WO2022055894A1 - Sars-cov-2 spike glycoprotein for virus generation and pseudotyping - Google Patents

Abstract

In various embodiments, a spike glycoprotein pseudotyped non-replicative viral particle is provided. The viral particle comprises a modified SARS-CoV-2 spike glycoprotein. In certain embodiments, the viral particle is capable of specifically infecting ACE2 expressing cells. In certain embodiments, the viral particle finds utility in neutralization studies, vaccine development, drug screening, antibody testing, and the like.

Description

SARS-COV-2 SPIKE GLYCOPROTEIN FOR VIRUS GENERATION

AND PSEUDOTYPING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of USSN 63/076,227, filed on September 9, 2020, and USSN 63/075,703, filed on September 8, 2020, both of which are incorporated herein by reference in their entirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

[ Not Applicable 1

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

[0002] [ Not Applicable ]

BACKGROUND

[0003] Human coronaviruses (CoV) are enveloped, positive stranded RNA viruses of the family of Coronaviridae (V’kovski et al., 2020). Three coronaviruses within the past two decades were transmitted from animals to humans to cause severe respiratory diseases in afflicted individuals: the 2002 severe acute respiratory syndrome coronavirus (SARS-CoV), the 2012 Middle East respiratory syndrome coronavirus (MERS-CoV), and most recently, the 2019 SARS- CoV-2 (V’kovski et al., 2020). Although researchers have raced to develop safe and efficacious vaccines to prevent SARS-CoV-2 spread (see, e.g., Poland et al., 2020), the virus has resulted in over 1.5 million deaths worldwide since the inception of its pandemic spread.

[0004] The spike (S) glycoprotein of coronavirus mediates viral entry into target cells after engaging with the cell surface receptor angiotensin converting enzyme 2 (ACE2) (Hoffmann et al., 2020). In addition to the prevalence of ACE2 receptors, various factors affect the potency of SARS-CoV-2 entry and transmission. One such factor includes the availability of proteases in target cells, such as the transmembrane protease serine 2 (TMPRSS2) (Hoffmann et al., 2020) or furin. TMPRSS2 facilitates S protein priming of SARS-Cov-2 to promote fusion of viral and cellular membranes to the target cell. Similarly, furin, a ubiquitous protease that activates a variety of viruses such as influenza A, HIV, Ebola, or measles (Bestie et al., 2020), cleaves SARS-CoV-2 S protein at the S1/S2 site to facilitate infection. A viral factor affecting potency includes the D614G substitution in the spike glycoprotein, which has quickly become the most prevalent form of SARS-Cov-2. The dominance of the D614G variant is attributed to its increased viral infectivity and transmission (Yurkovetskiy et al., 2020).

[0005] Despite the progressive development of vaccines and therapeutics to treat SARS-CoV-2, there is an unmet need for a safer alternative to infectious SARS-CoV-2 virus for research studies quantifying neutralizing antibody (or other therapeutic) activity, developing high-throughput drug screens, or performing animal studies. By “pseudotyping” a virus, one can effectively replace the envelope protein of a virus with that of another virus to limit or broaden its targeting capabilities (Verhoeyen et al., 2004). Recent studies demonstrated that the spike glycoprotein from SARS- CoV-2 can be “pseudotyped” onto replication defective viral particles such as HIV-based lentiviral particles, MLV-based retroviral particles, or Vesicular Stomatitis Virus (VSV) (see, e.g., Crawford et al., 2020). As a result, generating S glycoprotein pseudotyped non-replicative viral particles capable of specifically infecting ACE2 expressing cells will avoid the need for a biosafety-level-3 facility and expand the capabilities for studies of SARS-CoV-2.

[0006] A major hurdle with this approach, however, stems from the low titer of S- pseudotyped vectors (see, e.g., Nie et al., 2020; Tandon et al., 2020).

SUMMARY

[0007] Previous groups have shown that modifications to the cytoplasmic tail (CT) domain of viruses can increase their lentiviral vector pseudotype efficiency (Girard Gagnepain et al., 2014; Christodoulopoulos and Cannon, 2001; Sandrin et al., 2002; Schnierle et al., 1997; Sandrin and Cosset, 2006). For example, researchers have attempted to increase the infectious titers of lentiviral vectors pseudotyped by the gibbon ape leukemia virus (GaLV) by adjusting its CT (Sandrin et al., 2002; Tomas et al., 2019). The GaLV envelopes were modified to harbor the CT from MLV-A thereby increasing infectious titers by 25 -fold.

[0008] Described herein is a strategy to generate an S glycoprotein, with a modified CT, capable of pseudotyping a non-replicative 3rd generation HIV-1 lentiviral vector with a titer 3 -logs greater than its unmodified counterpart.

[0009] In various embodiments, a spike glycoprotein pseudotyped non-replicative viral particle is provided. The viral particle comprises a modified SARS-CoV-2 spike glycoprotein. In certain embodiments, the viral particle is capable of specifically infecting ACE2 expressing cells. In certain embodiments, the viral particle finds utility in neutralization studies, vaccine development, drug screening, antibody testing, and the like.

[0010] Various embodiments provided herein may include, but need not be limited to, one or more of the following:

[0011] Embodiment 1 : An S glycoprotein capable of pseudotyping a non-replicative

3rd generation HIV-1 lend viral vector.

[0012] Embodiment 2: The S glycoprotein of embodiment 1 , wherein said nucleic acid is codon optimized for expression in mammalian cells.

[0013] Embodiment 3: The S glycoprotein according to any one of embodiments 1-2, wherein said S glycoprotein comprises a glycine at position 614 of the spike protein instead of aspartic acid (D614G).

[0014] Embodiment 4: The S glycoprotein according to any one of embodiments 1-3, wherein spike protein comprises a cytoplasmic tail wherein said tail (aa 1240-1273) is replaced with that of the influenza hemagglutinin cytoplasmic tail (amino acids: NGSLQCRICI, SEQ ID NOG).

[0015] Embodiment 5: The S glycoprotein according to any one of embodiments 1-4, wherein spike protein comprises the amino acid sequence of SEQ ID NOG.

[0016] Embodiment 6: The S glycoprotein according to any one of embodiments 1-5, wherein said spike protein is disposed on a lentiviral vector.

[0017] Embodiment 7: The S glycoprotein of embodiment 6, wherein said lentiviral vector comprises a non-replicative 3rd generation HIV-1 lentiviral vector.

[0018] Embodiment 8: An expression cassette comprising a nucleic acid that encodes a modified SARS-CoV-2 spike protein.

[0019] Embodiment 9: The expression cassette of embodiment 8 , wherein said nucleic acid is codon optimized for expression in mammalian cells.

[0020] Embodiment 10: The expression cassette according to any one of embodiments 8-9, wherein said nucleic acid encodes glycine at position 614 of the spike protein instead of glycine (D614G).

[0021] Embodiment 11: The expression cassette according to any one of embodiments 8-10, wherein said nucleic acid encodes a spike protein wherein the cytoplasmic tail of the spike glycoprotein (aal240-1273) is replaced with that of the influenza hemagglutinin cytoplasmic tail (amino acids: NGSLQCRICI, SEQ ID NO: 3).

[0022] Embodiment 12: The expression cassette according to any one of embodiments 8-11, wherein said nucleic acid encodes a spike protein comprising the amino acid sequence of SEQ ID NO: 5.

[0023] Embodiment 13: The expression cassette of embodiment 12, wherein said nucleic acid sequence comprises the spike-encoding nucleic acid sequence shown in Figure 2 (the spike-encoding region of SEQ ID NO:1).

