Vero CCL81 and transduced 293T cells were used for VNAs. Sera previously shown to be negative, weakly positive, or strongly positive for CoV2pp neutralizations were selected to be pooled in equal volumes. These were subsequently used for VNAs. Notably, these VNAs were performed in the absence of exogenous trypsin or spinoculation.
[0042] FIGs. 9A and 9B illustrate the robust and efficient generation of an EGFP-reporter replication-competent VSV bearing SARS-CoV-2 spike (rcVSV-CoV2-S). FIG. 9A. Schematic of the rcVSV-CoV2-S genomic coding construct and the virus rescue procedure. The maximal T7 promoter (T7prom) followed by a hammer-head ribozyme (HhRbz) and the HDV ribozyme (HDVRbz) plus T7 terminator (T7term) are positioned at the 3’ and 5’ ends of the viral cDNA, respectively. An EGFP(E) transcriptional unit is placed at the 3’ terminus to allow for high level transcription. SARS-CoV-2-S is cloned in place of VSV-G using the indicated restriction sites designed to facilitate easy exchange of spike variant or mutants. FIG. 9B. For virus rescue, highly permissive 293T cells stably expressing human ACE2 and TMPRSS2 (293T- [ACE2+TMPRSS2], F8-2 clone) cells were transfected with the genome coding plasmid, helper plasmids encoding CMV-driven N, P, M, and L genes, and pCAGS encoding codon-optimized T7-RNA polymerase(T7opt). 48-72 hpi, transfected cells turn EGFP+ and start forming syncytia. Supernatant containing rcVSV-CoV2-S are then amplified in Vero-TMPRSS2 cells at the scale shown. The blue arrowsat the bottom indicate the timeline for production of each sequence verified stock.
[0043] FIGs. 10A and 10B illustrate the generation of replication-competent VSV bearing SARS-CoV-2 spike (rcVSV- CoV2-S). FIG. 10A. Representative images of de novo generation of rcVSV-CoV2-S, carrying an EGFP reporter, in transfected 293T- ACE2+TMPRSS2 (F8-2) cells as described in FIG. 9. Single GFP+ cells detected at 2-3 days post-transfection (dpt) form a foci of syncytia by 4 dpt. Images are taken by Celigo imaging cytometer (Nexcelom) and are computational composites from the identical number of fields in each well. White bar is equal to 1 millimeter. FIG. 10B. Entry efficiency of rcVSV-CoV2- S in parental 293T cells, 293T stably expressing ACE2 alone (293T-ACE2) or with TMPRSS2 (293T-ACE2+TMPRSS2). Serial dilutions of virus stocks amplified on Vero- TMPRSS2 cells were used to infect the indicated cell lines in 96-well plates in triplicates. GFP signal was detected and counted by a Celigo imaging cytometer (Nexcelom) 10 hours post-infection. Symbols are individual data points from triplicate infections at the indicated dilutions. Bars represent the average of 3 replicates with errorbars indicating standard deviation. A two-way ANOVA was used to compare the differences between cell lines at any given dilution. Adjusted p values from Tukey’s multiple comparisons test are given (ns; not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
[0044] FIGS. 11A and 11B illustrate the results of a neutralization activity of antibody responses elicited by the Sputnik V vaccine. FIG. 11A. Neutralization activity of individual serum samples against rcVSV- CoV2-S with the WT, variant (B.1.1.7 or B.1.351), or mutant E484K spike proteins. Neutralization is represented by the reciprocal 50% inhibitory dilution factor (I/IC50). Sera samples with no appreciable neutralization against a given virus were assigned a defined l/ICsovalue of 1.0, as values <1 are not physiological (Grey shaded area). Dashed line indicates the lowest serum dilution tested (1/IC50 = 20). Geometric mean titers (GMT and 95% Cl) for the neutralizing activity of all vaccine sera are indicated below each of the viral spike proteins examined. NS; not significant, *; p<0.05, p < 0.01; ** are adjusted p values from non- parametric one-way ANOVA with Dunn’s multiple comparisons test. FIG. 11B. For each serum sample, the fold-change in IC50 (reciprocal inhibitory dilution factor) against the indicated variant and mutant spike proteins relative to its IC50 against wild- type (WT) spike (set at 1) is plotted. Adjusted p values were calculated as in FIG. 11 A. Medians are represented by the bars and whi skersdem arcate the 95 % Cl. Neutralization dose- response curves were performed in triplicates, and the mean values from each triplicate experiment are shown as the single data points for each sera sample.
DETAILED DESCRIPTION
[0045] Standardized virus neutralization assay (VNA) can be used to understand and assess humoral immunity in acute and recovered COVID-19 patients, and to screen for therapeutic entry inhibitors, such as small molecules, monoclonal antibodies, or convalescent sera. Most importantly, such assays are needed to screen for vaccine induced responses, in animals as well as in humans, as the world rushes to develop a multitude of vaccines against SARS-CoV2.
[0046] A VNA that is robust, high throughput (>100,000 infections/week), easily adapted to kit format, and can generate absolute virus neutralization titers (VNT) allows for meaningful comparisons across different labs. In addition to helping evaluate the myriad vaccine candidates, use of a standardized VNA to report VNT in absolute units can crowd- source the immense effort being expended by multiple labs across the globe to better understand the basis of the marked variation in VNT seen in COVID-19 recovered patients.
[0047] The SARS-CoV-2 spike glycoprotein (S) is embedded in the viral envelope and facilitates bothreceptor recognition and membrane fusion. SARS-CoV-2-S is 1273 amino acids in length and, like other coronaviruses, is a trimeric class I fusion protein. The S glycoprotein contains two subunits, the N-terminal, SI subunit and the C-terminal, S2 subunit. The SI subunit contains the receptor-binding domain (RBD), which is responsible for host receptor binding. The S2 subunit contains the transmembrane domain, cytoplasmic tails, and machinery necessary for fusion, notably the fusion peptide and heptad repeats. Angiotensin-converting enzyme 2 (ACE2), a cell surface enzyme found in a variety of tissues, facilitates binding and entry of SARS-CoV-2. However, ACE2 alone is not sufficient for efficient entry into cells. While entry depends on the SI subunit binding ACE2, entry is further enhanced by proteolytic cleavage between the S1/S2 and S2’ subunits. For both SARS-CoV-1 and SARS-CoV-2, this cleavage-mediated activation of S-mediated entry is supported by the expression of cell-associated proteases, like cathepsins or transmembrane serine protease 2 (TMPRSS2), or the addition of exogenous proteases that mimic the various trypsin-like proteases present in the extracellular lung milieu. These proteases facilitate entry at the cell surface or via an endosomal route in a cell-type dependent manner. Extracellular proteases are thought to play a pathophysiogical role in the lung tissue damage caused by unabated MERS-CoV, SARS-CoV-1, and likely SARS-CoV-2 replication. Thus, in order to represent SARS-CoV-2 cell entry faithfully, a viral neutralization assay (VNA) should be sensitive not only to ACE2 binding but also to the proteolytic activation of spike. In addition to its role in receptor binding and entry, S is the primary surface glycoprotein and the major target of the neutralizing antibody response. Patients infected with SARS-CoV-2 typically seroconvert within two weeks of symptom onset, with about half developing antibodies within 7 days. Antibody titers appear to be durable at greater than 40 days post infection, but in the case of SARS-CoV-1, reductions in IgG positive titers begin around 4-5 months post infection and show a significant drop by 36 months. Although there are reports of SARS- CoV-2 infected individuals testing positive by RT-PCR weeks after being confirmed as recovered by two consecutive negative tests, these are more likely the result of false negatives than of reinfection. A better understanding of the durability and efficacy of the neutralizing antibody response in patients previously infected with SARS-CoV-2 is of paramount importance. Not only do IgG titers wane in the case of SARS-CoV-1, but reinfection is possible in other endemic human coronaviruses (HCoVs) such as 229E, NL63, and OC43 in as little as a year.
[0048] Humoral immune responses to the SARS-CoV-2 S protein are typically evaluated by enzyme-linked immunosorbent assays (ELISAs) and its many variants (CLIA, LFA, etc.). These serological binding assays rightfully play a central role in determining patient antibody responses and can complement diagnostics and sero-epidemiological studies, especially when combined with antibody subclass determination (IgM, IgA and IgG). Nonetheless, as many antibodies generated to the spike protein bind but do not block virus entry, ELISA-based assays that detect titers of spike-binding antibodies cannot always correlate perfectly with neutralizing antibody titers as measured by plaque reduction neutralization or microneutralization tests. Even a cleverly designed competitive ELISA set up to detect antibodies that block the binding of RBD to ACE2 cannot capture the universe of neutralizing antibodies targeted to conformationally dynamic trimeric spike on a virion. The gold standard for detecting antiviral antibodies remains the virus neutralizing assay. Assays that faithfully recapitulate entry of SARS-CoV-2 based on the compositions and methods disclosed herein that maximizing safety, speed, and scalability will be vital in the coming months and years. They will enable monitoring of patient neutralizing antibody response, efficacy of vaccines and entry inhibitors, and the screening of convalescent plasma from COVID-19 recovered patients.
[0049] Provided herein are compositions and methods utilizing a SARS-CoV-2 pseudotyped viral particle (CoV2pp) by using vesicular stomatitis virus bearing e fluorescent report egene (e.g., the Renilla luciferase gene) in place of its G glycoprotein (VSV \G-rLuc). These standardized CoV2pp can be used for ready-to-use “out of the box” VNAs. This assay can provide robust metrics (absIC50, absIC80, absIC90) for meaningful comparisons between labs.
[0050] Also provided are ultra-permissive 293T cell clones that stably express either ACE2 alone or ACE2+TMPRSS2 and methods of using these clones. These isogenic cell lines support either the late (293 T-ACE2) or early (293 T-ACE2/TMPRSS2) entry pathways that SARS-CoV-2 uses. These ultra-permissive 293T clones allow for the use of unpurified virus supernatant from the standard vims production batch, which can now provide for -150,000 infections per week (96-well format) with no further scale-up.
[0051] SARS-CoV-2 spike protein pseudotyped vesicular stomatitis virus (VSV) particles
[0052] In one aspect, provided herein are SARS-CoV-2 spike protein pseudotyped vesicular stomatitis virus (VSV) particles. In a one embodiment, provided herein are SARS- CoV-2 spike protein pseudotyped VSV particles comprising an encapsidated negative sense, single- stranded RNA genome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, and a nucleotide sequence encoding a reporter protein, wherein genome does not express VSV glycoprotein (G). In some embodiments, the reporter protein is luciferase. [0053] As used herein, the terms “SARS-CoV-2 spike protein” refers to a SARS-CoV-2 spike protein known to those of skill in the art. In certain embodiments, the spike protein comprises the amino acid or nucleic acid sequence found at GenBank Accession No. MN908947.3, MT291835.2, MT358639.1, MT079851, MT079848.1, or MT079845.1. A typical spike protein comprises domains known to those of skill in the art including an S 1 domain, a receptor binding domain, an S2 domain, a transmembrane domain and a cytoplasmic domain. See, e.g., Wrapp et al., 2020, Science 367: 1260-1263 for a description of SARS-CoV-2 spike protein (in particular, the structure of such protein). The spike protein may be characterized has having a signal peptide (e.g., a signal peptide of 1-14 amino acid residues of the amino acid sequence of GenBank Accession No. MN908947.3), a receptor binding domain (e.g., a receptor binding domain of 319-541 amino acid residues of GenBank Accession No. MN908947.3), an ectodomain (e.g., an ectodomain of 15-1213 amino acid residues of GenBank Accession No. MN908947.3), and a transmembrane and endodomain (e.g,. a transmembrane and endodomain of 1214-1273 amino acid residues of GenBank Accession No. MN908947.3). The spike protein may also be characterized as having an SI subunit and S2 subunit. In particular embodiments, the SARS-CoV-2 spike protein is full length.
[0054] In a specific embodiment, a SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:2. Due to the degeneracy of the code, any nucleotide sequence that encodes a SARS-CoV-2 spike protein (e.g., any nucleotide sequence encoding SEQ ID NO:2) may be used as described herein. In another specific embodiment, a nucleotide sequence encoding the SARS-CoV-2 spike protein is codon optimized for humans. In another specific embodiment, a SARS-CoV-2 spike protein is encoded by the nucleotide sequence set forth in SEQ ID NO:l.
[0055] VSV strains and genomic sequences are known in the art. For example, the ATCC offers VSV (ATCC VR-1238). In addition, nucleotide sequences for VSV and the proteins encoded by VSV may be found on, e.g., GenBank. See, e.g., GenBank Accession Nos. NC_001560.1 (GI: 9627229) and J02428.1 (GI: 335873).
[0056] In a one embodiment, the genome of the SARS-CoV-2 spike protein pseudotyped VSV particle further comprises a nucleotide sequence encoding a fluorescent protein. In some embodiments, the nucleotide sequence encoding the reporter protein and the nucleotide sequence encoding fluorescent protein are separated by a nucleotide sequence encoding a self-cleaving peptide (e.g., a 2 A self-cleaving peptide) such that a single polypeptide comprising the reporter protein, the self-cleaving peptide, and fluorescent protein is generated and may be cleaved to produce the reporter protein and the fluorescent protein.
[0057] In some embodiments, the fluorescent protein is a red fluorescent protein or green fluorescent protein. Additional examples of fluorescent proteins compatible with the compositions and methods disclosed herein include, include, but are not limited to, (3-F)Tyr- EGFP, A44-KR, aacuGFPl, aacuGFP2, aceGFP, aceGFP-G222E-Y220L, aceGFP-h, AcGFPl, AdRed, AdRed-C148S, aeurGFP, afraGFP, alajGFPl, alajGFP2, alajGFP3, amCyanl, amFP486, amFP495, amFP506, amFP515, amilFP484, amilFP490, amilFP497, amilFP504, amilFP512, amilFP513, amilFP593, amilFP597, anmlGFPl, anmlGFP2, anm2CP, anobCFPl, anobCFP2, anobGFP, apulFP483, AQ14, AQ143, Aquamarine, asCP562, asFP499, AsRed2, asulCP, atenFP, avGFP, avGFP454, avGFP480, avGFP509, avGFP510, avGFP514, avGFP523, AzamiGreen, Azurite, BDFP1.6, bfloGFPal, bfloGFPcl, BFP, BFP.A5, BFP5, bsDronpa (On), ccalGFPl, ccalGFP3, ccalOFPl, ccalRFPl, ccalYFPl, cEGFP, cerFP505, Cerulean, CFP, cFP484, cfSGFP2, cgfmKate2, CGFP, cgfTagRFP, cgigGFP, cgreGFP, CheGFPl, CheGFP2, CheGFP4, Citrine, Citrine2, Clomeleon, Clover, cp-mKate, cpCitrine, cpT-Sapphirel74-173, CyOFPl, CyPet, CyRFPl (CyRFPl), d-RFP618, DIO, dlEosFP (Green), dlEosFP (Red), d2EosFP (Green), d2EosFP (Red), deGFPl, deGFP2, deGFP3, deGFP4, dendFP (Green), dendFP (Red), Dendra (Green), Dendra (Red), Dendra2 (Green), Dendra2 (Red), Dendra2-M159A (Green), Dendra2-M159A (Orange), Dendra2-T69A (Green), Dendra2-T69A (Orange), dfGFP, dimerl, dimer2, dis2RFP, dis3GFP, dKeima, dKeima570, dLanYFP, DrCBD, Dreiklang (On), Dronpa (On), Dronpa-2 (On), Dronpa-3 (On), dsFP483, DspRl, DsRed, DsRed-Express, DsRed-Express2, DsRed- Max, DsRed.Ml, DsRed.T3, DsRed.T4, DsRed2, DstCl, dTFPO.l, dTFP0.2, dTG, dTomato, dVFP, E2-Crimson, E2-Orange, E2-Red/Green, EaGFP, EBFP, EBFP1.2, EBFP1.5, EBFP2, ECFP, ECFPH148D, ECGFP, eechGFPl, eechGFP2, eechGFP3, eechRFP, efasCFP, efasGFP, eforCP, EGFP, eGFP203C, eGFP205C, Emerald, Enhanced Cyan-Emitting GFP, EosFP (Green), EosFP (Red), eqFP578, eqFP611, eqFP611V124T, eqFP650, eqFP670, EYFP, EYFP-Q69K, fabdGFP, ffDronpa (On), FoldingReporterGFP, FP586, FPrfl2.3, FR-1, FusionRed, FusionRed-M, Gl, G2, G3, Gamillus (On), GamillusO.l, Gamillus0.2, Gamillus0.3, Gamillus0.4, GCaMP2, gfasGFP, GFP(S65T), GFP-151pyTyrCu, GFP- Tyrl51pyz, GFPmut2, GFPmut3, GFPxml6, GFPxml61, GFPxml62, GFPxml63, GFPxml8, GFPxml81uv, GFPxml8uv, GFPxml9, GFPxml91uv, GFPxml9uv, H9, HcRed, He Red-Tandem, HcRed7, hcriGFP, hmGFP, HriCFP, HriGFP, iFP1.4, iFP2.0, iLov, iq- EBFP2, iq-mApple, iq-mCerulean3, iq-mEmerald, iq-mKate2, iq-mVenus, iRFP670, iRFP682, iRFP702, iRFP713, iRFP720, IrisFP (Green), IrisFP (Orange), IrisFP-M159A (Green), Jred, Kaede (Green), Kaede (Red), Katushka, Katushka-9-5 , Katushka2S, KCY, KCY-G4219, KCY-G4219-38L, KCY-R1, KCY-R1-158A, KCY-R1-38H, KCY-R1-38L, KFP1 (On), KrkGRl (Green), KrkGRl (Red), KillerOrange, KillerRed, KO, Kohinoor (On), laesGFP, laGFP, LanFPl, LanFP2, lanRFP-AS831, LanYFP, laRFP, LSS-mKatel, LSS- mKate2, LSSmOrange, M355NA, mAmetrine, mApple, MaroonO.l, mAzamiGreen, mBanana, mBeRFP, mBlueberryl, mBlueberry2, mcl, mc2, mc3, mc4, mc5, mc6, McaGl, McaGlea, McaG2, mCardinal, mCarmine, mcavFP, mcavGFP, mcavRFP, mcCFP, mCerulean, mCerulean.B, mCerulean.B2, mCemlean.B24, mCemlean2, mCerulean2.D3, mCerulean2.N, mCemlean2.N(T65S), mCerulean3, mCherry, mCherry2, mCitrine, mClavGR2 (Green), mClavGR2 (Red), mClover3, mCyRFPl, mECFP, meffCFP, meffGFP, meffRFP, mEGFP, meleCFP, meleRFP, mEmerald, mEos2 (Green), mEos2 (Red), mEos2- A69T (Green), mEos2-A69T (Orange), mEos3.1 (Green), mEos3.1 (Red), mEos3.2 (Green), mEos3.2 (Red), mEos4a (Green), mEos4a (Red), mEos4b (Green), mEos4b (Red), mEosFP (Green), mEosFP (Red), mEosFP-F173S (Green), mEosFP-F173S (Red), mEosFP-M159A (Green), mEYFP, MfaGl, mGarnet, mGarnet2, mGeos-C (On), mGeos-E (On), mGeos-F (On), mGeos-L (On), mGeos-M (On), mGeos-S (On), mGingerl, mGinger2, mGrapel, mGrape2, mGrape3, mHoneydew, MiCy, mIFP, miniSOG, miniSOGQ103V, miniSOG2, miRFP, miRFP670, miRFP670nano, miRFP670vl, miRFP703, miRFP709, miRFP720, mlrisFP (Green), mlrisFP (Red), mK-GO (Early), mK-GO (Late), mKalamal, mKate, mKateM41GS158C, mKateS158A, mKateS158C, mKate2, mKeima, mKellyl, mKelly2, mKG, mKikGR (Green), mKikGR (Red), mKillerOrange, mKO, mK02, ihKOk, mLumin, mMaple (Green), mMaple (Red), mMaple2 (Green), mMaple2 (Red), mMaple3 (Green), mMaple3 (Red), mMaroonl, mmGFP, mMiCy, mmilCFP, mNectarine, mNeonGreen, mNeptune, mNeptune2, mNeptune2.5, mNeptune681, mNeptune684, Montiporasp.#20-9115, mOrange, mOrange2, moxBFP, moxCerulean3, moxDendra2 (Green), moxDendra2 (Red), moxGFP, moxMaple3 (Green), moxMaple3 (Red), moxNeonGreen, moxVenus, mPapaya, mPapaya0.7, mPlum, mPlum-E16P, mRaspberry, mRed7, mRed7Ql, mRed7QlSl, mRed7QlSlBM, mRFPl, mRFPl-Q66C, mRFPl-Q66S, mRFPl-Q66T, mRFPl.l, mRFP1.2, mRojoA, mRojoB, mRouge, mRtms5, mRuby, mRuby2, mRuby3, mScarlet, mScarlet-H, mScarlet-I, mStable, mStrawberry, mT-Sapphire, mTagBFP2, mTangerine, mTFP0.3, mTFP0.7 (On), mTFPl, mTFPl-Y67W, mTurquoise, mTurquoise2, muGFP, mUkG, mVenus, mVenus-Q69M, mVFP, mVFPl, mWasabi, Neptune, NijiFP (Green), NijiFP (Orange), NowGFP, obeCFP, obeGFP, obeYFP, OFP, OFPxm, oxBFP, oxCerulean, oxGFP, oxVenus, Pll, P4, P4-1, P4-3E, P9, PA-GFP (On), Padron (On), Padron(star) (On), Padron0.9 (On), PAmCherryl (On), PAmCherry2 (On), PAmCherry3 (On), PAmKate (On), PATagRFP (On), PATagRFP1297 (On), PATagRFP1314 (On), pcDronpa (Green), pcDronpa (Red), pcDronpa2 (Green), pcDronpa2 (Red), PdaCl, pdaelGFP, phiYFP, phiYFPv, pHluorin, ecliptic, pHluorin, ecliptic (acidic), pHluorin,ratiometric (acidic), pHluorin,ratiometric (alkaline), pHluorin2 (acidic), pHluorin2 (alkaline), pHuji, PlamGFP, pmeaGFPl, pmeaGFP2, pmimGFPl, pmimGFP2, Pp2FbFP, Pp2FbFPL30M, ppluGFPl, ppluGFP2, pporGFP, pporRFP, PS-CFP (Cyan), PS-CFP (Green), PS-CFP2 (Cyan), PS- CFP2 (Green), psamCFP, PSmOrange (Far-red), PSmOrange (Orange), PSmOrange2 (Far- red), PSmOrange2 (Orange), ptilGFP, R3-2+PCB, RCaMP, RDSmCherryO.l, RDSmCherryO.2, RDSmCherryO.5, RDSmCherryl, rfloGFP, rfloRFP, RFP611, RFP618, RFP630, RFP637, RFP639, roGFPl, roGFPl-Rl, roGFPl-R8, roGFP2, rrenGFP, RRvT, rsCherry (On), rsCherryRev (On), rsCherryRevl.4 (On), rsEGFP (On), rsEGFP2 (On), rsFastLime (On), rsFolder (Green), rsFolder2 (Green), rsFusionRedl (On), rsFusionRed2 (On), rsFusionRed3 (On), rsTagRFP (ON), Sandercyanin, Sapphire, sarcGFP, SBFP1,
