Production of high-titer recombinant vesicular stomatitis virus in suspension cell culture

Abstract

Methods for producing replication-incompetent recombinant vesicular stomatitis virus (rVSV) in suspension cell culture are disclosed. In some embodiments, the method includes inoculating a suspension cell culture medium with packaging cells, transfecting the packaging cells with a plasmid that includes a nucleic acid molecule encoding a viral envelope glycoprotein, introducing an rVSV lacking a gene encoding a functional envelope glycoprotein into the suspension cell culture medium, and isolating the rVSV produced from the packaging cells with the viral envelope glycoprotein incorporated into its viral envelope.

Description

[Technical field]

[0001] Sequence Listing Reference This application incorporates by reference a computer readable sequence listing in ST.26 XML format entitled 10976WO01_Sequence, created on Feb. 3, 2023, and containing 9,944 bytes.

[0002] The present invention relates to the production of recombinant viral particles, and in particular to a method for producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV) comprising viral envelope glycoproteins produced in transfected packaging cells. [Background technology]

[0003] The entry of all enveloped viruses requires a membrane fusion event mediated by one or more viral glycoproteins present on the surface of the viral lipid envelope. These envelope glycoproteins are responsible for binding the virus to the cell surface and for inducing the fusion of the viral envelope with either the plasma membrane of the host cell or with an internal membrane following endocytosis of the virion.

[0004] Vesicular stomatitis virus (VSV) is a prototypic, non-segmented, negative-stranded RNA virus that belongs to the Rhabdoviridae family. VSV has been widely used to study virus entry, replication, and assembly due to its broad host range and robust replication properties in a wide range of mammalian and insect cells. One notable property of VSV is that VSV virions are not particularly selective regarding the type of membrane protein that can be incorporated into the viral envelope. Early studies in which cells were coinfected with VSV and other enveloped viruses showed that VSV readily forms pseudotypes. Pseudotypes have the envelope protein of a heterologous virus assembled into the VSV membrane. The ability to form pseudotypes is likely due to a mechanism of VSV budding that has been shown not to require the VSV G protein. Studies on VSV assembly have led to the generation of a recombinant VSV in which the glycoprotein (G) gene has been deleted. This recombinant (rVSV-ΔG) was used to produce VSV pseudotypes containing the envelope glycoprotein of a heterologous virus. (Whitt, J Virol Methods, 169(2):365-374, 2010).

[0005] The production of rVSV mainly relies on anchorage-dependent cell culture technology.However, the scale-up of cell production using adhesion cell-based production presents many challenges, because adhesion-dependent culture processing generally requires more steps and time than suspension culture, resulting in more labor and higher production costs.Therefore, there is still a need to develop a scalable propagation method that produces recombinant VSV particles with sufficient yield for large-scale production. Summary of the Invention

[0006] The present disclosure is directed to a method for producing recombinant vesicular stomatitis virus (rVSV) in suspension cell culture that produces virus titers comparable to adherent cell culture processes. The method can be used, for example, to produce replication-incompetent rVSVs bearing the envelope glycoproteins of any one of a variety of enveloped viruses (e.g., SARS-CoV-2 or Ebola) for use as pseudoviruses in, for example, viral antibody neutralization assays or for use in vector-based vaccines.

[0007] In one aspect, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a suspension cell culture medium with a plurality of packaging cells; (b) transfecting the packaging cells in the suspension cell culture medium with a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein; (c) introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of 0.001 to 3 to infect the population of packaging cells expressing the viral envelope glycoprotein, wherein the rVSV has been engineered not to express a functional envelope glycoprotein; and (d) isolating the rVSV produced from the population of packaging cells 15 to 65 hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0008] In some embodiments, the MOI is between 0.001 and 1.0. In some embodiments, the MOI is between 0.005 and 0.05. In some cases, the MOI is between 0.008 and 0.012. In some cases, the MOI is about 0.01. In some cases, the MOI is between 0.01 and 2. In some cases, the MOI is between 0.01 and 1. In some cases, the MOI is between 0.8 and 1.2.

[0009] In some embodiments, the rVSV produced from the packaging cells is isolated 40-50 hours post-infection. In some cases, the rVSV produced from the packaging cells is isolated 43-47 hours post-infection. In some cases, the rVSV produced from the packaging cells is isolated 20-30 hours post-infection. In some cases, the rVSV produced from the packaging cells is isolated 22-26 hours post-infection.