[0024] Embodiment 14: The expression cassette according to any one of embodiments 8-13, wherein said nucleic acid encoding a modified SARS-CoV-2 spike protein is provided in a plasmid where said nucleic acid is downstream from a CMV promoter and P-globin intron and upstream of a P-globin polyA.

[0025] Embodiment 15: The expression cassette according to any one of embodiments 8-14, wherein said expression cassette is packaged into a lentiviral particle.

[0026] Embodiment 16: The expression cassette of embodiment 15, wherein said expression cassette is packaged into a 3rd generation HIV-1 lentiviral particle.

[0027] Embodiment 17: The expression cassette of embodiment 16, wherein said lentiviral particle further comprises a reporter gene.

[0028] Embodiment 18: The expression cassette of embodiment 17, wherein said lentiviral particle comprises a GFP reporter gene.

[0029] Embodiment 19: A spike glycoprotein pseudotyped non-replicative viral particle wherein:

[0030] said viral particle comprises a modified SARS-CoV-2 spike glycoprotein; and

[0031] said viral particle is capable of specifically infecting ACE2 expressing cells.

[0032] Embodiment 20: The viral particle of embodiment 19, wherein said viral particle is an HIV lentiviral particle.

[0033] Embodiment 21: The viral particle of embodiment 20, wherein said viral particle is a third generation HIV 1 lentiviral particle. [0034] Embodiment 22: The viral particle according to any one of embodiments 19-

21, wherein said viral particle comprises an expression cassette according to any one of embodiments 8-14.

[0035] Embodiment 23: The viral particle according to any one of embodiments 19-

22, wherein said viral particle further comprises a reporter gene.

[0036] Embodiment 24: The viral particle of embodiment 23, wherein said viral particle comprises a GFP reporter gene.

[0037] Embodiment 25 : A method of evaluating a therapeutic agent for efficacy against SARS-CoV-2 virus, said method comprising:

[0038] contacting cells expressing the cell surface receptor angiotensinconverting enzyme 2 (ACE2) with a pseudotyped virus according to any one of embodiments 19-24;

[0039] contacting said cells with said therapeutic agent; and

[0040] determining the amount and/or rate of infection of said cells by said pseudotyped virus where the amount and/or rate of infection provides a measure of the efficacy of said therapeutic agent where reduced amount and/or rate of infenction as compared to a control without said therapeutic agent indicates efficacy of said therapeutic agent.

[0041] Embodiment 26: The method of embodiment 25, wherein said reduced amount and/or rate of infection is a statistically significant reduced amount and/or rate to indicate efficacy.

[0042] Embodiment 27: The method according to any one of embodiments 25-26, wherein said determining comprise detecting a reporter gene expressed by said pseudotyped virus.

[0043] Embodiment 28: The method according to any one of embodiments 25-27, wherein said determining comprises visualizing expression of a reporter gene.

[0044] Embodiment 29: The method of embodiment 28, wherein said reporter gene comprises a GFP gene or an mCitrulline gene.

[0045] Embodiment 30: The method according to any one of embodiments 25-29, wherein said determining comprises quantifying said virus by PCR.

[0046] Embodiment 31 : The method according to any one of embodiments 25-30, wherein said therapeutic agent comprise an anti-SARS-CoV-2 antibody. [0047] Embodiment 32: The method according to any one of embodiments 25-31, wherein said therapeutic agent comprises plasma derived from a subject this is or that has been infected with SARS-CoV-2.

[0048] Embodiment 33: The method according to any one of embodiments 25-32, wherein said cells comprise mammalian cells transfected with a construct that expresses said angiotensin-converting enzyme 2 (ACE2).

[0049] Embodiment 34: The method of embodiment 33, wherein said cells are HEK293T cells.

[0050] Embodiment 35: A method of evaluating the efficacy of a vaccine directed against SARS-CoV-2, said method comprising:

[0051] contacting cells expressing the cell surface receptor angiotensinconverting enzyme 2 (ACE2) with a pseudotyped virus according to any one of embodiments 19-24;

[0052] contacting said cells with plasma derived from subjects inoculated with said vaccine; and

[0053] determining the amount and/or rate of infection of said cells by said pseudotyped virus where the amount and/or rate of infection provides a measure of the efficacy of said vaccine where reduced amount and/or rate of infenction as compared to a control without said vaccine indicates efficacy of said vaccine.

[0054] Embodiment 36: The method of embodiment 35, wherein said reduced amount and/or rate of infection is a statistically significant reduced amount and/or rate to indicate efficacy.

[0055] Embodiment 37: The method according to any one of embodiments 35-36, wherein said determining comprise detecting a reporter gene expressed by said pseudotyped virus.

[0056] Embodiment 38: The method according to any one of embodiments 35-37 wherein said determining comprises visualizing expression of a reporter gene.

[0057] Embodiment 39: The method of embodiment 38, wherein said reporter gene comprises a GFP gene or an mCitrulline gene.

[0058] Embodiment 40: The method according to any one of embodiments 35-39, wherein said determining comprises quantifying said virus by PCR. [0059] Embodiment 41: The method according to any one of embodiments 35-40, wherein said cells comprise mammalian cells transfected with a construct that expresses said angiotensin-converting enzyme 2 (ACE2).

[0060] Embodiment 42: The method of embodiment 41, wherein said cells are HEK293T cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 schematically illustrates construct encoding modified SARS-CoV-2 spike.

[0062] Figure 2 provides the nucleic acid sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO: 2) for the construct of Figure 1.

[0063] Figure 3 shows raw titer value of various spike pseudotypes.

[0064] Figure 4 shows % GPR 3 days post transduction.

[0065] Figure 5 illustrates a schematic of modifications used to generate each of our spike glycoprotein variants.

[0066] Figure 6 shows expression of spike lentiviral pseudotypes in ACE-293t and HEK-293t cells. 1E5 of ACE-293T and HEK-293T cells were transduced with a series of spike glycoproteins. Three days post transduction, cells were measure for %GFP and GFP MFI via FLOW Cytometry to assess pseudotype functionality and expression.

[0067] Figure 7 shows expression of combined modifications of spike lentiviral pseudotypes in ACE-293T and HEK-293T cells. 1E5 of ACE-293T and HEK-293T cells were transduced with a 1:10 dilution of raw virus from a series of spike glycoproteins. Three days post transduction, cells were measured for %GFP and GFP MFI via FLOW Cytometry to assess pseudotype expression.

[0068] Figure 8 shows infectious titer of combined modifications of spike lentiviral pseudotypes in ACE-293T and HEK-293T cells. 1E5 of ACE-293T and HEK-293T cells were transduced with a 1:10 dilution of raw virus from a series of spike glycoproteins. Three days post transduction, genomic DNA was extracted, and titer (transducing units/mL) was measured by quantifying VCN from GFP integrants via digital-droplet PCR.