SBFP2, SCFP1, SCFP2, SCFP3A, SCFP3B, scubGFPl, scubGFP2, scubRFP, secBFP2, SEYFP, sgll, sgl2, sg25, sg42, sg50, SGFP1, SGFP2, SGFP2(206A), SGFP2(E222Q), SGFP2(T65G), SHardonnay, shBFP, shBFP-N158S/L173I, ShG24, Sirius, SiriusGFP, Skylan-NS (On), Skylan-S (On), smURFP, SNIFP, SOPP, SOPP2, SOPP3, SPOON (on), stylGFP, SuperfolderGFP, SuperfoldermTurquoise2, SuperfoldermTurquoise2ox, SuperNovaGreen, SuperNovaRed, SYFP2, T-Sapphire, TagBFP, TagCFP, TagGFP, TagGFP2, TagRFP, TagRFP-T, TagRFP657, TagRFP675, TagYFP, td-RFP611, td-RFP639, tdimer2(12), tdKatushka2, TDsmURFP, tdTomato, tKeima, Topaz, TurboGFP, TurboGFP- V197L, Turbo RFP, Turquoise-GL, Ultramarine, UnaG, usGFP, Venus, VFP, vsfGFP-0, vsfGFP-9, W1C, W2, W7, WasCFP, Wi-Phy, YPet, zFP538, zoan2RFP, ZsGreen, ZsYellowl, aGFP, 10B, 22G, 5B, 6C, Ala, aacuCP, acanFP, ahyaCP, amilCP, amilCP580, amilCP586, amilCP604, apulCP584, BFPsol, Bluel02, CFP4, cgigCP, CheGFP3, Cloverl.5, cpasCP, Cyll.5, dClavGRl.6, dClover2, dClover2A206K, dhorGFP, dhorRFP, dPapayaO.l, Dronpa-C62S, DsRed-Timer, echFP, echiFP, EYFP-F46L, fcFP, fcomFP, Fpaagar, Fpag_frag, Fpcondchrom, FPmann, FPmcavgr7.7, Gamillus0.5, gdjiCP, gfasCP, GFPhal, gtenCP, hcriCP, hfriFP, KikG, LEA, mcFP497, mcFP503, mcFP506, mCherryl.5, mClavGRl, mClavGRl.l, mClavGRl.8, mCloverl.5, mcRFP, meffCP, mEos2-NA, meruFP, mKate2.5, mOFP.T.12, mOFP.T.8, montFP, moxEos3.2, mPA-GFP, mPapaya0.3, mPapaya0.6, mRFP1.3, mRFP1.4, mRFP1.5, mTFPO.4, mTFPO.5, mTFP0.6, mTFPO.8, mTFP0.9, mTFPl-Y67H, mTurquoise-146G, mTurquoise-146S, mTurquoise-DR, mTurquoise-GL, mTurquoise-GV, mTurquoise-RA, mTurquoise2-G, NpR3784g, PDM1-4, psupFP, Q80R, rfloGFP2, RpBphPl, RpBphP2, RpBphP6, rrGFP, RSGFP1, RSGFP2, RSGFP3, RSGFP4, RSGFP6, RSGFP7, Rtms5, scleFPl, scleFP2, spisCP, stylCP, sympFP, TeAPCa, tPapayaO.Ol, Trp-lessGFP, vsGFP, Xpa, yEGFP, YFP3, zGFP, and zRFP.
[0058] In some embodiments, the reporter protein is luciferase. In some embodiments, the luciferase is renilla luciferase, firefly luciferase or nano luciferase. See, e.g., England et al., 2016, Bioconjug. Chem 27(5): 1175-1187 for examples of luciferases, including nanoluciferase.
[0059] In a one embodiment, the genome of the SARS-CoV-2 spike protein pseudotyped VSV particles does not comprise a nucleotide sequence encoding VSV glycoprotein (G). In some embodiments, the genome of the SARS-CoV-2 spike protein pseudotyped VSV particles only comprises a fragment of the nucleotide sequence sequence encoding VSV glycoprotein (G) (e.g., 10, 15, 20, 25, 30 or so nucleotides of the sequence that would encode VSV glycoprotein). In another embodiment, the SARS-CoV-2 spike protein is one described in the Examples. In another specific embodiment, provided herein are SARS-CoV-2 spike protein pseudotyped VSV particles such as described in in the Examples.
[0060] In certain embodiments, the SARS-CoV-2 spike protein pseudotyped VSV particles are treated with trypsin. In some embodiments, the SARS-CoV-2 spike protein pseudotyped VSV particles are treated with trypsin and soybean inhibitor.
[0061] In a one embodiment, the SARS-CoV-2 spike protein pseudotyped VSV particles are unable to undergo multiple rounds of replication. In a particular embodiment, the SARS- CoV-2 spike protein pseudotyped VSV particles are only able to undergo a single round of replication. [0062] Compositions
[0063] In one aspect, provided herein are compositions comprising a SARS-CoV-2 spike protein pseudotyped VSV particle disclosed herein. In one embodiment, provided herein is a composition comprising a SARS-CoV-2 spike protein pseudotyped VSV particle described herein and a carrier. In certain embodiments, the carrier is phosphate buffered saline or another buffered saline solution. In some embodiments, the carrier is media (e.g., serum free media).
[0064] In another aspect, provided herein are compositions comprising a SARS-CoV-2 spike protein pseudotyped VSV particle described herein and trypsin, soybean inhibitor or both. In certain embodiments, provided herein is a composition comprising supernatant containing SARS-CoV-2 spike protein pseudotyped VSV particles described herein and trypsin, soybean inhibitor or both.
[0065] In another embodiment, provided herein is a composition comprising (1) SARS- CoV-2 spike protein pseudotyped VSV particles described herein (2) trypsin and (3) dextran (e.g., DEAE-dextran). In another embodiment, provided herein is a composition comprising (1) supernatant containing SARS-CoV-2 spike protein pseudotyped VSV particles described herein, (2) trypsin and (3) dextran (e.g., DEAE-dextran).
[0066] In some embodiments the trypsin concentration is between 5 and 1000, between 2 and 20, between 5 and 15, or between 6.25 and 12.5 pg/ml. In some embodiments the trypsin concentration is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 pg/ml.
[0067] In certain embodiments, the soybean inhibitor concentration is about 5, 10, 50,
100, 200, 300, 400, 500, 600, 700, 800, 1000 pg/ml, soybean inhibitor concentration is about 625 pg/ml.
[0068] Methods for generating SARS-CoV-2 spike protein pseudotyped VSV particles
[0069] In another aspect, provided herein are methods for generating SARS-CoV-2 spike protein pseudotyped VSV particles. Methods for generating SARS-CoV-2 spike protein pseudotyped VSV particles are, for example, described in the Examples.
[0070] In a specific embodiment, provided herein is a method for generating SARS-CoV- 2 spike protein pseudotyped VSV particle, comprising: (a) infecting cells (e.g., HEK293T cells) overexpressing SARS-CoV-2 spike protein with a recombinant VSV particle, wherein the recombinant VSV particle comprises an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding for a reporter protein (e.g., luciferase), wherein the genome does not express VSV glycoprotein (G), wherein the VSV particle is pseudotyped with VSV glycoprotein; and (b) purifying SARS-CoV-2 spike protein pseudotyped VSV particles from the supernatant of the cells. [0071] In a specific embodiment, the genome of the recombinant VSV particles further comprise a nucleotide sequence encoding a fluorescent protein. In some embodiments, the nucleotide sequence encoding the reporter protein (e.g., luciferase) and the nucleotide sequence encoding fluorescent protein are separated by a nucleotide sequence encoding a self-cleaving peptide (e.g., a 2 A self-cleaving peptide) such that a single polypeptide comprising the reporter protein (e.g., luciferase), the self-cleaving peptide, and fluorescent protein is generated and may be cleaved to produce comprising the reporter protein (e.g., luciferase) and the fluorescent protein.
[0072] In some embodiments, the cells are cultured in optiMEM containing anti-VSV-G antibody. The use of anti-VSV G neutralizing antibody may minimize the background sometimes seen with “bald” VSV pseudotypes.
[0073] The use of optiMEM media for production of SARS-CoV-2 spike protein pseudotyped VSV particles may be preferred over DMEM + 10% FBS because an increase of CoV-2 spike cleavage relative to DMEM + 10%FBS. In some embodiments, chemically defined serum free media is used to culture the cells.
[0074] Cells may be transiently or stably transfected with vector (e.g., plasmid) comprising a nucleotide sequence encoding SARS-CoV-2 spike protein. In a preferred embodiment, the cells do not express ACE-2 and do not support SARS-CoV-2 entry.
[0075] In some embodiments, the SARS-CoV-2 spike protein pseudotyped VSV particles are purified from the supernatant by low speed centrifugation. In some embodiments, the SARS-CoV-2 spike protein pseudotyped VSV particles are purified from the supernatant by low speed centrifugation to remove cell debris and concentrated via ultracentrifugation through a sucrose cushion, and/or Amicon and PEG concentration. [0076] Methods for infecting cells with SARS-CoV-2 spike protein pseudotyped VSV particles
[0077] In another aspect, provided herein are methods for infecting cells with SARS-CoV- 2 spike protein pseudotyped VSV particles described herein. Such methods are described, for example, in the Examples.
[0078] In one embodiment, provided herein is a method for infecting cells with SARS- CoV-2 spike protein pseudotyped VSV particles described herein, comprising: (a) contacting the SARS-CoV-2 spike protein pseudotyped VSV particles with trypsin for a certain period of time; and (b) infecting cells with the SARS-CoV-2 spike protein pseudotyped VSV particles.
[0079] In one embodiment, provided herein is a method for infecting cells with s SARS- CoV-2 spike protein pseudotyped VSV particle, comprising: (a) contacting the SARS-CoV-2 spike protein pseudotyped VSV particles with trypsin and soybean inhibitor for a certain period of time; and (b) infecting cells with the SARS-CoV-2 spike protein pseudotyped VSV particles.
[0080] In one embodiment, provided herein is a method for infecting cells with s SARS- CoV-2 spike protein pseudotyped VSV particle, comprising: (a) contacting the SARS-CoV-2 spike protein pseudotyped VSV particles with trypsin for a certain period of time; and (b) spinoulating the cells with the SARS-CoV-2 spike protein pseudotyped VSV particles. In certain embodiments, step (a) further comprises contacting the particles with soybean inhibitor. In some embodiments, step (a) further comprises contacting the particles with dextran (e.g., DEAE-dextran). As used herein, “spinoulation” refers to centrifugal inoculation.
[0081] Trypsin can be used to induce entry enhancement of SARS-CoV-2 spike protein pseudotyped VSV particles. In some embodiments the trypsin concentration is between 5 and 1000, between 2 and 20, between 5 and 15, or between 6.25 and 12.5 pg/ml. In some embodiments the trypsin concentration is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 pg/ml.
[0082] In certain embodiments, the soybean inhibitor concentration is about 5, 10, 50,
100, 200, 300, 400, 500, 600, 700, 800, 1000 pg/ml, soybean inhibitor concentration is about 625 pg/ml. [0083] In some embodiments, the certain period of time is 15 minutes. In some embodiments, low concentrations of trypsin and soybean inhibitor can be used to achieve maximal SAS-CoV-2 spike protein pseudotyped VSV particles entry with limited toxicity. [0084] In another aspect, provided herein are methods for detecting blood, sera or plasma that neutralizes SARS-CoV-2. Methods for detecting blood, sera or plasma that neutralizes SARS-CoV-2 are provided, for example, in the Examples. The SARS-CoV-2 spike protein pseudotyped VSV particles provide a surrogate for SARS-CoV-2 and allow neutralization assays to be conducted without the need for a biosafety level higher than level 2.
[0085] In one embodiment, a method for detecting sera that neutralizes SARS-CoV-2 comprising: (a) incubating with the SARS-CoV-2 spike protein pseudotyped VSV particles described herein with sera from a subject for a first period of time; (b) spinoculating cells expressing human ACE-2, TMPRSS2 or both with the sera-treated SARS-CoV-2 spike protein pseudotyped VSV particle for a second period of time; and (c) measuring the reporter protein (e.g., luciferase) activity after a third period of time, wherein a lower level of reporter protein (e.g., luciferase) activity is detected if the sera neutralizes SARS-CoV-2 spike protein pseudotyped VSV particle than if steps (b) to (c) are performed without performing step (a) or a negative control (e.g., an antibody that is known not neutralize SARS-CoV-2) is used in step (a) when performing steps (a) to (c), and the lower level of reporter protein (e.g., luciferase) activity indicates that the sera neutralizes SARS-CoV-2. Techniques known to one of skill in the art may be used to measure reporter protein (e.g., luciferase) activity. In the event that the SARS-CoV-2 spike protein pseudotyped VSV particles further express fluorescent protein, it may also be detected using techniques known in the art (e.g., cytometry).
[0086] In some embodiments, the first period of time is about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour or more. In certain embodiments, the first period of time is 10 to 45 minutes, 15 to 45 minutes, 15 to 60 minutes, or 30 to 60 minutes.
[0087] In some embodiments, the second period of time is about 30 minutes, 45 minutes,
1 hour, 1.25 hours, 1.5 hours, 1.75 hours, or 2 hours. In certain embodiments, the second period of time is 30 minutes to 1 hour, 45 minutes to 1.5 hours, or 1 hour to 2 hours.
[0088] In some embodiments, the third period of time is about 16 to 24 hours, about 18 to 20 hours, about 18 to 22 hours, or about 18 to 24 hours. [0089] In a specific embodiment, the subject is a human subject. In another specific embodiment, the serum is heat inactivated (e.g., the serum is incubated at 56° C for 30-60 minutes). In another specific embodiment, the serum is diluted in plain DMEM. In another specific embodiment, the serum is diluted in DMEM containing heat inactivated 10% heat inactivated FBS.
[0090] In certain embodiments, the method further comprises concurrently repeating steps (a) to (c) with a positive control antibody or sera that does not neutralize SARS-CoV-2. In some embodiments, the method comprises concurrently repeating steps (a) to (c) with a negative control antibody or sera that does not neutralize SARS-CoV-2. In certain embodiments, the cells overexpress human angiotensin-converting enzyme 2 (ACE-2), Transmembrane protease serine 2 (TMPRSS2), or both. Cells may be engineered to overexpress human ACE-2, TMPRSS2, or both using sequences and techniques known to one of skill in the art. For example, the cells may be transiently or stably transfected with vectors (e.g., plasmids) comprising nucleotide sequences encoding human ACE-2, TMPRSS2, or both. See, e.g., UniProtKB No. 15393 for the amino acid sequence of human TMPRSS2 and Gene ID: 59272 for human ACE-2 sequence information. See, e.g., Hoffmann et a , 2020, Cell 181: 271-280 for a description of the dependence of SARS-CoV- 2 on ACE-2 and TMPRSS2. In some embodiments, the neutralization assay is carried out in a high-throughput manner (e.g., using a 96 well microtiter plate). In certain embodiments, the sera is serially diluted. In some embodiments, between 1:4 and 1:10 dilution of the SARS-CoV-2 spike protein pseudotyped VSV particles (e.g., the dilution may be done in plain DMEM or DMEM containing 10% heat inactivated FBS) is used in the method. In some embodiments, the cells are Vero-CCL81 cells, 293T cells, or human ACE2, primary HAECs.
[0091] Methods for assessing the ability of an antibody to neutralize SARS-CoV-2 [0092] In another aspect, provided herein are methods for assessing the ability of an antibody to neutralize SARS-CoV-2. Methods for assessing the ability of an antibody to neutralize SARS-CoV-2 are provided, for example, in the Examples.
[0093] In one embodiment, provided herein is a method for assessing the ability of an antibody to neutralize SARS-CoV-2 comprising: (a) incubating with the SARS-CoV-2 spike protein pseudotyped VSV particles described herein with an antibody of interest for a first period of time; (b) spinoculating cells expressing human ACE-2, TMPRSS2 or both with the antibody-treated SARS-CoV-2 spike protein pseudotyped VSV particle for a second period of time; and (c) measuring the reporter protein (e.g., luciferase) activity after a third period of time, wherein a lower level of reporter protein (e.g., luciferase) activity is detected if the antibody neutralizes SARS-CoV-2 spike protein pseudotyped VSV particle than if steps (b) to (c) are performed without performing step (a) or a negative control (e.g., an antibody that is known not neutralize SARS-CoV-2) is used in step (a) when performing steps (a) to (c), and the lower level of reporter protein (e.g., luciferase) activity indicates that the antibody neutralizes SARS-CoV-2. In certain embodiments, the cells are spinoculated in the presence of serum free media (e.g., DMEM only). Techniques known to one of skill in the art may be used to measure reporter protein (e.g., luciferase) activity. If the SARS-CoV-2 spike protein pseudotyped VSV particles further express fluorescent protein, it may also be detected using techniques known in the art (e.g., cytometry).
[0094] In some embodiments, the first period of time is about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour or more. In certain embodiments, the first period of time is 10 to 45 minutes, 15 to 45 minutes, 15 to 60 minutes, or 30 to 60 minutes.
[0095] In some embodiments, the second period of time is about 30 minutes, 45 minutes,
1 hour, 1.25 hours, 1.5 hours, 1.75 hours, or 2 hours. In certain embodiments, the second period of time is 30 minutes to 1 hour, 45 minutes to 1.5 hours, or 1 hour to 2 hours.
[0096] In some embodiments, the third period of time is about 16 to 24 hours, about 18 to 20 hours, about 18 to 22 hours, or about 18 to 24 hours.