[0010] In some embodiments, the packaging cell is a mammalian cell. In some cases, the packaging cell is a primate cell. In some cases, the packaging cell is a human cell. In some embodiments, the packaging cell is a human embryonic kidney (HEK) cell. In some cases, the HEK cell is a HEK293 cell. In some embodiments, the HEK293 cell is a HEK293F cell, a HEK293T cell, a HEK293SF cell, or a HEK293S cell. In some cases, the HEK293 cell is a HEK293F cell.

[0011] In some embodiments, the suspension cell culture medium is a serum-free and protein-free medium.

[0012] In some embodiments, the isolated rVSV is at least 1×10 6 rVSV isolates are produced at infectious titers of at least 1 × 10 plaque-forming units (pfu) / mL. 7 pfu / mL. In some cases, rVSV isolates were produced at infectious titers of at least 1 × 10 8 pfu / mL. In some cases, rVSV isolates were produced at infectious titers of at least 5 × 10 8 pfu / mL. In some cases, rVSV isolates were produced at infectious titers of at least 1 × 10 9In some cases, the rVSV isolate is produced at a titer equivalent to the titer produced in the adherent cell-based control. In some cases, the rVSV isolate is produced at a titer at least two-fold higher than the titer produced in the adherent cell-based control. In some cases, the rVSV isolate is produced at a titer at least three-fold higher than the titer produced in the adherent cell-based control.

[0013] In some embodiments, the viral envelope glycoprotein is a vesicular stomatitis virus G protein. In some embodiments, the viral envelope glycoprotein is a class I fusion protein. In some embodiments, the viral envelope glycoprotein is a class II fusion protein. In some embodiments, the viral envelope glycoprotein is a class III fusion protein. In some embodiments, the viral envelope glycoprotein is a coronavirus spike protein. In some cases, the coronavirus spike protein is a spike protein of SARS-CoV-2. In some cases, the coronavirus spike protein comprises the amino acid sequence of SEQ ID NO:1. In some cases, the coronavirus spike protein comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the viral envelope glycoprotein is an Ebola virus glycoprotein. In some cases, the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0014] In some embodiments, the viral envelope glycoprotein is a viral envelope glycoprotein of a virus selected from the group consisting of a flavivirus, an alphavirus, a togavirus, a coronavirus, a herpesvirus, a hepadnavirus, a poxvirus, a paramyxovirus, a rhabdovirus, a bunyavirus, a filovirus, a retrovirus, a hepatitis virus, an influenza A virus, and an influenza B virus.

[0015] In some embodiments of the method, transfecting the packaging cells comprises use of a polyethylenimine transfection reagent. In some embodiments of the method, transfecting the packaging cells comprises use of a Lipofectamine transfection reagent or the proprietary transfection reagent FectoVIR®-AAV (Polyplus, France) transfection reagent.

[0016] In some embodiments, the cell culture medium contains at least 3×10 6 Includes cell density in packaging cells / mL.

[0017] In some embodiments of the method, transfecting the packaging cells comprises using a DNA concentration of at least 1.5 μg / mL. In some embodiments of the method, transfecting the packaging cells comprises using a transfection reagent to DNA ratio of about 2 to about 3. In some embodiments of the method, transfecting the packaging cells comprises using a transfection reagent to DNA ratio of about 2.5.

[0018] In some embodiments, the suspension cell culture medium is maintained at temperature during inoculation and transfection, and the temperature of the suspension cell culture medium is reduced to a lower temperature when introducing rVSV into the suspension cell culture medium. In some cases, the temperature is about 37° C. In some cases, the reduced temperature is about 32° C. to about 36.5° C. In some cases, the reduced temperature is about 35.5° C. In some cases, the reduced temperature is about 34° C.

[0019] In some embodiments of the methods discussed herein, the suspension cell culture medium is not changed prior to introducing the rVSV into the suspension cell culture medium.