[0069] Figure 9, panels A-E, illustrates the experimental overview and spike functionality. Schematic of the spike glycoprotein variants (panel A), vector packaging (panel B), and transduction (panels C-E) experiments. Panel A) The S glycoproteins were cloned into the pMD2.G plasmid backbone (Addgene plasmid #12259) which is driven by a CMV promoter and includes a beta-globin intron and beta-globin polyA. The three variants include codon optimization (IDT or Ou), an amino acid substitution (D614G), and cytoplasmic tail modifications (wildtype tail, mutated ALAYT, influenza HA tail, and murine leukemia virus MLV tail). Panel B) The spike (S) glycoprotein variants were packaged in PKR-/- HEK-293T cells alongside plasmids encoding for a 3^rd generation HIV-1 lentivirus and a plasmid encoding for an eGFP transgene (pCCL-MNDU3-eGFP, Dull et al. 1998 Logan et al., 2004). Raw viral supernatants were harvested 3 days post transfection and stored at -80C for future use. Panels C-E) 100,000 ACE2-expressing 293T cells (ACE-293T) and HEK-293T cells were transduced with raw virus at equal amounts of p24 lentiviral particles from a series of S pseudotyped lentiviral vectors containing an MNDU3-eGFP reporter cassette. Three days post transduction, cells were harvested and measured for %GFP (panel C) via flow cytometry to assess pseudotype functionality and expression. Panels D and E illustrate the infectivity of spike pseudotype vectors. To measure infectivity, genomic DNA was extracted, and vector copy number (VCN) was measured (panel D) by quantifying GFP integrants via QX200 Droplet Digital PCR System. The infectious titer (transducing units/mL) of each vector was determined (panel E) from the VCN shown in panel D. All data sets were compared to a non-transduced control (NTC) and a VSV-G pseudotyped lentiviral vector containing an MNDU3-eGFP reporter cassette. Data are represented as mean ± SD of biological triplicates from n=3 experiments. Statistical significance was analyzed using oneway ANOVA followed by multiple paired comparisons for normally distributed data (Tukey test). All statistical tests were two-tailed and a p value of < 0.05 was deemed significant (ns non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.)

[0070] Figure 10, panels A-B, illustrates the relationship between spike protein and infectious titer. Spike viral supernatant was used to quantify Spike protein and HIV-1 p24 lentiviral particles via an ELISA assay. To measure spike protein, a 1/1000 dilution of viral supernatant was bound to a pre-coated ELISA plate measuring the RBD domain of the spike glycoprotein (MyBioSource Catalog# MBS9141957). Following a series of steps described by MyBioSource, spike protein was quantified on a Tecan microplate reader (Tecan Trading AG, Mannedorf, Switzerland) by measuring optical density at 450nm. To measure HIV-1 p24 particles, a 1/1000 dilution of viral supernatant was bound to a Perkin Elmer Alliance HIV-1 p24 kit plate (cat# NEK050) to detect p24 antigen at a range of 0.31ng/mL to 4 ng/mL. P24 concentration across all pseudotyped lentiviral vectors contained a coefficient of variation of 10 percent. Panel A illustrates the ratio of Spike protein measured via ELISA to HIV-1 p24 particles measured via ELISA (panel A) after normalization. Panel B demonstrates the relationship of Spike protein/p24 to infectious titer. Statistical significance (panel B) was analyzed using an unpaired t-test. All statistical tests were two-tailed and a p value of < 0.05 was deemed significant. We detected no significance when comparing the Ratio of Spike/p24 to Titer (TU/mL).

[0071] Figure 11, panels A-B, illustrates the effect of TMPRSS2 expression on pseudotype functionality. 100,000 VeroE6, and VeroE6/TMPRSS2 cells were transduced with raw viral supernatant containing equal amounts of p24 lentiviral particles from a series of pseudotyped lentiviral vectors containing an MNDU3-eGFP reporter cassette. Panels A and B illustrate the effect of TMPRSS2 expression on spike pseudotype functionality. Three days post transduction, cells were harvested and measured for (panel A) GFP percentage and (panel B) vector copy number (VCN). GFP percentage was measured via flow cytometry. To measure infectivity, genomic DNA was extracted to measure VCN by quantifying GFP integrants via QX200 Droplet Digital PCR System. All data sets were compared to a nontransduced control (NTC) and a VSV-G pseudotyped lentiviral vector containing an MNDU3-eGFP reporter cassette. Data are represented as mean ± SD of biological triplicates from two experiments. We analyzed statistical significance using a two-way ANOVA followed by multiple paired comparisons for normally distributed data (Tukey test). All statistical tests were two-tailed and a p value of < 0.05 was deemed significant (ns nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.)

[0072] Figure 12, panels A-C, illustrates spike pseudotype concentration by tangential filtration. 100,000 ACE-293T and HEK-293T cells were transduced with a 1:10 dilution of raw virus or a 1:10,000 dilution of TFF concentrated virus from a VSV-G or Spike (Ou- D614G-HA) pseudotyped lentiviral vector containing an MNDU-3-eGFP reporter cassette. Three days post transduction, cells were harvested and measured for %GFP (panel A) via flow cytometry to assess pseudotype functionality and expression. Panels B and C illustrate the infectivity of spike pseudotype vectors. To measure infectivity, genomic DNA was extracted to measure vector copy number (VCN) (panel B) by quantifying GFP integrants via QX200 Droplet Digital PCR System. The infectious titer (transducing units/mL) of each vector was determined (panel C) from the VCN shown in panel B. All data sets were compared to a non-transduced control (NTC) and a VSV-G pseudotyped lentiviral vector containing an MNDU3-eGFP reporter cassette. Data are represented as mean ± SD of biological triplicates from one experiment. [0073] Figure 13, panels A-B, illustrates pseudotype expression with a UBC-mCitrine Reporter. 100,000 ACE2-expressing 293T cells (ACE-293T) and HEK-293T cells were transduced with raw virus at equal amounts of p24 lentiviral particles from a series of pseudotyped lentiviral vectors containing a UBC-mCitrine reporter cassette. Three days post transduction, cells were harvested and measured for (panel A) %GFP and (panel B) GFP mean fluorescent intensity (MFI) via flow cytometry to assess pseudotype functionality and expression. All data sets were compared to a non-transduced control (NTC) and a VSV- G pseudotyped lentiviral vector containing an UBC-GFP reporter cassette. Data are represented as mean ± SD of biological triplicates from one experiment.

DETAILED DESCRIPTION

[0074] Due to the requirement of infectious, replication-competent SARS-CoV-2, there remains an unmet need for a high-throughput method to quantify neutralizing antibody activity or the activity of other therapeutic agents against SAR-CoV-2.

[0075] To facilitate antibody (or other therapeutic agent) screening, provided herein are a spike glycoprotein pseudotyped nonreplicative viral particles capable of specifically infecting ACE2 expressing. The viral particles can be utilized in a variety of applications including, but not limited to quantifying the neutralization activity of human plasma and/or synthetic antibodies. More importantly, the pseudotyped viral particle described herein eliminate the need for a biosafety-level-3 requirement.

[0076] Described herein is a strategy to generate a spike glycoprotein capable of pseudotyping a nonreplicative 3rd generation HIV-1 lentiviral vector at higher viral titers (transducing units/mL) than that of its wildtype counterpart. Many groups have shown that modifications to the cytoplasmic tail domain of virus envelop proteins can increase their lentiviral vector pseudotype efficiency. As such, we constructed our spike glycoprotein as follows: First, the spike glycoprotein was codon optimized as denoted in Ou et al. (2020) Nat. Comm., 11: 1620 /doi.org/10.1038/s41467-020-15562-9 (see, e.g., spike-encoding region of SEQ ID NO:1 shown in Figure 2). Second, we substituted the aspartic acid at the 614th position of the protein to a glycine (D614G). Lastly, we replaced the cytoplasmic tail of the spike glycoprotein (aal240-1273) with that of the influenza hemagglutinin cytoplasmic tail (amino acids: NGSLQCRICI, (SEQ ID NOG).

[0077] We constructed a plasmid that includes this spike glycoprotein downstream of a CMV promoter and P-globin intron and upstream of a P-globin polyA. We packaged spike glycoprotein into 3rd generation HIV-1 lentiviral particles with a GFP reporter cassette and tested its transduction capability and specificity in HEK-293T cells expressing ACE2. Our construct has a 50-fold higher GFP expression than that of wildtype spike pseudotyped lend virus (see, e.g., Figure 4). Furthermore, this combination increases titer one log greater than wildtype, all while retaining high specificity to ACE2 expressing cells (see, e.g., Figure 3). The combination of these three modifications is important in further understanding the development of immunity against SARS-CoV-2 that harbors the D614G mutation, a dominant and highly infectious variant of the virus. Moreover, our pseudotyped vector eliminates the need for biosafety-level-3 laboratories when developing therapeutics against SARS-CoV-2. These therapeutics include, but are not limited to, neutralization studies, vaccine development, drug screening, and antibody testing.