[0097] In certain embodiments, the method farther comprises concurrently repeating steps (a) to (c) with a positive control antibody or sera that does neutralize SARS-CoV-2. In some embodiments, the method comprises concurrently repeating steps (a) to (c) with a negative control antibody or sera that does not neutralize SARS-CoV-2. In certain embodiments, the cells overexpress human angiotensin-converting enzyme 2 (ACE-2), Transmembrane protease serine 2 (TMPRSS2), or both. Cells may be engineered to overexpress human ACE-2, TMPRSS2, or both using sequences and techniques known to one of skill in the art. For example, the cells may be transiently or stably transfected with vectors (e.g., plasmids) comprising nucleotide sequences encoding human ACE-2, TMPRSS2, or both. See, e.g., UniProtKB No. 15393 for the amino acid sequence of human TMPRSS2 and Gene ID: 59272 for human ACE-2 sequence information. See, e.g., Hoffmann et al., 2020, Cell 181: 271-280 for a description of the dependence of SARS-CoV- 2 on ACE-2 and TMPRSS2. In some embodiments, the neutralization assay is carried out in a high-throughput manner (e.g., using a 96 well microtiter plate). In certain embodiments, the antibody is serially diluted. In some embodiments, between 1:4 and 1:10 dilution of the SARS-CoV-2 spike protein pseudotyped VSV particles (e.g., the dilution may be done in plain DMEM or DMEM ontaining 10% heat inactivated FBS) is used in the method. In some embodiments, the cells are Vero-CCL81 cells, 293T cells, or human ACE2, primary HAECs. In certain embodiments, RLUs >105 are achieved with a 1:10 dilution in SFM and spinoculation.
[0098] Also provided herein are methods for detecting neutralizing antibody or antibodies in sera, blood or plasma using a neutralization assay.
[0100] The particlednfectivity ratio may be calculated as A:B, where A is genome copies/ml and B is TCID50/ml. The SARS-CoV-2 SI incorporation may be determined by SI subunit of SARS-CoV-2 spike protein/VS V matrix ratio on Western blot. The percent cleavage of incorporated SI may be determined by SI subunit of SARS-CoV-2 spike protein/SARS-CoV-2 spike protein ratio on Western blot.
[0101] In another aspect, provided herein are methods for determining absolute IC50,
IC80, and IC90 values using SARS-CoV-2 spike protein pseudotyped VSV particles, such as described in the Examples.
[0102] Cells
[0103] In another aspect, provided herein are cells (e.g., 293T cells) that stably express ACE2 or ACE2 and TMPRSS2, such as the cells described in the Examples. The cells may be used in a neutralization assay described herein, including cell lines Vero-CCL81 TMPRSS2, HEK 293T-hACE2 (clone 5-7), and 293T-hACE2-TMPRSS2 (clone F8-2).
[0104] Kits
[0105] In another aspect, provided herein are kits comprising SARS-CoV-2 spike protein pseudotyped VSV particles described herein in a container. In a specific embodiment, provided herein is a kit comprising SARS-CoV-2 spike protein pseudotyped VSV particles in a container, and optionally instructions for performing a neutralization assay using the SARS- CoV-2 spike protein pseudotyped VSV particle. In another specific embodiment, the kit may further comprise a positive control antibody (e.g., an antibody known to neutralize SARS- CoV-2), a negative control antibody (e.g., an antibody known not to neutralize SARS-CoV- 2), or both. In another specific embodiment, the kit may further comprise one or more reagents need to detect reporter protein (e.g., lucif erase) activity.
[0106] Pseudotyped VSV particles
[0107] In another aspect, provided herein is a nucleic acid sequence comprising a VSV antigenome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding a reporter protein (e.g., luciferase), and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G).
[0099] In a specific embodiment, the nucleic acid sequence further comprises a T7 promoter, autocatalytic hammerhead ribozyme sequences, and a T7 terminator· See, e.g., Beaty et a , 2017, mSphere 2(2):e00376-16 for a description of the structure of the nucleic acid sequence (in particular, see, e.g., FIG. 4A of Beaty et ak).
[0100] In one embodiment, the nucleic acid sequence comprises an optimized T7 promoter and hammerhead ribozyme (HhRbz) just before the 5 ’ end of the viral genome. In one embodiment, the optimized T7 promoter comprises the sequence TAATACGACTCACTATAGGGAGA (SEQ ID NO:9). In one embodiment, the HhRbz sequence comprises the sequence
CTGATGAGTCCGTGAGGACGAAACGGAGTCTAGACTCCGTC (SEQ ID NO: 10) [0101] In one embodiment, the use of a codon-optimized T7 polymerase may alleviate the use of a vaccinia-driven T7 polymerase, resulting in higher rescue efficiency. In one embodiment, provided is a nucleic acid comprising the sequence of the codon-optimized T7 RNA polymerase comprises SEQ ID NO: 11. In one embodiment, the T7 RNA polymerase encoding sequence is provided by a helper plasmid. The sequence of a codon optimized T7 RNA polymerase has been deposited to Addgene (Cat no. 65974).
[0102] In a specific embodiment, the nucleotide sequence encoding the reporter protein (e.g., luciferase) and the nucleotide sequence encoding the fluorescent protein are separated by a nucleotide sequence encoding a self-cleaving peptide (e.g., a P2A self-cleaving peptide) so that a single polypeptide is produced that is cleaved into the reporter protein (e.g., luciferase) and fluorescent protein. In a specific embodiment, the nucleotide sequence encoding the fluorescent protein and the nucleotide sequence encoding the reporter protein (e.g., luciferase) are separated by a nucleotide sequence encoding a self-cleaving peptide (e.g., a P2A self-cleaving peptide) so that a single polypeptide is produced that is cleaved into fluorescent protein and the reporter protein (e.g., luciferase). In specific embodiments, the nucleic acid sequence is in a plasmid. In certain embodiments, the reporter protein is a luciferase such as a renilla luciferase, firefly luciferase or nano luciferase. In some embodiments, the fluorescent protein is a fluorescent protein disclosed herein.
[0103] Also provided are vectors comprising the nucleic acids disclosed herein.
[0104] In another aspect, provided herein is a kit comprising the nucleic acid sequence in a container and optionally instructions for generating pseudotyped VSV particles. In some embodiments, the kit further comprises a one, two, three or all of the following: (1) a first vector (e.g., a plasmid) comprising a nucleotide sequence encoding VSV M protein in a container, (2) a second vector (e.g., a plasmid) comprising a nucleotide sequence encoding VSV L protein in a container, (3) a third vector (e.g., a plasmid) encoding VSV N protein in a container, (4) a fourth vector (e.g., a plasmid) comprising a nucleotide sequence encoding VSV G protein in a container; (5) a fifth vector (e.g. a plasmid) comprising a nucleotide sequence encoding VSV P protein; (6) a sixth vector (e.g., a plasmid comprising a codon- optimized gene encoding T7 RNA polymerase. In certain embodiments, the kit may further comprise one or more ingredients to transfect cells with a plasmid.
[0105] In another aspect, provided herein are recombinant VSV particles pseudotyped with VSV glycoprotein comprising an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding a reporter protein (e.g., luciferase), and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G). In a specific embodiment, the reporter protein is a luciferase such as renilla luciferase, firefly luciferase or nano luciferase.
In one embodiment, the fluorescent protein fluorescent protein disclosed herein. In another aspect, provided herein is a method for generating pseudotyped VSV particles, comprising:
(a) infecting or spinoculating cells overexpressing a viral surface protein of interest (or other protein of interest) with the recombinant VSV particles; and (b) purifying viral surface protein pseudotyped VSV particles from the supernatant of the cells. In a specific embodiment, the viral surface protein is the SARS-CoV-2 spike protein.
[0106] In another aspect, provided herein is a method for generating recombinant VSV particles pseudotyped with VSV glycoprotein comprising an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding a reporter protein (e.g., luciferase), and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G), the method comprising techniques similar to those described in Beaty et al. or herein. In a specific embodiment, provided herein is a method for generating recombinant VSV particles pseudotyped with VSV glycoprotein comprising an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding a reporter protein (e.g., luciferase), and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G), the method comprising: (a) transfecting cells with a nucleic acid sequence described herein that comprises a VSV antigenome, a first vector (e.g., a plasmid) comprising a nucleotide sequence encoding VSV M protein, a second vector (e.g., a plasmid) comprising a nucleotide sequence encoding VSV L protein, a third vector (e.g., a plasmid) encoding VSV N protein, a fourth vector (e.g., a plasmid) comprising a nucleotide sequence encoding VSV G protein, and a fifth vector (e.g., a plasmid) comprising a nucleotide sequence encoding VSV P protein, optionally, a sixth vector (e.g., a plasmid comprising a codon-optimized gene encoding T7 RNA polymerase; and (b) purifying the recombinant VSV particles pseudotyped with VSV glycoprotein. On some embodiments the method further comprises transfecting the cells with a sixth vector comprising a codon-optimized sequence encoding a T7 polymerase. In some embodiments, the cells are 293T-ACE2 clone 5-7 or 293T-ACE2-TMPRSS2 clone F8-2.
[0107] SARS-CoV-2 Spike nucleotide sequence: [0108] ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGA
ACCTGACCACAAGAACCCAGCTGCCTCCAGCCTACACCAACAGCTTTACCAGAG
GCGTGTACTACCCCGACAAGGTGTTCAGATCCAGCGTGCTGCACTCTACCCAGGA
CCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGTCCG
GCACCAATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGG
TGTACTTTGCCAGCACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCAC
CACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGT
GGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGGCGTCTAC
TATCACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGC
GCCAACAACTGCACCTTCGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAG
GCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTCAAGAACATCGACG
GCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGTGCGGGATCTGCC
TCAGGGCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATC
ACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATA
GCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGC
CTAGAACCTTTCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGG
ATTGTGCTCTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGT
GGAAAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAATCCAT
CGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGGCGAGGTGTTCAATGCC
ACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTG
GCCGACTACTCCGTGCTGTACAACTCCGCCAGCTTCAGCACCTTCAAGTGCTACG
GCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAG
CTTCGTGATCCGGGGAGATGAAGTGCGGCAGATTGCCCCTGGACAGACAGGCAA
GATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCC
TGGAACAGCAACAACCTGGACTCCAAAGTCGGCGGCAACTACAATTACCTGTAC
CGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAG
ATCTATCAGGCCGGCAGCACCCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACT
TCCCACTGCAGTCCTACGGCTTTCAGCCCACAAATGGCGTGGGCTATCAGCCCTA
CAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGC
CCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAAC
GGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTC
CAGCAGTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAG ACACTGGAAATCCTGGACATCACCCCTTGCAGCTTCGGCGGAGTGTCTGTGATCA
CCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGACGTGAACT
GTACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGG
TGTACTCCACCGGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGC
CGAGCACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGGCATCTG
TGCCAGCTACCAGACACAGACAAACAGCCCCAGACGGGCCAGATCTGTGGCCAG
CCAGAGCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTA
CTCCAACAACTCTATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAG
ATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCG
GCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCA
GCTGAATAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAG
AGGTGTTCGCCCAAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGGACTTCG
GCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGA
GCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCAT
CAAGCAGTATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCC
CAGAAGTTTAACGGACTGACAGTGCTGCCTCCTCTGCTGACCGATGAGATGATCG
CCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGG
AGCTGGCGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAAC
GGCATCGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAAC
CAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGC
GCCCTGGGAAAGCTGCAGGACGTGGTCAACCAGAATGCCCAGGCACTGAACACC
CTGGTCAAGCAGCTGTCCTCCAACTTCGGCGCCATCAGCTCTGTGCTGAACGATA
TCCTGAGCAGACTGGACAAGGTGGAAGCCGAGGTGCAGATCGACAGACTGATCA
CCGGAAGGCTGCAGTCCCTGCAGACCTACGTTACCCAGCAGCTGATCAGAGCCG
CCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCT
GGGCCAGAGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTT
CCCTCAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACATACGTGCCCGCT
CAAGAGAAGAATTTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCAC
TTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACCCAGC
GGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAA
CTGCGACGTCGTGATCGGCATTGTGAACAATACCGTGTACGACCCTCTGCAGCCC
GAGCTGGACAGCTTCAAAGAGGAACTGGATAAGTACTTTAAGAACCACACAAGC CCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATC
CAGAAAGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCT
GATCGACCTGCAAGAACTGGGGAAGTACGAGCAGTACATCAAGTGGCCCTGGTA
CATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATG
CTGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCA
GCTGCTGCAAGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAAC
TGCACTACACCTGA (SEQ ID NO: 1)
[0109] SARS-CoV-2 Spike Amino Acid sequence:
[0110] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHST
QDFFFPFFSNVTWFHAIHVSGTNGTKRFDNPVFPFNDGVYFASTEKSNIIRGWIFGTT
FDSKTQSFFIVNNATNVVIKVCEFQFCNDPFFGVYYHKNNKSWMESEFRVYSSANN
CTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSAL
EPL VDLPIGINITRFQTLLALHRS YLTPGDS S S GWT AG AA A Y Y V GYLQPRTFLLKYNE
NGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGE
VFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVY
ADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYL
YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV
VVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGR
DIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIH
ADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRR
ARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYIC
GDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGF
NFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLT
VLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVL
YENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAIS
SVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSEC
VLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAH
FPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELD
SFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG
KYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDS
EPVLKGVKLHYT (SEQ ID NO:2)
[0111] Codon Optimized T7 Polymerase TGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCGCCACCATGAACACCAT
CAATATTGCCAAGAACGACTTTTCTGATATCGAGCTGGCCGCTATTCCATTCAAT
ACACTGGCTGACCACTACGGAGAGCGGCTGGCCCGCGAACAGCTGGCTCTGGAG
CATGAAAGCTATGAGATGGGAGAAGCCCGATTCAGGAAGATGTTTGAGAGGCAG
CTGAAAGCTGGGGAAGTGGCAGACAACGCAGCCGCTAAGCCACTGATTACCACA
CTGCTGCCCAAAATGATCGCCAGAATTAATGATTGGTTCGAGGAAGTGAAGGCA
AAACGAGGAAAGAGGCCTACCGCCTTCCAGTTTCTGCAGGAGATCAAGCCAGAA
GCTGTGGCATACATCACCATCAAGACTACCCTGGCATGCCTGACAAGCGCCGAC
AACACAACTGTGCAGGCAGTCGCTTCCGCAATCGGACGAGCTATTGAGGACGAA
GCACGCTTTGGGAGAATCCGGGATCTGGAGGCCAAGCACTTCAAGAAAAACGTG
GAGGAACAGCTGAACAAGAGGGTGGGGCATGTCTATAAGAAGGCCTTCATGCAG
GTGGTCGAGGCCGACATGCTGTCAAAGGGACTGCTGGGAGGAGAGGCCTGGAGC
TCCTGGCACAAAGAAGATAGCATCCATGTGGGAGTCCGCTGCATCGAGATGCTG
ATTGAATCTACTGGGATGGTGAGTCTGCACCGACAGAACGCCGGCGTGGTCGGA
CAGGACTCTGAGACAATCGAACTGGCTCCCGAGTATGCCGAAGCTATTGCAACT
AGAGCCGGAGCTCTGGCAGGGATCAGTCCAATGTTCCAGCCCTGCGTGGTCCCCC
CTAAGCCTTGGACTGGCATCACCGGGGGCGGATACTGGGCTAATGGAAGGAGAC
CACTGGCACTGGTGCGAACACACTCTAAGAAAGCCCTGATGAGATACGAGGATG
TCTATATGCCCGAAGTGTATAAGGCCATCAACATTGCTCAGAATACAGCATGGA
AAATTAACAAGAAAGTGCTGGCCGTCGCTAATGTGATCACTAAGTGGAAACATT
GTCCCGTGGAGGACATCCCTGCCATTGAACGGGAGGAACTGCCTATGAAGCCAG
AGGACATCGATATGAACCCTGAAGCTCTGACCGCATGGAAGCGCGCAGCCGCTG
CAGTGTACAGAAAGGATAAAGCCCGGAAGTCCCGGCGCATTTCTCTGGAGTTCA
TGCTGGAACAGGCCAACAAGTTTGCTAATCACAAAGCAATCTGGTTCCCCTACAA
CATGGACTGGCGCGGACGAGTCTATGCCGTGAGCATGTTCAACCCTCAGGGGAA
TGATATGACAAAGGGCCTGCTGACTCTGGCTAAGGGGAAACCAATTGGGAAGGA
GGGCTACTATTGGCTGAAAATCCACGGGGCCAATTGCGCTGGCGTCGACAAGGT
GCCATTCCCCGAGAGGATCAAGTTCATCGAGGAAAACCATGAAAATATTATGGC
ATGTGCCAAGTCTCCCCTGGAGAACACATGGTGGGCCGAACAGGATAGTCCTTTC
TGCTTTCTGGCCTTCTGTTTTGAGTACGCTGGAGTGCAGCACCATGGGCTGAGTT
ATAATTGCTCCCTGCCACTGGCCTTTGACGGCTCTTGTAGTGGAATCCAGCACTT
CTCCGCAATGCTGAGGGATGAGGTCGGAGGAAGAGCAGTGAACCTGCTGCCATC TGAGACAGTGCAGGACATCTACGGCATTGTCGCCAAGAAAGTGAATGAGATCCT
GCAGGCTGACGCAATTAACGGGACTGATAATGAGGTGGTCACCGTCACAGATGA
AAACACTGGCGAGATCAGCGAAAAGGTGAAACTGGGAACCAAGGCCCTGGCTG
GACAGTGGCTGGCATACGGAGTCACCCGCTCAGTGACAAAGCGAAGCGTGATGA
CCCTGGCTTATGGCAGCAAAGAGTTCGGCTTCAGGCAGCAGGTGCTGGAAGACA
CCATCCAGCCAGCCATTGATTCCGGAAAGGGGCTGATGTTTACACAGCCCAACC
AGGCCGCTGGCTACATGGCCAAGCTGATCTGGGAGTCAGTGAGCGTCACAGTGG
TCGCAGCCGTGGAAGCTATGAATTGGCTGAAGTCCGCTGCAAAACTGCTGGCCG
CTGAGGTGAAGGACAAGAAAACTGGCGAAATTCTGAGGAAAAGATGCGCCGTCC
ACTGGGTGACCCCTGATGGATTCCCAGTGTGGCAGGAGTATAAGAAACCCATCC
AGACCAGACTGAACCTGATGTTCCTGGGCCAGTTTCGGCTGCAGCCTACAATCAA
CACTAATAAGGACAGTGAGATTGATGCTCATAAACAGGAATCAGGGATTGCACC
TAATTTTGTGCACAGCCAGGACGGCTCCCATCTGCGGAAGACTGTGGTCTGGGCT
CACGAGAAATACGGCATCGAATCCTTCGCACTGATTCATGACTCTTTTGGAACCA
TCCCAGCCGATGCAGCCAACCTGTTCAAGGCTGTCCGCGAGACTATGGTGGACA
CCTACGAAAGTTGTGATGTGCTGGCCGACTTCTATGATCAGTTTGCTGACCAGCT
GCACGAGTCACAGCTGGATAAGATGCCCGCACTGCCTGCCAAAGGCAACCTGAA
TCTGAGAGACATCCTGGAGTCCGATTTCGCATTTGCCTGA (SEQ ID NO: 11)
EXAMPLES
[0112] Example 1: Material and Methods for Examples 3-10 [0113] Plasmids
[0114] SARS-CoV-2 spike is in a pCAGG backbone and expresses the codon optimized Wuhan-Hu-1 isolate (NCBI ref. seq. NC_045512.2).
[0115] SARS-CoV-2 sRBD (NCBI GenBank MT380724.1 from Krammer lab) is in a pCAGG backbone and expresses the codon optimized sequence from the Wuhan-Hu-1 isolate. sRBD-His used for neutralization studies was generated from this construct.