[0020] In one aspect, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293 cells into a suspension cell culture medium, wherein the temperature of the suspension cell culture medium is about 37° C.; (b) transfecting the HEK293 cells in the suspension cell culture medium with a polyethylenimine transfection reagent and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein; (c) inoculating the HEK293 cells into a suspension cell culture medium, wherein the temperature of the suspension cell culture medium is about 37° C.; (d) transfecting the HEK293 cells in the suspension cell culture medium with a polyethylenimine transfection reagent and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein; (d) reducing the temperature of the suspension cell culture medium to about 34°C and introducing rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.01 to infect a population of HEK293 cells expressing viral envelope glycoproteins, where the rVSV has been engineered not to express functional envelope glycoproteins; and (d) isolating the rVSV produced from the population of packaging cells 40-48 hours post-infection, where the viral envelope glycoproteins are incorporated into the viral envelope of the isolated rVSV.

[0021] In one aspect, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293 cells into a suspension cell culture medium, wherein the temperature of the suspension cell culture medium is about 37° C.; (b) transfecting the HEK293 cells in the suspension cell culture medium with a polyethylenimine transfection reagent and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein; (c) inoculating a plurality of HEK293 cells into a suspension cell culture medium, wherein the temperature of the suspension cell culture medium is about 37° C.; (a) reducing the temperature of the suspension cell culture medium to about 34°C and introducing rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 1 to infect a population of HEK293 cells expressing viral envelope glycoproteins, where the rVSV has been engineered not to express functional envelope glycoproteins; and (b) isolating the rVSV produced from the population of packaging cells 20-28 hours post-infection, where the viral envelope glycoproteins are incorporated into the viral envelope of the isolated rVSV.

[0022] In some embodiments, the viral envelope glycoprotein is a class I fusion protein, a class II fusion protein, or a class III fusion protein. In some embodiments, the viral envelope glycoprotein is a coronavirus spike protein. In some cases, the coronavirus is SARS-CoV-2. In some embodiments, the viral envelope glycoprotein is an Ebola virus glycoprotein. In some embodiments, the viral envelope glycoprotein is a viral envelope glycoprotein of a virus selected from the group consisting of flavivirus, alphavirus, togavirus, coronavirus, herpesvirus, hepadnavirus, poxvirus, paramyxovirus, rhabdovirus, bunyavirus, filovirus, retrovirus, hepatitis virus, influenza A virus, and influenza B virus.

[0023] In some embodiments, the cell culture medium contains at least 3×10 6 with a cell density of HEK293 cells / mL.

[0024] In some embodiments of the method, transfecting the HEK293 cells comprises using a DNA concentration of at least 1.5 μg / mL. In some embodiments of the method, transfecting the HEK293 cells comprises using a transfection reagent to DNA ratio of about 2.5.

[0025] In some embodiments, the HEK293 cells are HEK293F cells, HEK293T cells, HEK293SF cells, or HEK293S cells. In some cases, the HEK293 cells are HEK293F cells.

[0026] In various embodiments, any of the features or components of the embodiments described above or discussed herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value described above or discussed herein may be combined with another associated value described above or discussed herein to recite a range having values ​​representing the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure.

[0027] Other embodiments will be apparent from consideration of the detailed description that follows. [Brief description of the drawings]

[0028] [Figure 1] An exemplary suspension cell culture production schematic is shown in Figure 1. In the illustrated example, a recombinant VSV with a deleted glycoprotein (G) gene (rVSV-ΔG) is introduced into a suspension cell culture of packaging cells transfected with a plasmid containing a nucleic acid molecule encoding the VSV-G glycoprotein such that rVSV-ΔG incorporates the SV-G glycoprotein into the viral envelope during viral replication and budding.

[0029] [Diagram 2] FIG. 2 shows additional parameters (top to bottom on day 2: temperature, agitation rate, pH, and dissolved oxygen) of an exemplary production process in a small-scale bioreactor with parameters shown in Table 1 (Example 1).

[0030] [Diagram 3] Figure 3 shows the various parameters and the resulting virus titers at 24, 33, and 48 hours post-infection (hpi) in the control sample and seven samples that were varied by a single parameter. Highlighted cells indicate the parameter that was changed for each sample. Recombinant VSV-ΔG-Fluc / GFP stock virus (titer = 1.59E9) was used for infection. A comparator adherent cell system yielded a virus titer of 9.3E8 pfu / mL.