[0078] In certain embodiments uses of the pseudotype lentiviral particles are provided. Thus, for example, in certain embodiments, a method of evaluating a therapeutic agent (e.g., a putative therapeutic agent) for efficacy against SARS-CoV-2 virus is provided where the method involves contacting cells expressing the cell surface receptor angiotensinconverting enzyme 2 (ACE2) with a pseudotyped virus as described herein, contacting the cells with said therapeutic agent; and determining the amount and/or rate of infection of the cells by the pseudotyped virus where the amount and/or rate of infection provides a measure of the efficacy of said therapeutic agent where reduced amount and/or rate of infenction as compared to a control without said therapeutic agent indicates efficacy of said therapeutic agent.

[0079] In certain embodiments methods of using the pseudotyped lentiviral vectors described herein for evaluating the efficacy of a vaccine directed against SARS-CoV-2, are provided where the methods involve contacting cells expressing the cell surface receptor angiotensin-converting enzyme 2 (ACE2) with a pseudotyped virus described herein, contacting the cells with plasma derived from subjects inoculated with the vaccine; and determining the amount and/or rate of infection of said cells by said pseudotyped virus where the amount and/or rate of infection provides a measure of the efficacy of said vaccine where reduced amount and/or rate of infenction as compared to a control without said vaccine indicates efficacy of said vaccine.

[0080] Methods of conducting such assays are well known to those of skill in the art. Thus, for example, methods of performing viral entry and/neutralization assays are described by Capcha et al. (2021) Front. Cardiovasc. Med. 7: 381 doi.org/10.3389/fcvm.2020.618651, Khoury et al. (2020) Nat. Rev. Immunol., doi: 10.1038/s41577-020-00471-l; and the like. [0081] Using the pseudotyped viral particles described herein numerous assays will be available to one of skill in the art.

Kits.

[0082] In certain embodiments, kits are provided for use of the pseudotype viral particles described herrein. In certain embodiments the kits comprise a container containing an expression cassette that expresses a modified SARS-CoV-2 spike protien as described herein. In certain embodiments the kits comprise a container containing a viral vector (e.g., a lentiviral vector) pseudotypes with a modified spike protien as described herein. In certain embodiments the kits optionally further include cells (e.g., HEKT cells) that express a cell surface receptor angiotensin-converting enzyme 2 (ACE2).

[0083] In addition, the kits optionally include labeling and/or instructional materials providing directions (i.e., protocols) for the of the expression casssette and/or the pseudotyped viral particles described herein, e.g., in viral entry and/or neutralization assays.

[0084] While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

EXAMPLES

[0085] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1

Improved Spike Glycoproteins to Pseudotype Lentiviral Vectors.

Introduction:

[0086] The spike (S) glycoprotein of SARS-Cov-2 facilitates viral entry into target cells through the cell surface receptor angiotensin-converting enzyme 2 (ACE2). Third generation HIV-1 lentiviral vectors can be pseudotyped to replace the envelope protein of the virus and thereby either limit or expand the target cell population. We generated A pseudotyped lentiviral vector enveloped by a modified S glycoprotein of SARS-Cov-2 capable of infecting ACE2 expressing cells more efficiently than that of wildtype S glycoprotein.

Methods:

[0087] We generated a 3^rd generation HIV-1 pseudotyped lentiviral vector enveloped by Spike glycoprotein with an mCitrine transgene cassette. Prior research has suggested that multiple viral envelope proteins can pseudotype lentiviral vectors more effectively, such as that of influenza hemagglutinin (HA) and the murine leukemia virus cytoplasmic tail. As a result, many variables were considered when designing these new glycoproteins including 1) cytoplasmic tail modifications 2) mutations and 3) codon optimizations. Spike variants were packaged into lentiviruses with encoding for an mCitrine transgene cassette. ACE2 expressing cells were transduced with the spike pseudotyped lentiviral particles. The effectiveness of each glycoprotein variant was measured for its infectious particles, its efficiency of particle formation, and its particle functionality. These modifications led us to generate a candidate vector with better infectivity and expression than previous iterations.

Results:

[0088] Using our pseudotyped lentiviruses driving an mCitrine reporter, we demonstrated a variant capable of achieving 10-fold higher expression in ACE2 expressing cells than wildtype spike glycoprotein. Calu-3 and ACE-293T cells were transduced with each pseudotyped vector. Transducing units per mL (TU/mL) were measured by ddPCR to quantify the infectious particles of each variant (see, e.g., Figure 3). Furthermore, GFP percentage and MFI was measured by FACS to quantify vector functionality and expression. Finally, spike protein and p24 was measured by EEISA to quantify the efficiency of particle formations and to rule out bald particles (see, e.g., Figure 4).

Conclusion:

[0089] Our pseudotyped lentiviral vector with a modified spike glycoprotein demonstrated 10-fold higher expression than pseudotyped wildtype spike glycoprotein. This pseudotyped vector can therefore be used to preform neutralization studies and aid in development of vaccines or other treatments against SARS-Cov-2.

[0090] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

EXAMPLES

[0091] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1

Improved Spike Glycoproteins for Pseudotyping Lentiyral Vectors

[0092] The spike (S) glycoprotein of SARS-Cov-2 facilitates viral entry into target cells via the cell surface receptor angiotensin-converting enzyme 2 (ACE2). Third generation HIV-1 lenti viral vectors can be pseudotyped to replace the envelope protein of the virus and thereby either limit or expand the target cell population. As described in this Example, we generated a pseudotyped lentiviral vector enveloped by a modified S glycoprotein of SARS- Cov-2 capable of infecting ACE2 expressing cells. Through the modification of the cytoplasmic tail domain, and the inclusion of a mutant form of the spike protein (D614G), we generated a construct that achieved a titer (TU/mL) 2-log fold higher than that of its wildtype counterpart.

Methods.

[0093] To generate the most effective spike pseudotyped lentiviral vectorcapable of specifically transducing ACE2 expressing cells, we generated a series of glycoproteins with three main changes, codon optimizations, an amino acid substitution (D614G), and cytoplasmic tail (CT) modifications. The cytoplasmic tail modifications include mutating at the C terminus of the CT or replacing the wildtype CT with that of murine leukemia virus or influenza virus (Figure 5).

[0094] The spike (S) glycoprotein variants were packaged with plasmids encoding for a 3rd generation HIV-1 lentivirus and a plasmid encoding for a eGFP transgene (MND-eGFP, Addgene plasmid #36247). Raw viral supernatants were harvest 3 days post transduction and stored at -80°C for future use (see, e.g., Figure 9, panel B).

[0095] To quantify functionality and specificity of each pseudotype, we measured titer, GFP%, and GFP MFI of 293 T cells expressing ACE2 transduced with our lentiviral vectors. ACE-293T and HEK-293T cells were plated at le5 in a 6 well plate. 24 hours after plating, a 1:10 dilution of raw virus was used to transduce the cells. At 3 days post transduction, the cell pellet was harvested. Half the pellet was used to pseudotype functionality by measuring percent GFP and GFP MFI via flow cytometry (Figures 6, 7).

The other half of the pellet was used to assess titer (transducing units/mL) by measuring GFP integrants via digital-droplet PCR (Figure 8).

[0096] Raw Viral Supernatant was also used to measure p24 particles via an ELISA to quantify the efficiency of particle formation. After assessing particle formation, spike protein was measured from our raw viral supernatant to quantify spike protein relative to total lentiviral particles to quantify formation of empty particles, or those with an HIV capsid but no spike envelope protein.