[0116] VSV-G is in a pCAGG backbone and expresses wild type Indiana strain VSV-G (Genbank: ACK77583.1). [0117] ACE2 packaging construct (GeneCopoeia, cat no EX-U1285-Lvl05) uses a CMV promoter to express TMPRSS2 and bears a puromycin selection marker in the integrating cassette.
[0118] TMPRSS2 packaging construct (GeneCopoeia, cat no EX-Z7591-Lvl97) uses a CMV promoter to express TMPRSS2 and bears a blasticidin selection marker in the integrating cassette.
[0119] psPAX22nd generation lentiviral packaging plasmid (Addgene #12259) expresses HIV-1 Gag, Pol, and Pro proteins.
[0120] NiV-RBP is in a pCAGG backbone and expresses the HA-tagged codon optimized NiV receptor binding protein.
[0121] All plasmids listed here are ampicillin resistant. These constructs were transformed into competent cells, grown in bacterial growth media containing carbenicillin, prepared using Invitrogen’s midiprep kit, and sequence verified prior to use for experiments.
[0122] Maintenance and generation of cell lines
[0123] Vero-CCL81 and 293T cells were cultured in DMEM with 10% heat inactivated FBS at 37 °C with 5% C02. VSV-G pseudotyped lentiviruses packaging ACE2 or TMPRSS2 expression constructs were generated by using Bio-T (Bioland; BOl-01) to transfect 293T cells with the second-generation lentiviral packaging plasmid (Addgene; 12259), pCAGG- VSV-G, and the desired expression construct (i.e. ACE2 or TMPRSS2). The media was changed the next morning. Viral supernatant was collected 48 hours post transfection, clarified by centrifugation at 4000 rpm for 5mins, and aliquoted prior to storage at -80°C. Vero-CCL81 and 293T cells were transduced in a 6-well plate with the prepared lentiviral constructs. Two days after transduction, these cells were expanded into a 10cm plate and placed under selection with puromycin (for ACE2 transduced cells) or blasticidin (for TMPRSS2 transduced cells). 293T and Vero-CCL81 cells were selected with 2 or 10pg/mL of puromycin, respectively. For blasticidin, 293T were selected with 5pg/mL and Vero- CCL81 cells were selected with 15pg/ml. To generate ACE2 and TMPRSS2 expressing 293T cells, 293T-ACE2 cells were transduced with the VSV-G with 5pg/mL blasticidin. Low passage stock of each cell line generated were immediately frozen down using BamBanker (Fisher Scientific; NC9582225). Single cell, isogenic clones were isolated via serial dilution in a 96 well plate. Wells with only a single cell were grown up and eventually expanded while under selection.
[0124] Production of SARS-CoV-2 VSV-AG rLuc Pseudotvped particles [modified from HNVPP protocol]
[0125] I. Summary
[0126] Briefly, 293T producer cells were transfected to overexpress SARS-CoV-2 or VS V-G glycoproteins. For background entry with particles lacking a viral surface glycoprotein, pCAGG empty vector was transfected into 293T cells. Approximately 8 hours post transfection, cells were infected with the VSV \G-rLuc reporter virus for 2 hours, then washed with DPBS. Two days post infection, supernatants were collected and clarified by centrifugation at 1250 rpm for 5 mins. Upon collection, a small batch of VSVAG-rLuc particles bearing the CoV2pp were then treated with TPCK-treated trypsin (Sigma- Aldrich; T1426-1G ) at room temperature for 15 minutes prior to inhibition with soybean trypsin inhibitor (SBTI) (Fisher Scientific; 17075029). Particles were aliquoted prior to storage in - 80 °C to avoid multiple freeze-thaws.
[0127] II. Purpose
[0128] The following procedure should be used to generate VSV-AG particles pseudotyped with CoV-2 Spike (S) glycoprotein. These purified stocks can then be used to perform single-round infection in various cell types using BSL-2 conditions.
[0129] III. Biosafety
[0130] While all virus generated through this protocol is bio-contained by being limited to single-cycle infection, all aspects of this protocol should be carried out with standard sterile tissue culture technique, and BSL-2 precautions whenever handling VSV.
[0131] The VSV \G reporter backbone is from the Indiana lab-adapted strain which is cleared for use at BSL-2 (reviewed in PMID: 20709108). Bona fide recombination amongst negative sense RNA viruses (as opposed to positive sense RNA viruses) is exceedingly rare if not absent (reviewed in PMID: 21994784). Finally, the VSV-G provided in trans lacks the VSV-G gene start and gene stop signals present in the VSV \G backbone, making even the possibility of a productive homologous recombination event vanishingly small.
[0132] IV. Materials needed [0133] Sigma Poly-L-Lysine solution, 0.01% m/w cat no. 4707; Kerafast anti-VSV-G [8G5F11] antibody cat no. EB0010; Polysciences Transporter 5 (PEI) Transfection Reagent cat no. 26008-5; Promega Renilla Luciferase Assay System cat no. E2820; VSV[Rluc]- AG- G* parental stock, stored at -80° C and with known titer; sterile midi or maxiprep of surface glycoprotein(s) expression plasmids; 20% w/v sucrose in DPBS + lmM EDTA (sterilized); if trypsin treating particles: TPCK Trypsin and Soybean Trypsin Inhibitor.
[0134] V. Transfection of293T cells in 10 cm plates with surface glycoprotein construct [0135] Before seeding, 10 cm dishes were coated with poly-L-lysine (PLL). Alternatively, collagen coated plates can be used. For this PLL stock was diluted 1:10 with sterile ddH20. Approximately 5 mL PLL dilution were added to each plate. The plates were rotated and tilted until all surfaces are coated with liquid. The plates were incubated for > 15 min in a 37 °C incubator. The PLL was removed, the plates were washed with sterile DPBS and aspirated until dry.
[0136] At ~20 hours before transfection, 5 x 106 or 6 x 106293T cells were plated in PLL- coated 10 cm dishes.
[0137] At time 0 h (~20 hrs post-seeding), _the media was (optionally) replaced with 5 mL DMEM + 10% FBSi. The purpose of this media exchange is to reduce the volume that the transfection reagents are added to i.e. 5 mLs instead of 10 mLs. Then, 24 pg total DNA (glycoprotein expression plasmid) were transfect per plate. For this, 120 uL PEI reagent were diluted in 500 uL PBS. A total of 24 ug of DNA was diluted in a separate tube of 500 uL PBS. The PEI mix was added to the DNA mix and incubated at room temperature for 30 mins. The mixture was added drop wise to cells.
[0138] For viruses requiring only a single-entry glycoprotein, up to 12 pg of that plasmid were used along with 12 pg of empty vector to bring total DNA concentration up to 24pg total. For viruses requiring multiple entry glycoproteins, equal amounts of each glycoprotein were used. For example, Henipaviruses require a lusion and attachment glycoprotein. Accordingly, 12 pg of F and 12 pg of RBP were transfected into the 293T cells.
[0139] VI. Infection of transfected cells ~8 hours post transfection:
[0140] The timing of VSV-AG-G infection can be varied depending on the cell surface expression kinetics of the specific envelope protein. For example, 6, 8, 12, and 24 hpt can be used. optiMEM may be used for CoV2pp production, as this leads to an increase of CoV-2 spike cleavage relative to DMEM + 10% FBS. [0141] At ~8 hpt (see comment above), cells were infected with a VSV[Rluc]- AG-G* stock (~1 X 108 TCID50 units) in an inoculum volume of 5 mL per plate. The inoculum was incubated for 1-2 hours at 37 °C to permit infection. The inoculum was removed. The plates were washed 2x with dPBS to remove excess VSV-G particles that did not infect.
[0142] The incubation medium, opti-MEM (modification of Eagle's Minimum Essential Medium, buffered with HEPES and sodium bicarbonate, and supplemented with hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, and growth factors) containing a 1:20,000 dilution of anti- VSV-G antibody (8G5F11 from Kerafast), was prepared. This potently neutralizing antibody minimizes the background sometimes seen with “bald” VSV pseudotypes (293T cells transfected with 24pg empty vector, then infected as normal). Subsequently, 10 mL incubation medium was added to each plate of washed 293T cells. At 48 hpi the supernatant into a 50 mL conical tube was collected — roughly 10 mL per plate.
[0143] VII. Purification of pseudoparticles by clarification and ultracentrifusation (CoV2pp):
[0144] At this stage, CoV2pp were clarified, but not concentrated through a sucrose cushion. If desired, other concentration methods such as Amicon and Peg concentration may be used.
[0145] The pseudotype-containing supernatant of cell debris was clarified by centrifugation in a standard benchtop centrifuge at 1,250 rpm (approximately. 450 x g) for 5 min. The clarified supernatant was transferred to Seton Open-Top Polyclear Centrifuge Tubes for SW28 rotor. 15 mL serum- free media was added. A pipette containing 10.5 mL 20% sucrose was inserted to the bottom of the tube. 10 mL 20% sucrose were getnly added to the bottom of the tube to create cushion. The pipette was removed without disrupting the sucrose-media interface.
[0146] After ultracentrifugation for 2 hours at 25,000 rpm at 4 °C, the supernatant was removed. The pellet of concentrated pseudoparticles was resuspended in 600 pL DPBS. Four 110 pL aliquots for experiments and one -200 uL aliquot for TCID50 titration were prepared. [0147] VIII. Titrate by limiting dilution on susceptible cell line:
[0148] -24 h prior to infection, -20,000 or -25,000 Vero cells per well were seeded in a
96-well plate. [0149] At time 0 h, serial 1:10 dilutions of the virus stock in the desired media (i.e.
DMEM + 10% FBS for Vero or 293T cells) were prepared such that one was able to transfer a final volume of 100 uL inoculum per well. This dilution series was performed in 6 replicates in order to generate a TCID50 value.
[0150] At 18-22 hpi, Promega Renilla or Passive Lysis buffer was prepared by diluting the stock 1/5 in ddH20. The culture media/inoculum was removed. Each well was washed with 100 pL of DPBS.
[0151] The cells were lysed by adding 25 pL prepared lysis buffer to each well. From this point on, all samples can be handled safely outside the biosafety cabinet, as all living and infectious material has been inactivated. It is recommended that, in addition to using the passive lysis buffer provided in the kit, one freeze-thaw cycle is performed to release the rLuc. Alternatively, incubation on an orbital shaker for 15 mins at 500 rpm can be performed. [0152] After lysis the same plate was assayed for Renilla luciferase production on a plate reader using the Promega rLuc kit. For preparing the Renilla luciferase assay reagent, the assay buffer 1:1 was diluted with DPBS and a 1:200 dilution of assay substrate was used. [0153] When reading on the Cytation3, the following procedure was used: (1) delay of 5 seconds between each well, (2) dispense 40pL of assay reagent, (3) shake for 2 seconds, (4) delay for 2 seconds, (5) read luminescence, (6) quench the reaction by dispensing 50pL of 70% ethanol, (7), shake for 5 seconds and (8) proceed to the next well. The limit of detection for the Cytation3 is -300 RLUs. When calculating TCID50, the Spearman & Karber algorithm was used. Positive wells are those with >2x the average background signal i.e. for the Cytation3, this would be >600 RLUs. For other instruments, uninfected wells were assayed to determine the background signal.
[0154] IX. TPCK Trypsin treat CoV2pp to enhance entry:
[0155] Once a titer has been established for a batch (or in parallel with titering the untreated stock) of CoV2pp, a small batch (-400 - 500 pL) of CoV2pp was immediately treated with a serial dilution of TPCK trypsin and one concentration of soybean inhibitor. [0156] Trypsin treatment was as follows: (1) The desired concentration of trypsin was added, followed by incubation at room temperature for 15 mins. The desired concentration of soybean inhibitor was added, followed by storage at -80 °C
[0157] For two separate batches of trypsin, the following conditions were used: 1st batch: 625 pg/mL of trypsin with 625 pg/mL of soybean inhibitor; 2nd batch: 475 pg/mL of trypsin with 600 pg/mL of soybean inhibitor. Despite inactivation with soybean inhibitor, there is some “leaky” trypsin that retains activity until it interacts with cells to enhance/prime SARS- CoV-2 spike mediated infection. Certain trypsin might be particularly useful, such as concentrations between 6.25 pg/mL and 12.5 pg/mL, to induce this entry enhancement. Taken together, drastically lower concentrations of trypsin and soybean inhibitor can be used to achieve maximal CoV2pp entry with limited toxicity.
[0158] The remaining batch was treated at the desired trypsin concentrated, aliquot appropriately and store at -80 °C.
[0159] X. Example of an experimental timeline
[0160] Day -1 (pre-infection): PLL coating of 10 cm dishes, seeding of 293T cells.
[0161] Day 0: Transfection of 293T cells; infection of transfected cells with parental VSV[Rluc]- AG-G* stock.
[0162] Day 1 post-infection: Seeding of susceptible cells in 96-well plates for titration (optional).
[0163] Day 2 post-infection: Collection of supernatant from transfected/infected cells; clarification, and, if needed, concentration (i.e ultracentrifugation, Amicon filter, or PEG) of supernatant; if needed, trypsin-treatment of CoV2pp, aliquotting and freezing; tittering of new pseudotyped virus stocks on susceptible cells.
[0164] Day 3 post-infection: Assaying of Renilla luciferase for titer of pseudotyped stocks.
[0165]
[0166] Pseudovirus titering [0167] I. Summary
[0168] To titer the pseudo viruses, 20,000 Vero-CCL81 cells were seeded in a 96 well plate 20-24 hrs prior to infection. A single aliquot of BALDpp, CoV2pp, and VSV-Gpp were used for infections and titrations were performed in technical triplicates. At 18-22 hours post infection, the infected cells were washed with DPBS, lysed with passive lysis buffer, and processed for detection of Renilla luciferase. The Cytation3 (BioTek) was used to read luminescence.
[0169] II. Materials
[0170] Vero CCL81 cells or isogenic cells (293T-ACE2 clone 5-7 or 293T- ACE2+TMPRSS2 clone F8), all maintained in DMEM + 10% FBS, DMEM with 10% FBS; Promega Renilla luciferase assay system (100 assays-E2810; 1000 assays-E2820); CoV2pp: VSV \G-Rluc bearing SARS-CoV-2 Spike glycoprotein; VSV-Gpp: VSV \G-Rluc bearing VSV-G entry glycoprotein; BALDpp: VSV-G-Rluc bearing no protein (produced in parallel with samples above)
[0171] III. Safety notes
[0172] The CoV2pp are a VSV pseudotyped particle (pp) system that do not encode any viral glycoprotein in the VSV genome and can be worked with under Bio-Safety Level 2 (BSL2) conditions.
[0173] IV. Protocol for Titering CoV2pp by limiting dilution on susceptible cell lines [0174] ~24 hours prior to infection, 20,000 Vero CCL81 cells were seeded per well in a
96-well plate. If using 293T clone 5-7 or F8 cell lines, -35,000 cells were seeded per well. [0175] Cells were grown in standard DMEM + 10% FBS.
[0176] At time 0 hrs, the desired serial dilution of virus was prepared in Serum Free Media (SFM; DMEM only) such that one was able to transfer a final volume of 100 pL/well. The media was removed from the Vero cells. Starting from the lowest dilution, 100 pL from the titration plate were carefully transferred to the cells, which were then incubated at 37 °C. [0177] At 18-22 hrs post infection, the Promega Renilla lysis buffer was prepared by diluting the stock 1 :5 in ddH20. The culture media/inoculum was removed. Each well was washed by adding 100 pL of DPBS, then removing this volume with a multichannel pipette. [0178] The cells were lysed by adding 25 pL prepared lysis buffer to each well. It is recommended that, in addition to using the passive lysis buffer provided in the kit, one freeze-thaw cycle is performed to release the rLuc. Alternatively, incubation on an orbital shaker for 15 mins at 500 rpm can be performed.
[0179] After lysis the same plate was assayed for Renilla luciferase production on a plate reader using the Promega rLuc kit. For preparing the Renilla luciferase assay reagent, the assay buffer 1:1 was diluted with DPBS and a 1:200 dilution of assay substrate was used. [0180] When reading on the Cytation3, the following procedure was used: (1) delay of 5 seconds between each well, (2) dispense 40pL of assay reagent, (3) shake for 2 seconds, (4) delay for 2 seconds, (5) read luminescence, (6) quench the reaction by dispensing 50pL of 70% ethanol, (7), shake for 5 seconds and (8) proceed to the next well. The limit of detection for the Cytation3 is -300 RLUs. [0181] For calculating TCID50, the Spearman & Karber algorithm was used. Positive wells are those with >3x the average background signal of uninfected wells *For the Cytation3, the limit of detection is 300 RLUs. When calculating TCID50, positive wells as those with >1000 RLUs were considered. For other instruments, multiple uninfected wells can be assayed to determine the background signal.
[0182] V. Protocol for neutralization studies with monoclonal antibodies or patient sera: [0183] 24 hours prior to infection, 20,000 Vero CCL81 cells were seeded per well in a 96- well plate
[0184] At time 0 hrs, a dilution of the monoclonal antibody or sera in DMEM + 10%FBS in a V bottom 96 well plate was prepared as follows: If using a 4-fold serial dilution, 28.75 pL media were added to all wells except the top well. To the top well, 38.34 pL of the entry inhibitor were added to the well containing the starting dilution. Then 9.58 pL were transferred for a 4-fold serial dilution. This ensured that there were 28.7 5pL of the entry inhibitor (e.g. sera, monoclonal antibody or small molecule) in each well, which will be further diluted 1:4 after the addition of virus.
[0185] The virus stock was diluted in DMEM + 10%FBS and, using a multichannel and a sterile basin, the desired amount of virus was transferred to each well of a V bottom 96 well plate. For example, between a 1:4 and 1:50 dilution of the CoV2pp can be used. When using Vero-CCL81 cells, RLUs -105 (> 100 x signalmoise) were achieved with a 1:4 dilution of CoV2pp. When using either the 293T-ACE2 clone 5-7 or 293T-ACE2-TMPRSS2 clone F8- 2, the same RLUs can be achieved with a 1:20-1:50 dilution. All CoV2pp batches were tittered first prior to use for viral neutralization assays or entry inhibition assays. Each point of the neutralization curve was performed in triplicate.
[0186] When using a 4-fold serial dilution of the entry inhibitor, 86.25 pL of virus was transferred to each well. This further dilutes the entry inhibitor 4 x. For patient sera, a final dilution range of 1 : 10 to 1 :40,960 was used, which covers a wide neutralization range.