[0031] [Figure 4] Figure 4 shows time course titer data (plaque forming units / mL as a function of time (hours post infection)) corresponding to various production parameters (e.g., transfection, infection, culture, and harvest conditions) in 15 different runs shown in Table 2 (Example 2).

[0032] [Figure 5A] Figures 5A and 5B show the effect of various parameters of the suspension cell culture production process on virus titer (pfu / mL), including (i) the absence or presence of medium exchange prior to infection, (ii) multiplicity of infection (MOI) rate, (iii) type of transfection reagent, (iv) temperature shift during infection, and (v) harvest time at hours post infection (hpi). [Figure 5B]Figures 5A and 5B show the effect of various parameters of the suspension cell culture production process on virus titer (pfu / mL), including (i) the absence or presence of medium exchange prior to infection, (ii) multiplicity of infection (MOI) rate, (iii) type of transfection reagent, (iv) temperature shift during infection, and (v) harvest time at hours post infection (hpi).

[0033] [Figure 6] 6 shows an exemplary suspension cell culture production schematic for producing pseudotyped recombinant VSV (e.g., having a coronavirus spike glycoprotein in place of the VSV-G glycoprotein). In the illustrated example, a recombinant VSV with a deleted glycoprotein (G) gene (rVSV-ΔG) is introduced into a suspension cell culture of packaging cells transfected with a plasmid containing a nucleic acid molecule encoding the coronavirus spike glycoprotein such that rVSV-ΔG incorporates the coronavirus spike glycoprotein into the viral envelope during viral replication and budding.

[0034] [Figure 7] 7 shows the results of a luciferase assay demonstrating production of spike glycoprotein-pseudotyped rVSV (VSV-spike) at titers greater than three-fold higher than those produced by adherent cell culture controls (adherent control D614G) and mock transfection (background). As shown, at 24 hours post-infection, the adherent control exhibited a titer of 1430 RLU, while VSV-spike exhibited a titer of approximately 5300 RLU. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.As used herein, the term "about" when used in relation to a specific referenced numerical value means that the value can vary by 1% or less from the referenced value.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0037] As used herein, the terms "include", "includes", and "including" are intended to be open-ended and are understood to mean "comprise", "comprises", and "comprising", respectively.

[0038] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0039] Selected Abbreviations VSV - Vesicular stomatitis virus

[0040] rVSV - recombinant vesicular stomatitis virus

[0041] rVSV-ΔG - recombinant vesicular stomatitis virus with a deletion of the glycoprotein (G) gene

[0042] VSV-G - Vesicular stomatitis virus envelope glycoprotein (G)

[0043] HEK Cells - Human Embryonic Kidney Cells

[0044] PFU - plaque forming unit

[0045] MOI - Multiplicity of infection

[0046] VLPs - Virus-like particles

[0047] HPI - time post infection

[0048] SARS-CoV-2 - Severe Acute Respiratory Syndrome Coronavirus 2

[0049] definition "VSV" refers to any strain or variant of VSV, including rVSV. VSV encompasses replication-incompetent VSV, such as VSV lacking the G glycoprotein.

[0050] "VSV-G" refers to the type III viral fusion protein that mediates fusion between the viral envelope of VSV and the host cell membrane, releasing the viral genome into the host cell.

[0051] "Replication-incompetent VSV" refers to a VSV in which the gene encoding the G envelope glycoprotein has been deleted or mutated to produce a non-functional protein.

[0052] As used herein, the term "vector" or "plasmid" refers to a polynucleotide construct designed for transduction / transfection of one or more cell types. A vector or plasmid may be, for example, an "expression vector" or "expression plasmid" designed for expression of a nucleotide sequence in a host cell (e.g., a packaging cell).

[0053] The terms "polynucleotide" and "nucleic acid" as used interchangeably herein refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include single-stranded, double-stranded, or triple-stranded DNA, genomic DNA, cDNA, genomic RNA, mRNA, DNA-RNA hybrids, or polymers that contain purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide can contain sugar and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. In addition, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing a complementary strand and annealing the strands under appropriate conditions, or by synthesizing a new complementary strand using DNA polymerase with appropriate primers.

[0054] The term "heterologous" refers to a combination of elements that do not naturally occur in a virus or cell. For example, heterologous DNA refers to DNA that is not naturally located in a cell or at a chromosomal site of a cell. Heterologous DNA may include genes that are foreign to the cell. In the context of VSV, "heterologous glycoprotein" refers to a glycoprotein that is not present in the envelope of wild-type VSV.