Conclusions

[0097] The combination of codon optimization, D614G mutation, and influenza HA cytoplasmic tail increases titer nearly 1 log greater than wildtype, all while retaining high specificity to ACE2 expressing cells. The combination of these three modifications is critical in further understanding the development of immunity against SARS-CoV-2 that harbors the D614G mutation. Moreover, our pseudotyped vector eliminates the need for biosafety-level- 3 laboratories when developing therapeutics against SARS-CoV-2. These therapeutics include, but are not limited to, neutralization studies, vaccine development, drug screening, and antibody testing.

Example 2 Improved Spike Glycoproteins for Pseudotyping Lentiviral Vectors

[0098] The spike (S) glycoprotein of SARS-Cov-2 facilitates viral entry into target cells via the cell surface receptor angiotensin-converting enzyme 2 (ACE2). Third generation HIV-1 lentiviral vectors can be pseudotyped to replace the native CD4 tropic envelope protein of the virus and thereby either limit or expand the target cell population. As described in this example, we generated a modified S glycoprotein of SARS-Cov-2 to pseudotype lentiviral vectors which efficiently transduced ACE2- expressing cells with high specificity and contain minimal off-target transduction of ACE-2 negative cells. By utilizing optimized codons, modifying the S cytoplasmic tail domain, and including a mutant form of the spike protein (D614G), we generated an expression plasmid encoding the optimized protein that produces S pseudotyped lentiviral vectors at an infectious titer (TU/mL) 1000- fold higher than the unmodified S protein. S pseudotyped replication-defective lentiviral vectors eliminate the need for biosafety-level-3 laboratories required when developing therapeutics against SARS-CoV-2 with live infectious virus. Furthermore, S pseudotyped vectors with high activity and specificity may be used as tools to understand the development of immunity against SARS-CoV-2, to develop assays of neutralizing antibodies and other agents that block viral binding, and to allow in vivo imaging studies of ACE-2 expressing cells.

Results.

[0099] To achieve the most effective S pseudotyped lentiviral vector, we modified the SARS-CoV-2 S glycoprotein gene to generate a series of envelope expression plasmids for transfection experiments. The modifications included various codon optimizations, an amino acid substitution, and CT modifications (Figure 9, panel A). As such, the S glycoprotein was codon optimized using the Integrated DNA Technologies algorithm, denoted “IDT,” or using the codon optimization described by a Ou et al., denoted “Ou” (Ou et al., 2020). We also modified the constructs to include an aspartic acid to glycine substitution at the 614^th amino acid position of the S protein. This mutation reproduces the D614G SARS-CoV-2 variant that has rapidly become the dominant form around the world ( Yurko vetskiy et al., 2020). Recent research has demonstrated that this strain exhibits increased competitive fitness and infectivity thereby increasing transduction of ACE2 expressing cells.

[0100] We modified the spike CT to introduce amino acid substitutions into its C terminus or replace it with the CT of the influenza A or murine leukemia virus (Figure 9, panel A). Various groups have shown that mutating the five most C-terminal residues of the S glycoprotein eliminates the endoplasmic reticulum retention signal and improves surface expression of S pseudovirions (Mcbride et al., 2006; Crawford et al., 2020). This mutant, termed “AEAYT,” replaces K1269 and H1271 with alanines (A). Next, we replaced the spike CT with that of the influenza A virus hemagglutinin (HA) glycoprotein. Given that SARS-Cov-2, influenza HA, and HIV-1 are homotrimeric class I fusion glycoproteins (Du et al., 2019; V’kovski et al., 2020; Tortorici et al., 2019), we reasoned that the influenza CT may pseudotype HIV-1 lentiviral vectors more effectively than that of SARS-CoV-2.

Eentiviral vectors pseudotyped with GAEV, RD114, or baboon envelopes have also shown increased stability and transduction when their native CTs are replaced with the MLV cytoplasmic tail (Sandrin et al., 2002; Girard Gagnepain et al., 2014). As a result, we replaced the spike CT with that from the murine leukemia virus (MLV) glycoprotein.

[0101] We generated the SARS-CoV-2 pseudotyped virus using a 3^rd generation HIV- 1 packaging system (9, panel B). We transfected PKR -I- 293T cells (Han et al., 2020) with plasmids encoding for HIV-1 proteins, Gag- Pol and Rev; a plasmid encoding for the spike glycoprotein envelope; and a lentiviral transfer plasmid containing an eGFP reporter cassette. The S glycoprotein on the produced lentivirus should improve specificity towards ACE2 expressing cells. Infected cells can be measured by means of the eGFP reporter driven by the MNDU3 promoter, an enhancer/synthetic promoter that contains the U3 region of the Myeloproliferative Sarcoma Virus long terminal repeat (Logan et al., 2004).

[0102] We first examined the effect of glycoprotein modifications on viral expression and functionality. ACE2 expressing 293T cells (ACE-293T) or parental 293T cells that do not express ACE2 (HEK- 293T) were transduced with the series of lentiviral vectors. Three days post transduction cells were harvested to quantify vector expression and infectivity. All spike pseudotyped lentiviral vectors exhibited specificity to ACE2 expressing cells with essentially no off-target transduction of the parental HEK-293T cells that lack expression of ACE2 (9, panel C). Furthermore, Ou codon optimized variants presented greater infectivity than their IDT counterparts. The inclusion of the D614G mutant or the HA tail increased infectivity by 10-fold, whereas the ALAYT amino acid modifications had no impact on pseudotyping efficiency.

[0103] When combined, the D614G mutant and HA tail achieved a 50-fold synergistic effect of the percentage of cells expressing GFP, a 1000-fold increase of vector copy number, and a mid-10⁷ titer - 3 logs greater than its unmodified spike counterpart (Figure 9, panels C-E). In contrast, the MLV tail impeded pseudotyping efficiency. When combined with the D614G mutant, the MLV tail reduced expression and infectivity of the pseudotyped virus in comparison to the D614G mutant alone (Figure 9, panels C and E). The S pseudotyped vector also supports large scale concentration (Figure 12) to nearly 1000-fold by means of tangential flow filtration (Cooper et al., 2011). Ultimately, these data demonstrate that the combination of the Ou codon optimization, the D614G mutation, and the HA cytoplasmic tail drastically increased pseudotyping efficiency of lentiviruses with the spike glycoprotein.

[0104] It is also worth noting the reduction in transduction activity assessed by GFP expression across all spike pseudotyped variants compared to the VSV-G control (Figure 9, panel C). Even with a VCN of 0.1, various pseudotyped lentiviruses exhibited negligible GFP percentage (<5%) and GFP mean fluorescent intensity (data not shown) measured by flow cytometry. This reduced expression may be attributed to the MNDU3 promoter driving the GFP reporter, as it is a relatively weak expressing promoter within 293T cell lines. This is evidenced by the increase in expression when we packaged the spike pseudotyped vectors with a GFP reporter cassette driven by the ubiquitin C promoter (Figure 13). These UBC- mCitrine spike pseudotyped vectors had greater GFP expression in ACE-293T cells in comparison to the MNDU3-eGFP pseudotyped variants after transduction at equal p24.