[0187] Subsequently, the media from was removed from the cells.
[0188] Starting from the lowest serial dilution, 100 pL of the mix were transferred to the cells. Optionally, plates were spinoculated by centrifugation at 1250 rpm for 1 h at 37 °C. [0189] Plates were then incubated at 37 °C. After 18-22 hrs, the plates were lysed and read as described as above. [0190] Collection of producer cells and concentration of pseudotyped particles
[0191] Cell lysates were collected from producer cells with lOmM EDTA in DPBS. Cells were subsequently lysed with RIPA buffer (Thermo Scientific, 89900) containing protease inhibitor (Thermo Scientific, 87785) for 30 minutes on ice. Lysates were centrifuged at 25,000 x g for 30 minutes at 4°C, and the supernatants were collected and stored at -80°C. Total protein concentrations were determined by the Bradford assay. For viral pseudoparticles, 10 mL of designated viral particles was concentrated via 20% Sucrose cushion (20% Sucrose in DPBS), Amicon Ultra centrifugal filter (100 kDa cutoff, Millipore Sigma, UFC910024), or PEG precipitation (Abeam, abl02538). Concentrated viral particles were resuspended in 300 pL of PBS or Opti-MEM for further analysis.
[0192] Western blots
[0193] All protein samples were ran under reduced conditions by dilution in 6X SDS containing DTT and 5% Beta-Mercaptoethanol (Fisher Scientific; ICN19483425). The protein was subsequently incubated in a heating block at 95 °C for 15mins, run on a 4-15% SDS-PAGE gel, and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked with Phosphate-Buffered Saline Blocking Buffer (LI-COR, 927- 700001), and then probed with the indicated antibodies. Antibodies against SARS-CoV2 (2B3E5 from Dr. Thomas Moran and GTX632604 from GeneTex), ACE2 (66699-1-Ig from Proteintech and Rb abl08252 from abeam), VSV-G (A00199 from Genescript), VSV-M (EB0011 from Kerafast), anti-HA (NB600-363 from Novus), and CoX IV (926-42214 from LI-COR) were used. For secondary staining, membranes were washed and incubated with the appropriate Alexa Fluor 647-conjugated anti- mouse antibody or Alexa Fluor 647-conjugated anti-rabbit antibody. Alexa Fluor 647 was detected using the ChemiDoc MP imaging system (Bio-Rad). Relative ACE2 or TMPRSS2 abundance was calculated by First normalizing abundance relative to GAPDH expression, then normalizing to wild type expression.
[0194] RNA extraction and qPCR for ACE2 and TMPRSS2 expression [0195] Total RNA was extracted from cells using Direct-zolTM RNA Miniprep kit (Zymol, R2051), and reverse transcription (RT) was performed with the TetroTM cDNA Synthesis kit (Bioline, BIO- 65043) and random hexamers. RT PCR was performed with the SensiFASTTM SYBR & Fluorescein Kit (Bioline, BIO-96005). For qPCRs, HPRT forward (5’- ATTGTAATGACCAGTCAACAGGG-3 ’ , SEQ ID NOG) and reverse (5’- GCATTGTTTTGCCAGTGTCAA- 3’, SEQ ID NO:4) primers, ACE2 forward (5’- GGCCGAGAAGTTCTTTGTATCT-3 ’ , SEQ ID NOG) and reverse (5’- CCCAACTATCTCTCGCTTCATC-3 ’ , SEQ ID NO:x6) primers, and TMPRSS2 forward (5’- CCATGGATACCAACCGGAAA-3 ’ , SEQ ID NO:7) and reverse (5’- GGATGAAGTTTGGTCCGTAGAG-3’, SEQ ID NO:8) primers were utilized. Samples were read on the CFX96 Touch Real-Time PCR Detection System (Biorad). For qPCR forward and reverse primers were utilized. The qPCR was performed in duplicates for each sample and results were calculated using 2-AACT with normalization to the HPRT housekeeping gene control and further normalization to the 293T parental cells.
[0196] Sera acquisition, ELISAs and Live Virus Neutralization [0197] All patient sera were ethically acquired by the respective groups involved in this study. Spike ELISAs for patient sera from the Krammer lab were performed in a clinical setting using the two-step protocol previously published. Briefly, this involves screening patient sera (at a 1:50 dilution) with sRBD and samples determined to be positive were further screened at 5 dilutions for reactivity to Spike ectodomain. All 36 samples were screened in this manner, but a subset of 15 samples were further screened for IgG and IgM binding antibodies to Spike Ectodomain. The protocol from Stadlbauer et all04 was modified slightly to start from a 1:300 and end at a 1:24300 dilution of sera. IgG and IgM antibodies were detected with secondary antibodies conjugated to HRP (Millipore AP101P for anti- Human IgG and Invitrogen A18841 for anti-Human IgM).
[0198] Background was subtracted from the OD values, samples were determined to be positive if >3 fold over the negative control and AUC was calculated in PRISM. ELISAs performed by the LSUHS group utilized sRBD with a 1:50 dilution of patient sera to screen all samples followed by Spike ectodomain with patient sera at a 1:100 dilution. Background subtracted OD values are reported for both sets of ELISAs. ELISAs performed by the COVIDAR group utilized a mixture of sRBD and spike ectodomain for samples serially diluted from 1 :50 to 1 :6400. AUC were calculated as described above.
[0199] All live virus neutralizations were performed at Biosafety-Level-3 (BSL-3) using the USA-WA/2020 isolate of SARS-CoV-2. Briefly, -600 TCID50 of virus was incubated with a serial dilution of patient sera for lhr at 37°C prior to infection of Vero-E6 cells. Forty- eight hours post infection, cells were fixed in 10% PFA and stained with mouse anti-SARS- CoV nucleoprotein antibody. This was subsequently detected by the addition of HRP- conjugated goat anti-mouse IgG and SIGMAFAST OPD. The BioTek Synergy 4 plate reader was used to measure OD490, which was subsequently used to calculate microneutralization (MN) titers. The samples with live vims MN titers were a part of a larger study looking at the longitudinal dynamics of the humoral immune response. A random subset of sera samples and their associated MN titers for validation studies with the CoV2pp based virus neutralization assay were used
[0200] Neutralization studies with patient sera, soluble RBD. or Nafamostat-mesylate [0201] De-identified sera were obtained with IRB approval to use for research purposes. Unless otherwise noted, all patient sera were heat inactivated at 56°C for 30 minutes, and serially diluted in DMEM + 10% FCS when performing virus neutralization assays (VNAs). For the VNAs performed in lab 1 (ISMMS-1), a pre-titrated amount of pseudotyped particles (diluted to give approximately 105 RLU) was incubated with a 4-fold serial dilution of patient sera for 30 minutes at room temperature prior to infection of Vero-CCL81 cells seeded the previous day. For sRBD or Nafamostat inhibition, a pre-titrated amount of pseudotyped particle dilution was mixed with the protein or compound and added to cells immediately after. Approximately 20 hours post infection, cells were processed for detection of luciferase activity as described above. Raw luminometry data were obtained from labs that volunteered VNA results from at least 12 patient samples and analyzed as indicated below.
[0202] Method modifications from the three contributing labs [0203] Serum neutralizations by LSUHS (Kamil and Ivanov) were performed by first diluting 4-fold in 100 pi total volume then diluting via a 3-fold serial dilution. Cell lysates were transferred to a white walled 96 well plate, then the Promega Renilla luciferase assay kit was utilized to detect luciferase. Plates were read on a Tecan SPARK plate reader by collecting total luminescence signal for 10 seconds. ISMMS- 2 (Hioe) began neutralizations at a 10-fold dilution and proceeded with a 4-fold serial dilution.
[0204] Plates were read on a black walled 96 well plate using the Renilla Glo substrate (Promega, E2720) with a 1 second signal integration time. COVID-19 samples were provided to ISMMS-2 by the Clinical Pathology Laboratory at ISMMS or from an IRB-approved study at the James J. Peters VA Medical Center. COVIDAR (Gamarnik) began at either an 8-fold or 16-fold dilution then continuing with either a 3-fold or 2-fold serial dilution respectively. White, F-bottom Lumitrac plates (Greiner, 655074) plates were read via the GloMax® Navigator Microplate Luminometer (Promega, GM200) using the ONE-GloTM Luciferase Assay System (Promega, E6110).
[0205] Inhibitory Concentration Calculations and other R packages used [0206] Relative inhibitory concentrations (IC) values were calculated for all patient sera samples by modeling a 4-parameter logistic regression with drm in the R drc package.110 For examples, a relative inhibitory concentration of 50% (IC50) is calculated as the midway point between the upper and lower plateaus of the curve. Absolute inhibitory concentration (absIC) was calculated as the corresponding point between the 0% and 100% assay controls. For example, the absIC50 would be the point at which the curve matches inhibition equal to exactly 50% of the 100% assay control relative to the assay minimum (0%).lll As a result, sera samples that are non-neutralizing or minimally neutralizing may have lower plateaus indicating they cannot reach certain absolute inhibitory concentrations, such as an absIC90 or absIC99.
[0207] Example 2: Material and Methods for Example 11 [0208] Cell lines
[0209] Vero-CCL81 TMPRSS2, HEK 293T-hACE2 (clone 5-7), and 293T-hACE2- TMPRSS2 (clone F8-2) cells were maintained in DMEM + 10% FBS. The HEK 293T- hACE2-TMPRSS2 cells were plated on collagen coated plates or dishes. BSR-T7 cells 52, which stably express T7 -polymerase were maintained in DMEM with 10% FBS.
[0210] VSV-eGFP-CoV2 spike (A21aa) genomic clone and helper plasmids [0211] The VSV-eGFP sequence was cloned into the pEMC vector (pEMC-VSV-eGFP), which includes an optimized T7 promoter having the sequence TAATACGACT CACTATAGGG AGA (SEQ ID NO: 9) and hammerhead ribozyme having the sequence CTGATGAGTC CGTGAGGACG AAACGGAGTC TAGACTCCGT C (SEQ ID NO: 10) just before the 5’ end of the viral genome (see FIG. 4A of Beaty et a , 2017, mSphere 2(2):e00376-16). [0212] pEMC-VSV-eGFP-CoV2-S (Genbank Accession: MW816496) was generated as follows: the VSV-G open reading frame of pEMC-VSV-eGFP was replaced with the SARS- CoV-2 S, truncated to lack the final 21 amino acids 54. A Pac-I restriction enzyme site was introduced just after the open reading frame of S transcriptional unit, such that the S transcriptional unit is flanked by Mlul / Pad sites. SARS-CoV-2 S is from pCAGGS-CoV-2-
5 55, which codes the codon optimized S from the Wuhan Hu-1 isolate (NCBI ref. seq. NC_045512.2) with a point mutation of D614G, resulting in B.l lineage. The B.1.1.7 Spike used carries the mutations found in GISAID Accession Number EPI_ISL 668152: del 69-70, del 145, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H. The B.1.351 Spike carries the mutations D80A, D215G, del242-244, K417N, E484K, N501Y, D614G, and A701V (from EPI_ISL_745109). The Spike sequences of WT, B.l.1.7, B.1.351, and E484K are available at Genbank (Accession Numbers: MW816497, MW816498, MW816499, and MW8 16500
[0213] Sequences encoding the VSV N, P, M, G, and L proteins were also cloned into a pCI vector to make expression plasmids for virus rescue, resulting in plasmids: pCI-VSV-N, pCI-VSV-P, pCI-VSV-M, pCI- VSV-G, and pCI-VSV-L.
[0214] Generation of VSV-CoV2 spike from cDNA
[0215] 4 x 105293T-ACE2-TMPRSS2 cells per well were seeded onto collagen-I coated
6 well plates. The next day, 2000 ng of pEMC-VSV-EGFP-CoV2 spike, 2500 ng of pCAGGS-T7opt 56, 850 ng of pCI-VSV-N, 400 ng of pCI-VSV-P, 100 ng of pCI-VSV-M, 100 ng of pCI-VSV-G, 100 ng of pCI-VSV-L were mixed with 4 mL of Plus reagent and 6.6 mL of Lipofectamine LTX (Invitrogen). 30 min later, transfection mixture was applied to 293T- hACE2-TMPRSS2 cells in a dropwise fashion. Cells were maintained with medium replacement every day for 4 to 5 days until GFP positive syncytia appeared. Rescued viruses were amplified in Vero-CCL81 TMPRSS2 cells 40, then titered and used for the assay.
[0216] Virus neutralization assay
[0217] 5 x 10E4293T-hACE2-TMPRSS2 cells per well were seeded onto collagen- coated 96 well cluster plates one day prior to use in viral neutralization assays. Virus stocks were mixed with serially diluted serum for 10 minutes at room temperature, then infected to cells. Note: all sera assayed in this study were previously heat inactivated by degrees for 30 min before use in any viral neutralization studies. At 10 h post infection, GFP counts were counted by Celigo imaging cytometer (Nexcelom). Each assay was done in triplicate. For calculation of IC50, GFP counts from “no serum” conditions were set to 100%; GFP counts of each condition (serum treated) were normalized to no serum control well. Inhibition curves were generated using Prism 8.4.3 (GraphPad Software) with ‘log (inhibitor) vs normalized response - variable slope’ settings.
[0218] Example 3: Production of VSVAG-rLuc bearing SARS-CoV-2 spike glycoprotein
[0219] The initial objective was to produce SARS-CoV-2 PsV sufficient for 10,000 infections/week at -1:100 signaknoise ratio when performed in a 96- well format. A VSV- based rather than a lentiviral PsV system was used as lentiviruses are intrinsically limited by their replication kinetics particle production rate (104-106/ml for lentiviruses versus 107-109/ml for VSV without concentration). The production of the VSVAG-rLuc pseudotyped viral particles (pp) bearing the SARS-CoV-2 spike glycoprotein was optimized as diagramed in FIG. 1A.
[0220] In the first step, cells overexpressing SARS-CoV-2 spike protein are infected with VSV[Rluc] AG-G* at a low MOI and grown in the presence of anti-VSV-G monoclonal antibody. SARS-CoV-2 spike protein pseudotyped particles bud out from the infected cells. Those SARS-CoV-2 pseudotyped particles (at a certain concentration, e.g., genome copies/ml) may then be used infect target cells. The purified pseudotyped particles may be trypsin treated and spinoculation may be performed to enhance entry of the pseudotyped particles. The particle: infectivity ratio and SARS-CoV-2 spike protein incorporation may be determined as well as the percent cleavage of incorporated spike protein.
[0221] Notably, this protocol involves infecting producer cells at a low multiplicity of infection (MOI) of stock VSVAG-G*, incubating producer cells with an anti-VSV-G monoclonal antibody and generating the pseudotyped particles in Opti-MEM media. The first two measures effectively eliminated the background signal from residual VSV-G while the last measure allowed for more cleavage of SARS-CoV-2pp in producer cells. While truncating the cytoplasmic tail (CT) of SARS-CoV-2-S is typically required for greater functional incorporation into heterologous viral cores, here pseudotyping was optimized with full-length SARS-CoV-2 spike. CT truncations in many other class I viral fusion proteins, including other ACE2- using coronaviruses (HCoV-NL63 and SARS-CoV-1) can affect ectodomain conformation and lunction. As such surrogate assay was developed that reflects the biology of the full-length virus spike.
[0222] BALDpp, NiV-RBPpp, CoV2pp, and VSV-Gpp were produced using the VSVAG- rLuc reporter backbone and titered them on Vero-CCL81 cells FIG. IB). High background problems have resulted in low signahnoise ratios when using VSV-based PsV, especially for viral envelope proteins that do not mediate efficient entry. Here two different negative controls were used, BALDpp and NiV-RBP, to show that the background issue was resolved. BALDpp lacks any surface glycoprotein while NiV-RBPpp incorporates the NiV receptor binding protein (RBP), which binds to the broadly expressed ephrin-B2 with sub-nanomolar affinity. However, the NiV fusion (L) glycoprotein necessary for viral entry is absent. NiV- RBPpp without NiV-L should not fuse and effectively serves as a stricter and complementary negative control. Under the conditions shown, neither BALDpp nor NiV-RBPpp gives any background even at the highest concentration of virus particles used. These constructs were used to infect Vero-CCL81 cells and, as expected, an average of <500 RLUs of entry was observed with the BALDpp and NiV-RBPpp negative controls. These levels of entry were comparable to the “cells only” signal, providing confidence in any infection signals 10-fold over background.
[0223] Undiluted CoV2pp entry resulted in luciferase values of over 50,000 RLUs; greater than 100-fold over background BALDpp signals (Fig. IB). VSV-Gpp gave several logs higher infectivity as expected.
[0224] FIG. 1C shows the genome copy number and particle to infectivity ratio for BALDpp, NiV-G only, CoV2pp, or VAV-Gpp. The genome copy number was assessed using primers against the VSV-L protein as previously described (Pryce, Azarm CedV-Bl usage). The particle to infectivity ratio was calculated as a fraction of number of genomes to TCID50.
[0225] Western blots of the producer cells demonstrated effective expression of cleaved, SARS-CoV-2 spike glycoproteins (FIG. ID, left panel). Cleaved CoV-2 spike products (SI, S2, and S2’) all appear to be incorporated into the VSVAG pseudotyped particles (Fig. ID, right panel). To ensure that entry of CoV2pp is SARS-CoV-2 spike-mediated, it was shown that the homologous soluble spike receptor binding domain (sRBD) competitively inhibits CoV2pp (Fig. IE).
[0226] Example 4: CoV2pp entry is enhanced by trypsin treatment and spinoculation [0227] The relative signal of CoV2pp infections was enhanced to effectively increase the number of infections one can provide or perform per batch of CoV2pp. Next, the effect of typsin treatment and spinoculation on CoV2pp entry into target cells was determined.
[0228] CoV2pp stocks were treated with the indicated range of trypsin concentrations for 15 min at room temperature (FIG. 2A). In order to mitigate the effects of trypsin-dependent cytotoxicity, 625 pg/mL of soybean trypsin inhibitor (SBTI) were added to all samples before titrating the trypsin-treated CoV2pp onto Vero-CCL81 cells. CoV2pp treated with the highest concentration of trypsin (625pg/mL) resulted in ~100-fold enhancement of entry (FIG. 2A), but this trypsin-dependent enhancement was only apparent when comparing entry of undiluted trypsin-treated CoV2pp. A greater than 50-fold reduction in entry (RLUs) was observed after a 10-fold serial dilution, which nullified any entry enhancement effects of trypsin. Indeed, the role of trypsin in enhancing SARS-CoV-2 entry has not been fully determined. Trypsin may be acting to prime CoV2pp to facilitate better entry upon spike- receptor interactions and/or assist to proteolytically activate spike protein at or after receptor binding. The remaining uninhibited trypsin-dependent effect, which must be present at the highest trypsin concentration, might have inadvertently been neutralized by diluting the trypsin-treated CoV2pp in Dulbecco’s modified Eagle Medium (DMEM) +10% fetal bovine serum (FBS), which is the standard infection media for titrating CoV2pp.
[0229] To test this, CoV2pp and trypsin-treated CoV2pp were diluted 1:10 in three different media conditions before infecting Vero-CCL81 cells. For trypsin-treated CoV2pp, dilution in DMEM alone (serum free media, SFM) produced the highest signalmoise ratio, almost 1000-fold over BALDpp (FIG. 2B). As a result, CoV2pp treated with 62 5pg/mL of TPCK-treated trypsin was chosen, then 625 pg/mL of SBTI, diluted in SFM as the standard treatment condition. Furthermore, spinoculation at 1,250 rpm for 1 hr enhanced entry 3-5 fold (compare signalmoise in FIG. 2B to FIG. 2C).
[0230] The finding above suggest that the uninhibited trypsin-dependent enhancing effect was acting at the point of infection when CoV2pp is interacting with the host cell receptor.
To investigate further, additional SBTI was spiked in onto cells at the time of infection using particles produced under the standard treatment condition as above. Additional SBTI (25 pg/mL) added directly to cells at the point of infection was able to inhibit trypsin-dependent entry enhancement (FIG. 2D). The data suggest that some trypsin was not inhibited by the first 625 pg/mL of SBTI and enough remained to enhance entry at the point of infection. [0231] FIG. 2E shows that certain trypsin can improve CoV2pp activation. Supernatant containing CoV2pp were treated with different concentrations of trypsin for 15 minutes, then used to infect Vero-CCL81 cells.
[0232] Example 5: Entry of CoV2pp is independently enhanced by stable expression of ACE2 and TMPRSS2 in cells already permissive for SARS-CoV-2 entry and replication
[0233] To further characterize the determinants of CoV2pp entry, Vero-CCL81 cell lines were generated stably expressing human ACE2 or human TMPRSS2. Vero-CCL81 cells are already highly permissive for SARS-CoV-2 entry and replication. The indicated cells were infected with CoV2pp or trypsin-treated CoV2pp diluted in serum-free media (standard treatment) and enhanced entry in both stable cell lines was observed (Fig. 3). However, the entry enhancement of trypsin-treated CoV2pp in Vero-CCL81 + TMPRSS2 overexpressing cells was subdued relative to untreated CoV2pp. This suggests that the presence of exogenous trypsin during CoV2pp entry can substitute, in part, for the role played by cell surface TMPRSS2, an endogenous protease known to facilitate entry into physiological relevant cell types in vivo. The relationship between ACE2 and TMPRSS2 expression — with regard to their effect on enhancing SARS-CoV-2 spike mediated entry — is not straightforward. As ACE2 itself is a substrate for TMPRSS2, the right stoichiometry of receptor/protease expression appears to be the main driver of entry efficiency rather than the absolute expression of one or the other.
[0234] Example 6: Standardizing the parameters that impact CoV2pp-based virus neutralization assay
[0235] Having established that exogeneous trypsin can serve as a physiologically relevant substitute for endogenous proteases known to enhance entry of CoV2pp, such as TMPRSS2, the parameters that might affect the performance CoV2pp VNA were characterized. Conditions tested included heat-inactivation of sera and the infection media used to dilute human sera samples. Representative spike ELISA positive or negative sera were used to serve as positive and negative controls, respectively. When first diluted in SFM, negative sera could have alarming amounts of neutralizing activity that appeared specific for CoV2pp as the same sera did not neutralize VSV-Gpp entry (compare FIG. 4A with FIG. 4B). This CoV2pp serum neutralizing factor was somewhat reduced but not completely diminished by heat inactivation for 1 hr at 56 °C. Notably, the effect of this neutralizing factor from negative sera was preempted by diluting the trypsin treated CoV2pp in DMEM containing 10% FBS (FIG. 4B). Importantly, recombinant sRBD neutralization was not affected by the dilution of CoV2pp in Serum Free Media or DMEM + 10% FBS (FIG. 4C). Regardless, for standardizing the CoV2pp-based VNA, all subsequent patient sera were heat inactivated for at least 30 mins prior to use an serially diluted in DMEM + 10% FBS, which also served as the infection media. Despite the data from FIG. 2 implicating a trypsin-inhibitor- like activity in FBS, the marked inhibition of CoV2pp entry by seronegative human sera is a greater limiting factor that prevents the robust determination of true SARS-CoV-2 Nab titers. To achieve the same signalmoise ratio while performing the VNA in the presence of 10% FBS, the concentration of CoV2pp used per infection was increased.
[0236] Example 7 : Performance characteristics of the standardized CoV2pp virus neutralization assay
[0237] An initial set of patient sera for validation of CoV2pp VNA was used. These sera were screened according to a previously described two-stage ELISA protocol in which 1 :50 dilutions of patient sera were first screened for reactivity against sRBD. Subsequently, the presumptive RBD-positive patient sera were used to assess reactivity to the trimer stabilized ectodomain of spike at five different dilutions (1:80, 160, 320, 960, and 2880). These samples were used for neutralization studies with CoV2pp (FIG. 5). From the 36 patient sera tested, 6 were found to be negative for SARS-CoV-2 spike binding in the ELISA described above. All of those 6 sera samples also showed no neutralization of CoV2pp. The remaining 30 spike positive sera had 50% neutralizing titers that span 2 orders of magnitude (160 - 10,240). For a more quantitative assessment, the total IgG and IgM spike binding activity was determined (ELISA AUC) of a representative subset of fifteen sera samples and compared them with their reciprocal absIC50 and absIC80 values calculated from the CoV2pp neutralization curves (FIG. 5). Spike binding antibodies (IgG + IgM ELISA AUC) demonstrated a significant, positive correlation with neutralizing antibody (nAb) titers (reciprocal absIC50 and absIC80) as determined by CoV2pp VNA. Moreover, these Nab titers against CoV2pp also correlated well with live virus microneutralization titers (MN absIC50, MN absIC80). AbsIC80 appeared to be a more stringent measure of nAb activity, as some sera that have respectable MN absIC50 titers never achieve an absIC80. In this respect, the CoV2pp VNA has a larger dynamic range and was more sensitive in its ability to sort out sera samples that can reach their respective absIC80 values. Notably, sera samples with potent absIC50 titers do not always display potent absIC80 values.
[0238] Example 8: Independent validation of CoV2pp VNA with geographically distinct and ethnically diverse COVID-19 patient cohorts
[0239] To assess the robustness of the standardized CoV2pp VNA, CoV2pp was shared with different labs for use in various screens for nAbs. The raw virus neutralization data provided by three independent groups at the Icahn School of Medicine at Mount Sinai (ISMMS-2), Louisiana State University Health Sciences Center Shreveport (LSUHS), and Argentina (COVIDAR) are provided. In sera or plasma neutralization studies, similar absIC50, absIC80, and absIC90 distributions were observed. The LSUHS and ISMMS-2 cohorts represent data from 25 and 28 seropositive as well as 10 and 11 seronegative samples, respectively, while the COVIDAR consortium assessed neutralization from an initial set of 13 seropositive patient samples.
[0240] The seronegative control samples from all groups revealed no CoV2pp neutralization. Rare, but notable, seropositive samples from LSUHS also showed no neutralization. ISMMS-2 performed their analysis on confirmed convalescent plasma donors. While all donors had detectable nAb titers, their titers were highly variable and ranged across 2-3 logs. AbsIC80s were calculated for all samples shown and a moderate, but significant, positive correlation between various spike ELISA metrics and absIC80 was observed.
[0241] Notably, a Gaussian distribution of reciprocal absIC80s from all groups (n=89) was observed. The descriptive statistics from this aggregated data set reveals reciprocal absIC8025th percentile of 68.5, median of 170.8, and 75th percentile of 343.4. Descriptive statistics for reciprocal 387absIC50 and absIC90 were also calculated and are reported in Table 1. Table 1 Descriptive statistics for CoV2pp neutralization across four groups
Figure imgf000055_0001
[0242] Using the absIC80 descriptive statistics above and the ELISA endpoint titers from the initial 36 sera samples, 0% of the samples displaying an ELISA endpoint titer of 320 have an absIC50 greater than the median IC50. Perhaps not surprisingly, over 90% of samples with ELISA endpoints of 2880 have IC50s at or beyond the 75th percentile (Table 2).
Table 2 Comparison of ELISA endpoint titers to CoV2pp neutralization
Figure imgf000055_0002
[0243] Although absIC80 also generally follows this trend, differences in the ranked order of absIC50 and absIC80 values calculated for all sera samples were observed. This difference is more pronounced when comparing the absIC50 and absIC90 graphs further highlighting the need for a neutralization assay with a broad dynamic range. Additionally, the samples from each of the 4 groups show no statistical difference when absIC50, 80, or 90 calculations are compared (FIG. 6). Altogether, these data support the robustness of the CoV2pp VNA and suggest that absIC80 is a more stringent and meaningful measure of Nab titers.
[0244] Example 9 : Ultra-permissive 293T-ACE2 and 293T-ACE/TMPRSS2 clones allow for use of CoV2pp in VNA at scale
[0245] Although the standardized VNA is robust, the requirement for exogenous trypsin and spinoculation to achieve the optimal signalmoise limits the scalability of the VNA. Therefore, the untreated CoV2pp was used to screen for ultrapermissive cell lines that would allow for CoV2pp VNA to be performed with dilutions of virus supernatant without any trypsin treatment, virus purification, or spinoculation.
[0246] Three different 293T cell lines were generated stably expressing ACE2 and/or TMPRSS2 via lentiviral transduction. Then, these cells were infected with CoV2pp.
Increased expression of TMPRSS2 alone (293T-TMPRSS2) did not significantly improve entry (FIG. 7A), likely due to the low to undetectable ACE2 expression levels. However, expression of ACE2 significantly increased the entry of CoV2pp, which was further increased in 293T-ACE2+TMPRSS2 cells, suggesting the synergistic activity of TMPRSS2 and ACE2 (FIG. 7A). Western blot analysis confirmed the increased expression of ACE2 in the singly and doubly transduced 293T cells. Additionally, increased expression of both ACE2 and TMPRSS2 was confirmed by qPCR. Interestingly, ACE2 expression appeared to be decreased by >50% in 293T-ACE2+TMPRSS2 cells relative to 293T-ACE2 cells. These observations highlight the complex roles that receptor binding and protease activation play in SARS-CoV-2 entry, especially since ACE2 is a known substrate for TMPRSS2, and TMPRSS2 is also known to undergo autocatalytic cleavage.
[0247] Given how TMPRSS2 can enhance ACE2 dependent virus entry in a non-linear fashion, BALDpp, CoV2pp, and VSV-Gpp were used to screen 19 single cell clones derived from 293T-ACE2 or 293T-ACE2+TMPRSS2 or Vero-ACE2 bulk transduced cells. The latter (FIG. 3) served as an additional control in a naturally permissive cell line for SARS-CoV-2 entry and replication. All three bulk transduced cell lines resulted in significant increases in entry of CoV2pp relative to the parental 293T and Vero CCL81 cells (FIG. 7B). However, only a subset of the single cell clones performed better than bulk transduced cells. This is especially notable in single cell clones derived from 293T-ACE2+TMPRSS2 parentals, where only two of eight single cell clones show greater entry than the bulk transduced cells (FIG. 7B). One particular clone, F8-2 (FIG. 7B) showed a nearly ten-fold increase in CoV2pp entry relative to the bulk transduced cells. Using F8-2 to titer untreated CoV2pp without spinoculation, a dramatic increase was observed in signaknoise relative to Vero- CCL81 WT cells and even the most permissive 293T-ACE2 clone 5-7 (FIG. 7C) such that RLU signals were consistently 100-200 fold over BALDpp even at 1:50 dilution. TMPRSS2 was determined to be the main driver of this entry enhancement in the F8-2 cells as treatment with Nafamostat, a serine protease inhibitor, potently inhibited entry. However, this entry inhibition plateaued at 90% of maximal infection and the remaining 10% is nearly equivalent to the raw RLU values seen with bulk 293Ts stably expressing ACE2 alone (FIG 7D), suggesting a TMPRSS2-independent mechanism of entry. Entry into 293T-ACE2 cells was not inhibited by Nafamostat, once again highlighting that CoV2pp can enter by both the early and late entry pathways that have differential protease requirements.
[0248] Example 10: Diverse cell lines maintain similar kinetics in CoV2pp viral neutralization assays:
[0249] Sera samples were identified from 15 patients shown in FIG. 5 and tiered them into three groups: negative for CoV2pp neutralization (negative), weakly positive for CoV2pp neutralization (low positive), or strongly positive for CoV2pp neutralization (high positive) (FIG. 8A). Equal volumes of each set of samples were pooled and CoV2pp neutralization assays were performed on Vero-CCL81 WT, 293T-ACE2 clone 5-7, 293T- ACE2+TMPRSS2 bulk transduced, and the 293 T - ACE2+TMPRS S2 clone F8-2. Even in the case of varying levels of ACE2 and TMPRSS2 expression, CoV2pp neutralization assays show consistent patterns of neutralization, exhibiting the robust nature of the assay in tandem with its sensitivity in detecting relative differences in neutralizing titer (FIG. 8B). Patterns of neutralization as well as the calculated absIC50 and absIC80 reveal a large dynamic range between low and high neutralizing patient sera across cell lines (FIG. 8B).
[0250] Example 11: A replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system for virus neutralization assays (VNAs)
[0251] To generate rcVSV-CoV2-S containing different variants or mutants on demand, without the need for extensive passaging, a robust reverse genetics system and VNA was developed. The system uses a replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system uses rcVSV-CoV2-S, which encodes S from SARS coronavirus 2 in place of VSV-G, and coupled with a clonal HEK-293T ACE2 TMPRSS2 cell line optimized for highly efficient S-mediated infection. These rcVSV-CoV2-S can be used in BSL-2 compatible virus neutralization assays (VNAs), which correlate very well with VNAs using live SARS-CoV-2 (Spearman’s r > 0.9 across multiple studies).
[0252] The rcVSV-CoV2-S genomic coding construct comprises a hammerhead ribozyme immediately upstream of the 3’ leader sequence which cleaves in cis to give the exact 3’ termini (FIG. 9A). The system further uses a codon-optimized T7 -polymerase which alleviates the use of vaccinia-driven T7 -polymerase, and a highly permissive and transfectable 293T-ACE2+TMPRSS2 clone (F8-2) (FIG. 9B). A 6-plasmid transfection into F8-2 cells results in GFP+ cells 2-3 days post-transfection (dpt), which turn into foci of syncytia by 4-5 dpt indicating virus replication and cell-to-cell spread (Fig. 10A). Transfer of F8-2 cell supernatant into interferon-defective Vero-TMPRSS2 cells allowed for rapid expansion of low-passage viral stocks that maintain only the engineered Spike mutations. Clarified viral supernatants from Vero-TMPRSS2 cells were aliquoted, sequenced verified, then titered on F8-2 cells to determine the linear range of response (Fig. 10B).
[0253] Example 12: Exemplary use of the replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system
[0254] The emergence of SARS-CoV-2 ‘variants of concern’ (VOC) across diverse geographic locales have prompted re-evaluation of strategies to achieve universal vaccination. All three officially designated VOC carry Spike (S) polymorphisms thought to enable escape from neutralizing antibodies elicited during initial waves of the pandemic, including the ensemble of S mutations present in VOC lineages B.1.1.7 (501Y.V1) and B.1.351 (501Y.V2). The S genes of B.1.351 and P.l viruses each carry a number of mutations, but include three in the receptor binding domain (RBD) that are particularly notable, the S: N501 Y substitution, found in B.1.1.7, alongside polymorphisms at positions 417 and 484, K417N/T and E484K. The P.2 lineage, originally detected in Rio de Janeiro, carries only the E484K mutation in the RBD and has spread to other parts of South America, including Argentina laboratories to confer escape from convalescent sera and monoclonal antibodies.
[0255] The assay described in Example 11, may, for example be used to neutralizing activity of vaccine sera. For this, isogenic rcVSV-CoV2-S were generated expressing the B.1.1.7 (UK SARS-CoV-2 lineage), B.1.351 or E484K S to evaluate the neutralizing activity of Sputnik V vaccine sera from Argentina.
[0256] At one-month post-completion of the two-dose regimen, the Sputnik V vaccine generated respectable virus neutralizing titers (VNT) against rcVSV-CoV2-S bearing the WT (D614G) and B.1.1.7 spike proteins (Fig. 11A). The geometric mean titer (GMT) and 95% Cl for WT (1/IC50 GMT 49.4, 23.4 - 105) in the cohort of vaccine recipients was remarkably similar to that reported in the phase III Sputnik vaccine trial (GMT 44.5, 31.8 - 62.2). However, GMT against B.1.351 and E484K was reduced by a median 6.1- and 2.8-fold, respectively compared to WT (Fig. 11B). Even more revealing is their dose-response curves (not shown). When extrapolated to full serum strength, half of the sera samples failed to achieve an IC80 and only 1 out 12 achieved an IC90. One serum had little to no detectable neutralizing activity against B.1.351, E484K and even WT, but neutralized B.1.1.7 effectively.
[0257] Altogether, these data suggest vaccines that do not use the 2P stabilized Spike appear to generate more variable neutralizing responses that make it difficult to establish immune correlates of protection, especially against emerging VOC/VOI that contain the recurrent E484K mutation.
[0258] The foregoing is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the antibodies and methods provided herein and their equivalents, in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
[0259] All references cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Claims