[0105] We next assessed how the different variants of the S glycoprotein altered S protein concentration in pseudotyped lentiviral virions and, subsequently, how these changes in S protein concentrations affected infectivity. The lentiviral supernatants containing each pseudotyped construct were used to quantify S protein content. The S receptor binding domain was detected within viral supernatant via an ELISA assay and normalized to the levels of HIV-1 p24 Gag protein (Figure 10, panel A). Concentration of p24 across all pseudotyped lentiviral vectors contained a coefficient of variation of 10 percent (data not shown), showing that the different S pseudotypes had minimal effects on vector particle release during packaging. In general, IDT codon optimized constructs contained higher levels of spike protein per p24 than the Ou codon optimized constructs. Furthermore, the IDT-HA and IDT-MLV modifications contained nearly 2-fold greater spike/p24 than the other constructs. However, when plotted against infectious titer, the increase of S protein concentration did not correlate with infectivity (Figure 10, panel B). These data suggest that greater spike protein concentration of pseudotyped lentiviral vectors does not lead to greater infectivity; rather, some intrinsic property of the S proteins determines the relative infectivity of the pseudotyped vectors.

[0106] Finally, we determined whether increasing levels of TMPRSS2 protease in target cells could affect infectivity by the S pseudotyped lentiviral vectors. VeroE6 cells, which lack TMPRSS2 expression, or VeroE6 cells transfected to express TMPRSS2 (VeroE6/TMPRSS2) were used to titer the vectors. VeroE6 and VeroE6/TMPRSS2 cells were transduced with IDT, IDT-D614G-HA, Ou, and Ou-D614G-HA pseudotyped lentiviral vectors. T hree days post transduction, cells were harvested to quantify vector expression and infectivity. For both the IDT-D614G-HA and Ou-D614H-HA pseudotyped vectors, transduction was 5-fold higher on the VeroE6/TMPRSS2 cells, demonstrating their dependence on this protease for enhanced transduction (Figure 11, panels A & B).

Discussion.

[0107] Despite the continued progression of vaccines and therapeutics for the treatment of SARS-CoV- 2, the methods to support high-throughput neutralization studies and drug screens have remained suboptimal. Previous groups have attempted to pseudotype viral vectors with the S glycoprotein (Crawford et al., 2020; Hoffmann et al., 2020; Nie et al., 2020), by replacing the S cytoplasmic tail with the influenza HA cytoplasmic tail or by modifying its amino acids composition. Those pseudotyped vectors, however, failed to achieve infectious titers high enough for use in downstream assays such as in vivo studies. Therefore, in this report, we generated modified S pseudotyped lentiviral vectors capable of infecting ACE2-expressing cells at greater levels than the unmodified counterpart.

[0108] It has been shown for several envelope glycoproteins, such as those of HIV-1, GaLV, RD 114, and the baboon envelope retroviral glycoprotein, that the CT domain of the envelope determines pseudotyping constraints (Girard Gagnepain et al., 2014;

Christodoulopoulos and Cannon, 2001; Sandrin and Cosset, 2006; Schnierle et al., 1997; Stitz et al., 2000). Ultimately, the CT modulates the envelope incorporation into viral particles, potentially through interactions with Gag, GagPol or cellular cytoplasmic proteins (Lucas et al., 2010).

[0109] We investigated whether various CT modifications to the spike glycoprotein could affect pseudotype efficiency. We utilized previously published codon optimizations (Crawford et al., 2020; Ou et al., 2020) and CT modifications (Crawford et al., 2020;

Mcbride et al., 2006; Sandrin et al., 2002; Girard Gagnepain et al., 2014) as well as the D614G mutant of SARS-Cov- 2. We showed that the influenza HA cytoplasmic tail and the D614G mutant could increase infectious titer by 10-fold over their unmodified S counterpart. In combination, however, these modifications exhibited a 1000-fold synergistic increase in infectious titer. The Ou-D614G-HA vector exhibited an infectious titer in the mid-10⁷ TU/mL range in ACE2 expressing 293T cells, which is only 1-log lower than the titer of VSV-G pseudotypes, the gold-standard in lentiviral pseudotyped vectors. This improved pseudotyped lentiviral vector can provide a means for studying both the neutralizing antibodies in recovered or symptomatic patients and the potency of antibody responses from current vaccine candidates. Furthermore, the ability to concentrate the spike pseudotyped vectors by means of tangential flow filtration (Figure 12; Cooper et al., 2011) should support in vivo studies and the infection of difficult to transduce cell lines and primary cells.

[0110] In contrast to the HA cytoplasmic tail, the murine leukemia virus (MLV) CT hampered pseudotyping efficiency, as evidenced by the reduction in GFP expression and titer of D614G- MLV variants in comparison to D614G alone. It has been shown that within the MLV cytoplasmic tail, several elements regulate the envelope’s incorporation into the virion and aid with fusogenicity into host cell membranes. The C-terminus of the MLV cytoplasmic tail, known as the R peptide, includes a conserved leucine- valine dipeptide cleavage site and a tyrosine (YXXL, SEQ ID NO:4) motif that has been implicated in promoting endocytosis of the envelope glycoprotein (Blot et al., 2006; Kubo et al., 2007; Loving et al., 2008; Lucas et al., 2010). Although not further studied, it remains a possibility that these motifs contained within the MLV tail - but not the HA tail - may hinder particle formation or membrane fusion of spike pseudotyped vectors.

[0111] To determine how each glycoprotein modification affected the relative spike concentration in virus particles, we performed an ELISA assay to quantify spike glycoprotein in relation to HIV-1 p24 particles. Resulting data demonstrated that the vectors pseudotyped with IDT codon optimized variants contained, on average, greater surface spike protein in comparison to the Ou codon optimized variants. The spike protein contains three hinges used to scan the host cell surface for attachment to ACE2 and viral entry (Turonova et al., 2020). While unknown, it is possible that the increase in S protein concentration of the IDT variants hinders attachment and fusion of pseudotyped vectors to ACE2 targets through steric hinderance or other unknown mechanisms.

[0112] Further, the addition of the D614G mutant decreased spike protein concentration in every glycoprotein variant. It is possible that the conformational change caused by the D614G variant (Yurkovetskiy et al., 2020) affected antibody binding for spike protein quantification; however, further studies are necessary for confirmation. We next studied the effect spike protein concentration had on infectivity of pseudotyped vectors. Results suggested, however, that an increase in spike concentration did not correlate with an increase in vector infectivity.

[0113] Various groups have indicated the importance of the TMPRSS2 protease for S protein priming and subsequent infectivity (Hoffmann et al., 2020; Ou et al., 2020;

Yurkovetskiy et al., 2020). To evaluate the effect of TMPRSS2 expression on the infectivity of S pseudotyped lentiviral vectors, we transduced VeroE6 cells, which lack TMPRSS2 expression, or VeroE6 cells transfected to express TMPRSS2 (VeroE6/TMPRSS2) with the modified S pseudotyped lentiviral vectors. The expression of TMPRSS2 increased the transduction of IDT-D614G-HA or Ou-D614G- HA variants by 5-fold. S pseudotyped lentiviral vectors depend on TMPRSS2 for successful transduction, indicating similar protein priming and entry as the SARS-CoV-2 virus. As a result, the pseudotyped vectors can be utilized for drug screens against SARS-CoV-2 or as models for in vivo imaging studies of ACE-2 expressing cells.

[0114] Given the non-replicative nature of pseudotyped lentiviral vectors, they have significant value in studying the biology of pathogenic viruses, such as SARS-CoV-2, due to their lower biosafety requirements. The highly infectious nature of SARS-CoV-2 requires biosafety level 3 (BSL-3) equipment within laboratories to appropriately handle and study the pathogenesis or treatment of the virus. (Bain et al., 2020). By designing an efficient spike pseudotyped HIV-1 lenti viral vector with greater potency than the unmodified S spike pseudotype, the need for BSL-3 laboratories can be avoided for many studies of SARS-CoV- 2. Given the rampant, global spread of the virus, the shift from BSL-3 to BSL-2 laboratories will facilitate screening of patients’ serum for neutralizing antibodies in a high-throughput fashion without risk of infection. Furthermore, this pseudotyped vector expands the capacity of research to help investigators study the effectiveness of current vaccine candidates, establish new treatments via high-throughput drug screens, examine lung pathology via infection of animal models or air- liquid interface cultures, and even explore the possible applications to gene therapy for treatment of lung diseases such as cystic fibrosis.

Materials and methods.

Cell lines and culture

[0115] HEK-293T (#CRL-3216; American Type Culture Collection [ATCC], Manassass, VA), PKR -/- HEK 293T (in house; Han et al., 2020), ACE-293T (ACE2 expressing 293T cells generously provided by Dr. Lili Yang (UCLA) and Dr. Pin Wang (USC)), VeroE6 (generously provided by Dr. Jocelyn Kim (UCLA)), and VeroE6/TMPRSS2 (#JCRB1819; JCRB Cell Bank) cells were cultured in DMEM (#10- 017-CV; Coming Inc., Coming, NY) supplemented with 10% fetal bovine serum (#100-106; GeminiBio, Calabasas, CA) and 1% penicillin/streptomycin/L-glutamine (#400-110; GeminiBio). Calu-3 cells (HTB-55; ATCC) were cultured in DMEM:F12(#11320033; ThermoFisher Scientific, Waltham, MA) and supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin/L-glutamine. Cell counts were measured with a Vi-CELL XR automated cell counter (Beckman Coulter, Indianapolis, IN).

Generation of Spike Glycoprotein Envelope Plasmids

[0116] The spike pseudotype backbone was generated by PCR amplification using the pMD.G-VSVG (Naldini et al., 1996) and primers oPAF147 and oPAR43 (see Supplemental materials). Ou (Ou et al., 2020) and IDT (Crawford et al., 2020) codon optimized spike glycoproteins were ordered as double stranded DNA fragments (Integrated DNA Technologies [IDT], Coralville, IA) with the inclusion of wildtype, HA (Crawford et. al, 2020), ALAYT (Mcbride et al., 2006), and MLV (Sandrin et al., 2002) cytoplasmic tails for downstream cloning. The following constructs were generated using the New England Biosciences (NEB) Gibson Assembly workflow with double- stranded DNA fragments and the spike backbone: IDT (oPAG40 and oPAG41), IDT-ALAYT (oPAG40 and oPAG42), IDT-HA (oPAG40 and oPAG43), IDT-MLV (oPAG40 and oPAG44), Ou (oPAG45 and oAPG46), and Ou-MLV (oPAG45 and oPAG47). The Q5 Site-Directed Mutagenesis Kit (#E0554S, NEB, Ipswich, MA) - with primers oPAF176 and oPAR56 or oPAF177 and oPARF - was used to generate the D614G mutants from each respective plasmid. Ou-D614G- HA spike glycoprotein was PCR amplified and cloned from the Ou-D614G plasmid using primers oPAF184 and oPAR63. All plasmids were mini prepped using the PureLink™ Quick Plasmid Miniprep Kit (#K210010; Invitrogen, Carlsbad, CA). All plasmids were maxi prepped using NucleoBond Xtra Maxi Kit (#740414; Machery-Nagel Inc., Duren, Germany).

Vector Packaging and Titration

[0117] Spike pseudotyped lentiviruses were packaged by transient transfection of PKR -/- 293T (Han et al., 2020) cells with fixed amounts of HIV Gag/Pol, Rev, and Lentiviral envelope (VSV-G or Spike) expression plasmids and equimolar amounts MNDU3-eGFP transfer plasmid using TransIT-293 (Minis Bio, Madison, WI) as described in Cooper et al. (2011). Viral supernatants were then directly used for titer determination or concentrated by tangential flow filtration, as described by Cooper et al., 2011. Briefly, ACE-293T, HEK- 293T, VeroE6, or VeroE6/TMPRSS2 cells were transduced at equal amounts of p24 lentiviral particles with either a 1:10 dilution of raw or a 1:10,000 dilution of concentrated vector. To calculate titers, we harvested cells and determined VCNs by ddPCR approximately 72 h post transduction.

Transduction of cell lines with spike pseudotyped lentivirus

[0118] ACE-293T, Calu-3, HEK-293T, VeroE6, or VeroE6/TMPRSS2 cells, 1 x 10⁵ per sample, were collected by trypsinization, centrifuged at 90g for 10 minutes and resuspended in 2mL of culture medium for plating in a 6-well plate (#3516; Coming Inc.). 24 hours after plating, cells were transduced with equal amounts of p24 lentiviral particles; culture medium was replaced with a 1: 10 dilution of viral supernatant in ImL of culture medium. 24 hours after transduction, culture medium was refreshed on all wells. 72 hours after transduction, cells were harvested for downstream analyses. Cell counts were measured with a Vi-CELL XR automated cell counter. Cells were assayed for GFP expression with a BD LSRFortessa or BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed with FlowJo (Tree Star, Ashland, OR). Digital Droplet PCR for VCN and Titer (TU/mL) Quantification

[0119] Genomic DNA from transduced cells was extracted using PureLink Genomic DNA Mini Kit (KI 82002; Invitrogen). VCN was calculated by using the vector GFP gene (primers eGFP616F and eGFP705R; probe eGFP653Pr) and an endogenous human diploid gene control (SCD4 [Human Syndecan 4] primers oPAF-SDC4 and oPAR-SDC4; probe oPAP-SDC4) as a reference. Reaction mixtures of 22uL volume, comprising 1 x Digital droplet (dd)PCR Master Mix (#1863010; BioRad, Hercules, CA), 400 nmol/L primers and 100 nmol/L probe for each set, 40 U Dral (R0129S; NEB) and 30- 100 g of the gDNA to study, were prepared and incubated at 37°C for 1 h. Droplet generation was performed as described in Hindson et al. [9] with 20 L of each reaction mixture. The droplet emulsion was then transferred with a multichannel pipet to a 96-well TWIN.TEC® real-time PCR Plates (Eppendorf, Hamburg, Germany), heat sealed with foil, and amplified in a conventional thermal cycler (T100 Thermal Cycler, Bio-Rad). Thermal cycling conditions consisted of 95°C 10 min, (94°C 30 s and 60°C 1 min) (55 cycles), 98°C 10 min (1 cycle) and 12°C hold. After PCR, the 96-well plate was transferred to a droplet reader (Bio-Rad). Acquisition and analysis of the ddPCR data was performed with the QuantaSoft software (BioRad), provided with the droplet reader. Vector Titer (TU/mL) was calculated as TU - VCN x (cell count at day of transduction) x virus dilution.

P24 Assay

[0120] p24 antigen concentration in vector supernatants were measured by the

UCLA/CFAR (Center for AIDS Research) Virology Core using the Alliance HIV-1 p24 Antigen ELISA Kit (#NEK050, PerkinElmer, Waltham, MA), following the manufacturer's manual.

Spike Protein Quantification

[0121] Spike viral supernatant was used to quantify Spike protein via an ELISA assay. To measure spike protein, a 1/1000 dilution of raw viral supernatant was bound to an ELISA plate pre-coated with a monoclonal antibody recognizing the RBD domain of the spike glycoprotein (#MBS9141957; MyBioSource San Diego, CA). The assay was performed as described by MyBioSource. In short, the pre-coated plate was washed 3 times before the addition of spike pseudotyped lentiviral supernatant. The viral supernatant was washed off after an incubation for 2 hours at 37°C. A biotin conjugated antibody recognizing the SI subunit of the spike protein was added and incubated for 1 hour at 37°C. Unbound conjugated antibody was washed before the addition of Streptavidin-HRP. After incubation for 30 minutes at 37°C, a 3,3 ‘,5,5 ‘-Tetramethylbenzidine (TMB) substrate was added and the plate was incubated for 15 minutes at 37°C (protected from light). A stop solution was added, and spike protein was then quantified on a Tecan Infinite 200 Pro microplate reader (Tecan Trading AG, Mannedorf, Switzerland) by measuring optical density at 450nm.