What is claimed is:
1. A SARS-CoV-2 spike protein pseudotyped vesicular stomatitis virus (VSV) particle comprising an encapsidated negative sense, single- stranded RNA genome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, and a nucleotide sequence encoding luciferase, wherein genome does not express VSV glycoprotein (G).
2. The SARS-CoV-2 spike protein pseudotyped VSV particle of claim 1, wherein the luciferase is renilla luciferase or nanoluciferase.
3. The SARS-CoV-2 spike protein pseudotyped VSV particle of claim 1 or 2, wherein the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:2.
4. The SARS-CoV-2 spike protein pseudotyped VSV particle of claim 3, wherein the SARS-CoV-2 spike protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 1.
5. The SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 4, wherein the genome does not comprise a nucleotide sequence sequence encoding VSV glycoprotein (G).
6. The SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 5, wherein the genome further comprises a nucleotide sequence encoding a fluorescent protein.
7. The SARS-CoV-2 spike protein pseudotyped VSV particle of claim 6, wherein the fluorescent protein is red fluorescent protein or enhanced green fluorescent protein.
8. The SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 8, which has been treated with trypsin.
9. The SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 8, which has been treated with trypsin and soybean inhibitor.
10. A composition comprising the SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 9 and a carrier.
11. The composition of claim 10, wherein the carrier is serum free media.
12. The composition of claim 10, wherein the carrier is phosphate buffered saline.
13. A composition comprising the SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 7 and trypsin.
14. A composition comprising the SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 7 and trypsin and soybean inhibitor.
15. A method for generating SARS-CoV-2 spike protein pseudotyped VSV particles, comprising:
(a) infecting cells overexpressing SARS-CoV-2 spike protein with a recombinant VSV particle, wherein the recombinant VSV particle comprises an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding luciferase, wherein the genome does not express VSV glycoprotein (G), wherein the VSV particle is pseudotyped with VSV glycoprotein; and
(b) purifying SARS-CoV-2 spike protein pseudotyped VSV particles from the supernatant of the cells.
16. The method of claim 15, wherein the cells are cultured in optiMEM containing anti-VSV-G antibody.
17. The method of claim 15 or 16, wherein the SARS-CoV-2 spike protein pseudotyped VSV particles are purified from the supernatant by low speed centrifugation.
18. The method of any one of claims 15 to 17, wherein the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:2.
19. The method of claim 18, wherein the SARS-CoV-2 spike protein is encoded by the nucleotide sequence set forth in SEQ ID NO:l.
20. The method of any one of claims 15 to 19, wherein the luciferase is renilla luciferase.
21. A method for infecting cells with a SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 7, comprising:
(a) contacting the SARS-CoV-2 spike protein pseudotyped VSV particle with trypsin for a certain period of time; and
(b) infecting cells with the SARS-CoV-2 spike protein pseudotyped VSV particle.
22. A method for infecting cells with a SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 7, comprising:
(a) contacting the SARS-CoV-2 spike protein pseudotyped VSV particle with trypsin and soybean inhibitor for a certain period of time; and
(b) infecting cells with the SARS-CoV-2 spike protein pseudotyped VSV particle.
23. A method for infecting cells with a SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 7, comprising:
(a) contacting the SARS-CoV-2 spike protein pseudotyped VSV particle with trypsin for a certain period of time; and
(b) spinoculating the cells with the SARS-CoV-2 spike protein pseudotyped VSV particle.
24. The method of claim 23, wherein step (a) further comprises contacting the particle with soybean inhibitor.
25. The method of any one of claims 21 to 24, wherein the certain period of time is 15 minutes.
26. A method for detecting sera that neutralizes SARS-CoV-2 comprising:
(a) incubating with the SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 7 with sera from a subject for a first period of time;
(b) spinoculating cells expressing human ACE-2, TMPRSS2 or both with the sera-treated SARS-CoV-2 spike protein pseudotyped VSV particle for a second period of time; and
(c) measuring the luciferase activity after a third period of time, wherein a lower level of luciferase activity is detected if the sera neutralizes SARS-CoV-2 spike protein pseudotyped VSV particle than if steps (b) to (c) are performed without performing step (a), and the lower level of luciferase activity indicates that the sera neutralizes SARS-CoV-2.
27. The method of claim 26, wherein the first period of time is about 30 minutes.
28. The method of claim 26 or 27, wherein the second period of time is about 1 hour.
29. The method of any one of claims 26 to 28, wherein the third period of time is about 18 to 22 hours.
30. The method of any one of claims 26 to 29, wherein the subject is a human subject.
31. The method of any one of claims 26 to 30, wherein the sera is heat inactivated.
32. The method of any one of claims 26 to 31, wherein the sera is diluted in plain DMEM or DMEM and 10% heat inactivated fetal bovine serum.
33. The method of any one of claims 26 to 32, which further comprises concurrently repeating steps (a) to (c) with a positive control antibody or sera that does neutralize SARS-CoV-2.
34. The method of any one of claims 26 to 33, which further comprises concurrently repeating steps (a) to (c) with a negative control antibody or sera that does not neutralize SARS-CoV-2.
35. A method for assessing the ability of an antibody to neutralize SARS-CoV-2 comprising:
(a) incubating with the SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 7 with an antibody of interest for a first period of time;
(b) spinoculating cells expressing human ACE-2, TMPRSS2 or both with the antibody-treated SARS-CoV-2 spike protein pseudotyped VSV particle for a second period of time; and
(c) measuring the luciferase activity after a third period of time, wherein a lower level of luciferase activity is detected if the antibody neutralizes SARS-CoV-2 spike protein pseudotyped VSV particle than if steps (b) to (c) are performed without performing step (a), and the lower level of luciferase activity indicates that the antibody neutralizes SARS-CoV-2.
36. The method of claim 35, wherein the first period of time is about 30 minutes.
37. The method of claim 35 or 36, wherein the second period of time is about 1 hour.
38. The method of any one of claims 35 to 37, wherein the third period of time is about 18 to 22 hours.
39. The method of any one of claims 35 to 38, wherein the cells overexpress human ACE-2, TMPRSS2, or both.
40. A kit comprising the SARS-CoV-2 spike protein pseudotyped VSV particle of any one of claims 1 to 7, and optionally instructions for performing a neutralization assay using the SARS-CoV-2 spike protein pseudotyped VSV particle.
41. A nucleic acid sequence comprising a VSV antigenome that comprises a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding for luciferase, and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G).
42. The nucleic acid sequence of claim 41, which further comprises a T7 promoter, autocatalytic hammerhead ribozyme sequences, and a T7 terminator, optionally wherein the hammerhead ribozyme sequences is immediately upstream of the 3 ’ leader sequence.
43. The nucleic acid sequence of claim 41 or 42, wherein the luciferase is renilla luciferase or nanoluciferase.
44. The nucleic acid sequence of any one of claims 41 to 43, wherein the fluorescent protein is red fluorescent protein or enhanced green fluorescent protein.
45. A recombinant VSV particle pseudotyped with VSV glycoprotein comprising an encapsidated genome comprising a nucleotide sequence encoding for VSV nucleoprotein (N), a nucleotide sequence encoding for VSV matrix (M) protein, a nucleotide sequence encoding for VSV large (L) protein, a nucleotide sequence encoding for VSV phosphoprotein (P) proteins, a nucleotide sequence encoding for luciferase, and a nucleotide sequence encoding a fluorescent protein, wherein genome does not express VSV glycoprotein (G).
46. The recombinant VSV particle pseudotyped with VSV glycoprotein of claim 45, wherein the luciferase is renilla luciferase or nanoluciferase.
47. The recombinant VSV particle pseudotyped with VSV glycoprotein of claims 45 or 46, wherein the fluorescent protein is red fluorescent protein or enhanced green fluorescent protein.
48. A method for generating the recombinant VSV particle pseudotyped with VSV glycoprotein of any one of claims 45 to 47, comprising:
(a) transfecting cells with the nucleic acid sequence of any one of claims 41 to 44, a first vector comprising a nucleotide sequence encoding VSV M protein, a second vector comprising a nucleotide sequence encoding VSV L protein, a third vector encoding VSV N protein, a fourth vector comprising a nucleotide sequence encoding VSV G protein, and a fifth vector comprising a nucleotide sequence encoding VSV P protein; (b) purifying the recombinant VSV particle pseudotyped with VSV glycoprotein.
49. A method for generating pseudotyped VSV particle, comprising:
(a) infecting or spinoculating cells overexpressing viral surface protein of interest with the recombinant VSV particle of any one of claims 45 to 47; and
(b) purifying viral surface protein pseudotyped VSV particles from the supernatant of the cells.
50. The method of claim 48, the method further comprising transfecting the cells with a sixth vector comprising a codon-optimized sequence encoding a T7 polymerase.
51. The method of any one of clai s 48-50, wherein the cells are 293T-ACE2 clone 5-7 or 293T-ACE2-TMPRSS2 clone F8-2.
PCT/US2021/033713 2020-05-22 2021-05-21 Sars-cov-2 spike protein pseudotyped vsv-delta g particles and uses thereof WO2021237124A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202063029217P 2020-05-22 2020-05-22
US63/029,217 2020-05-22
US202063063041P 2020-08-07 2020-08-07
US63/063,041 2020-08-07