Following quantification, spike protein was normalized to HIV-1 p24 particles.

Statistical Analysis

[0122] All data are reported as mean ± SD unless otherwise stated All statistical analyses were carried out using statistical software SAS version 9.4 (SAS Institute, 2013) and GraphPad Prism version 8.4.0 (GraphPad Software, San Diego, CA, USA). The statistical significance between two averages was established using unpaired t test. When the statistical significance between three or more averages were evaluated, a one-way ANOVA was applied followed by multiple paired comparisons for normally distributed data (Tukey test).

Pearson's correlation was used to correlate the ratio of Spike protein/p24 to the titer of the pseudotyped LVs. A two-way ANOVA was utilized to assess the significance between TMPRSS2 expression and pseudotype modifications on GFP percentage or infectious titer. All statistical tests were two-tailed and a p value of < 0.05 was deemed significant.

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[0155] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. INFORMAL SEQUENCE LISTING

Sequence ID No: (SEQ ID NO : 5 ) modified spike protein.

Claims

CLAIMS What is claimed is:

1. An S glycoprotein capable of pseudotyping a non-replicative 3rd generation HIV-1 lentiviral vector.

2. The S glycoprotein of claim 1 , wherein said nucleic acid is codon optimized for expression in mammalian cells.

3. The S glycoprotein according to any one of claims 1-2, wherein said S glycoprotein comprises a glycine at position 614 of the spike protein instead of aspartic acid (D614G).

4. The S glycoprotein according to any one of claims 1-3, wherein spike protein comprises a cytoplasmic tail wherein said tail (aa 1240-1273) is replaced with that of the influenza hemagglutinin cytoplasmic tail (amino acids: NGSLQCRICI, SEQ ID NOG).

5. The S glycoprotein according to any one of claims 1-4, wherein spike protein comprises the amino acid sequence of SEQ ID NOG.

6. The S glycoprotein according to any one of claims 1-5, wherein said spike protein is disposed on a lentiviral vector.

7. The S glycoprotein of claim 6, wherein said lentiviral vector comprises a non-replicative 3rd generation HIV-1 lentiviral vector.

8. An expression cassette comprising a nucleic acid that encodes a modified SARS-CoV-2 spike protein.

9. The expression cassette of claim 8 , wherein said nucleic acid is codon optimized for expression in mammalian cells.

10. The expression cassette according to any one of claims 8-9, wherein said nucleic acid encodes glycine at position 614 of the spike protein instead of glycine (D614G).

11. The expression cassette according to any one of claims 8-10, wherein said nucleic acid encodes a spike protein wherein the cytoplasmic tail of the spike glycoprotein (aal240-1273) is replaced with that of the influenza hemagglutinin cytoplasmic tail (amino acids: NGSLQCRICI, SEQ ID NO:3).

12. The expression cassette according to any one of claims 8-11, wherein said nucleic acid encodes a spike protein comprising the amino acid sequence of SEQ ID NO:5.

13. The expression cassette of claim 12, wherein said nucleic acid sequence comprises the spike-encoding nucleic acid sequence shown in Figure 2 (the spikeencoding region of SEQ ID NO:1).

14. The expression cassette according to any one of claims 8-13, wherein said nucleic acid encoding a modified SARS-CoV-2 spike protein is provided in a plasmid where said nucleic acid is downstream from a CMV promoter and P-globin intron and upstream of a P-globin polyA.

15. The expression cassette according to any one of claims 8-14, wherein said expression cassette is packaged into a lentiviral particle.

16. The expression cassette of claim 15, wherein said expression cassette is packaged into a 3rd generation HIV-1 lentiviral particle.

17. The expression cassette of claim 16, wherein said lentiviral particle further comprises a reporter gene.

18. The expression cassette of claim 17, wherein said lentiviral particle comprises a GFP reporter gene.

19. A spike glycoprotein pseudotyped non-replicative viral particle wherein: said viral particle comprises a modified SARS-CoV-2 spike glycoprotein; and said viral particle is capable of specifically infecting ACE2 expressing cells.

20. The viral particle of claim 19, wherein said viral particle is an HIV lentiviral particle.

21. The viral particle of claim 20, wherein said viral particle is a third generation HIV 1 lentiviral particle.

22. The viral particle according to any one of claims 19-21, wherein said viral particle comprises an expression cassette according to any one of claims 8-14.

23. The viral particle according to any one of claims 19-22, wherein said viral particle further comprises a reporter gene.

24. The viral particle of claim 23, wherein said viral particle comprises a GFP reporter gene.

25. A method of evaluating a therapeutic agent for efficacy against SARS- CoV-2 virus, said method comprising: contacting cells expressing the cell surface receptor angiotensinconverting enzyme 2 (ACE2) with a pseudotyped virus according to any one of claims 19-24; contacting said cells with said therapeutic agent; and determining the amount and/or rate of infection of said cells by said pseudotyped virus where the amount and/or rate of infection provides a measure of the efficacy of said therapeutic agent where reduced amount and/or rate of infenction as compared to a control without said therapeutic agent indicates efficacy of said therapeutic agent.

26. The method of claim 25, wherein said reduced amount and/or rate of infection is a statistically significant reduced amount and/or rate to indicate efficacy.

27. The method according to any one of claims 25-26, wherein said determining comprise detecting a reporter gene expressed by said pseudotyped virus.

28. The method according to any one of claims 25-27, wherein said determining comprises visualizing expression of a reporter gene.

29. The method of claim 28, wherein said reporter gene comprises a GFP gene or an mCitrulline gene.

30. The method according to any one of claims 25-29, wherein said determining comprises quantifying said virus by PCR.

31. The method according to any one of claims 25-30, wherein said therapeutic agent comprise an anti-SARS-CoV-2 antibody.

32. The method according to any one of claims 25-31, wherein said therapeutic agent comprises plasma derived from a subject this is or that has been infected with SARS-CoV-2.

33. The method according to any one of claims 25-32, wherein said cells comprise mammalian cells transfected with a construct that expresses said angiotensinconverting enzyme 2 (ACE2).

34. The method of claim 33, wherein said cells are HEK293T cells.

35. A method of evaluating the efficacy of a vaccine directed against SARS-CoV-2, said method comprising: contacting cells expressing the cell surface receptor angiotensinconverting enzyme 2 (ACE2) with a pseudotyped virus according to any one of claims 19-24; contacting said cells with plasma derived from subjects inoculated with said vaccine; and determining the amount and/or rate of infection of said cells by said pseudotyped virus where the amount and/or rate of infection provides a measure of the efficacy of said vaccine where reduced amount and/or rate of infenction as compared to a control without said vaccine indicates efficacy of said vaccine.

36. The method of claim 35, wherein said reduced amount and/or rate of infection is a statistically significant reduced amount and/or rate to indicate efficacy.

37. The method according to any one of claims 35-36, wherein said determining comprise detecting a reporter gene expressed by said pseudotyped virus.

38. The method according to any one of claims 35-37 wherein said determining comprises visualizing expression of a reporter gene.

39. The method of claim 38, wherein said reporter gene comprises a GFP gene or an mCitrulline gene.

40. The method according to any one of claims 35-39, wherein said determining comprises quantifying said virus by PCR.

41. The method according to any one of claims 35-40, wherein said cells comprise mammalian cells transfected with a construct that expresses said angiotensinconverting enzyme 2 (ACE2).

42. The method of claim 41, wherein said cells are HEK293T cells.