Publications (1)

Publication Number Publication Date
WO2021237124A1 true WO2021237124A1 (en) 2021-11-25

Family

ID=78707608

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/033713 WO2021237124A1 (en) 2020-05-22 2021-05-21 Sars-cov-2 spike protein pseudotyped vsv-delta g particles and uses thereof

Country Status (1)

Country Link
WO (1) WO2021237124A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060121580A1 (en) * 2003-07-22 2006-06-08 Crucell Binding molecules against SARS-coronavirus and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060121580A1 (en) * 2003-07-22 2006-06-08 Crucell Binding molecules against SARS-coronavirus and uses thereof

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BERGER RENTSCH MARIANNE, ZIMMER GERT: "A Vesicular Stomatitis Virus Replicon-Based Bioassay for the Rapid and Sensitive Determination of Multi-Species Type I Interferon", PLOS ONE, vol. 6, no. 10, 5 October 2011 (2011-10-05), pages e25858, XP055881124, DOI: 10.1371/journal.pone.0025858 *
ENGLAND CHRISTOPHER G ET AL: "NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence.", BIOCONJUGATE CHEMISTRY, vol. 27, no. 5, 18 May 2016 (2016-05-18), pages 1175 - 1187, XP002778324, ISSN: 1520-4812, DOI: 10.1021/acs.bioconjchem.6b00112 *
HOFFMANN MARKUS; KLEINE-WEBER HANNAH; SCHROEDER SIMON; KRüGER NADINE; HERRLER TANJA; ERICHSEN SANDRA; SCHIERGENS TOBIAS S.; H: "SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor", CELL, ELSEVIER, AMSTERDAM NL, vol. 181, no. 2, 5 March 2020 (2020-03-05), Amsterdam NL , pages 271, XP086136225, ISSN: 0092-8674, DOI: 10.1016/j.cell.2020.02.052 *
NIE ET AL.: "Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2", EMERG MICROBES INFECT, vol. 9, 24 March 2020 (2020-03-24), pages 680 - 686, XP055818011, DOI: 10.1080/22221751.2020.1743767 *
OGUNTUYO KASOPEFOLUWA Y., STEVENS CHRISTIAN S., HUNG CHUAN-TIEN, IKEGAME SATOSHI, ACKLIN JOSHUA A., KOWDLE SHREYAS S., CARMICHAEL : "Quantifying absolute neutralization titers against SARS-CoV-2 by a standardized virus neutralization assay allows for cross-cohort comparisons of COVID-19 sera", MEDRXIV, 27 August 2020 (2020-08-27), XP055881140, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430605/pdf/nihpp-2020.08.13.20157222.pdf> [retrieved on 20220119], DOI: 10.1101/2020.08.13.20157222 *
SHANNON M. BEATY, ARNOLD PARK, SOHUI T. WON, PATRICK HONG, MICHAEL LYONS, FREDERIC VIGANT, ALEXANDER N. FREIBERG, BENJAMIN R. TENO: "Efficient and Robust Paramyxoviridae Reverse Genetics Systems", MSPHERE, vol. 2, no. 2, 26 April 2017 (2017-04-26), pages e00376 - 16, XP055468664, DOI: 10.1128/mSphere.00376-16 *
WHITT ET AL.: "Generation of VSV pseudotypes using recombinant AG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines", J VIROL METHODS, vol. 169, 13 August 2010 (2010-08-13), pages 365 - 74, XP027338746 *

Similar Documents

Publication Publication Date Title
Hu et al. D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity
Neerukonda et al. Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2
Hu et al. Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2
Schmidt et al. Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses
Yu et al. Deletion of the SARS-CoV-2 spike cytoplasmic tail increases infectivity in pseudovirus neutralization assays
Oguntuyo et al. Quantifying absolute neutralization titers against SARS-CoV-2 by a standardized virus neutralization assay allows for cross-cohort comparisons of COVID-19 sera
Le Tortorec et al. Antagonism to and intracellular sequestration of human tetherin by the human immunodeficiency virus type 2 envelope glycoprotein
Qi et al. Rab11-FIP1C and Rab14 direct plasma membrane sorting and particle incorporation of the HIV-1 envelope glycoprotein complex
Ao et al. Importin α3 interacts with HIV-1 integrase and contributes to HIV-1 nuclear import and replication
Jiang et al. The interdomain linker region of HIV-1 capsid protein is a critical determinant of proper core assembly and stability
Yang et al. Second-site suppressors of HIV-1 capsid mutations: restoration of intracellular activities without correction of intrinsic capsid stability defects
Morozov et al. Single mutations in the transmembrane envelope protein abrogate the immunosuppressive property of HIV-1
Oguntuyo et al. In plain sight: the role of alpha-1-antitrypsin in COVID-19 pathogenesis and therapeutics.
Murray et al. A low-molecular-weight entry inhibitor of both CCR5-and CXCR4-tropic strains of human immunodeficiency virus type 1 targets a novel site on gp41
Mishra et al. SARS CoV-2 nucleoprotein enhances the infectivity of lentiviral spike particles
Diehl et al. Identification of postentry restrictions to Mason-Pfizer monkey virus infection in New World monkey cells
CN112138160A (en) Application of griffithsin in preparation of novel coronavirus infection medicine
WO2021237124A1 (en) Sars-cov-2 spike protein pseudotyped vsv-delta g particles and uses thereof
Moreno et al. A novel circulating tamiami mammarenavirus shows potential for zoonotic spillover
Ruiz et al. BST-2 mediated restriction of simian–human immunodeficiency virus
Afonso et al. Absence of accessory genes in a divergent simian T-lymphotropic virus type 1 isolated from a bonnet macaque (Macaca radiata)
WO2007122517A2 (en) Influenza h5 pseudotyped lentiviruses and uses thereof
Douglas et al. A comparative mutational analysis of HIV-1 Vpu subtypes B and C for the identification of determinants required to counteract BST-2/Tetherin and enhance viral egress
Stevens et al. Alpha-1-antitrypsin and its variant-dependent role in COVID-19 pathogenesis
Amberg et al. Expanded tropism and altered activation of a retroviral glycoprotein resistant to an entry inhibitor peptide

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21808471

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21808471

Country of ref document: EP

Kind code of ref document: A1