[0055] As used herein, the terms "cell," "host cell," "cell culture," and the like are intended to include any individual cell or cell culture that can be or has been a recipient of a virus, vector, or incorporation of an exogenous nucleic acid molecule, polynucleotide, and / or protein. They are also intended to include the progeny of a single cell. Cells are preferably eukaryotic cells, but may be prokaryotic cells, including, but not limited to, bacterial cells, yeast cells, animal cells, and mammalian cells (e.g., mouse, rat, monkey, or human).

[0056] "Expression" includes transcription and / or translation.

[0057] "Encoded by" or "encoding" refers to a nucleic acid sequence that codes for the polypeptide sequence (ie, the amino acid sequence) of a polypeptide encoded by the nucleic acid sequence.

[0058] "Replication" and "growth" are used interchangeably and refer to the ability of the virus of the present invention to reproduce or grow. These terms are well understood in the art. For the purposes of the present invention, replication includes the production of VSV proteins and generally refers to the reproduction of VSV. Replication can be measured using standard assays in the art. "Replication" and "growth" include any activity directly or indirectly involved in the virus manufacturing process, including but not limited to viral gene expression, production of viral proteins, nucleic acids, or other components, packaging of viral components into complete viruses, and cell lysis.

[0059] The term "multiplicity of infection" or "MOI" refers to the average number of viral particles that infect a single cell. "MOI" is calculated by dividing the total number of viral plaque-forming units (PFU) by the total number of cells infected.

[0060] The terms "plaque" or "viral plaque" alternatively refer to distinct, often round patches of lysed cells in an opaque cell culture layer. "Plaque-forming unit" or "PFU" refers to the average number of infectious virus particles per unit volume. For example, if a virus solution has 100 PFU / ml, this means that every milliliter of the virus solution has 100 virus particles that can each form a plaque. PFU / mL is the conventional means of referring to the concentration of a plaque-forming virus preparation. However, PFU is generally used interchangeably with "infectious unit" or "IU" to represent the unit of infectious virus in a virus preparation.

[0061] The terms "culture medium," "cell culture fluid," "cell culture media," "cell culture medium," "culture medium," and / or "bioreactor fluid" are used interchangeably and refer to the medium or solution in which cell cultures are grown.

[0062] A cell suspension, suspension culture, or suspension cell culture is a type of cell culture in which single cells or small aggregates of cells can function and grow in an agitated growth medium, thus forming a suspension. Suspension cell cultures are in contrast to adherent cell cultures, in which cells grow while fixed to a physical substrate.

[0063] As used herein, the term "growing" or "propagation" refers to the in vitro propagation of viruses in various types of cells. Growing / propagating viruses in cells in the laboratory involves inoculating cells with the virus, followed by incubation to allow virus production, and then harvesting the cell culture medium containing the virus. Virus-infected cells are usually grown in a growth medium in a culture vessel (such as a flask or bioreactor), and the culture is maintained in a cell incubator with a specified temperature, humidity, and gas composition. However, culture conditions may vary depending on the cell type and may be altered to induce changes in the cells or to support or enhance virus production by the cells.

[0064] As used herein, the term "harvesting" refers to the collection of cells or cell culture medium following infection of a cell or cell line with any of the viral strains or serotypes described herein in preparation for virus isolation and purification.

[0065] The terms "isolated," "isolating," "purified," or "purifying" mean that a material is removed from the production environment. Thus, an isolated or purified virus, or the process of doing so, refers to the removal of the virus from the cell culture and packaging cells in which it was produced.

[0066] As used in the context of the present invention, a "gene" is a sequence of nucleotides in a nucleic acid molecule (such as a chromosome, a plasmid, etc.) with which a genetic function is associated. A gene can code for an expressed product, such as a polypeptide or a polynucleotide (e.g., a tRNA). Typically, a gene includes coding sequences, such as a polypeptide-encoding sequence, and non-coding sequences, such as introns or regulatory sequences.

[0067] The term "genetic modification" or "recombination" generally refers to the introduction of one or more mutations or deletions in the genome of a virus by any means known to those of skill in the art.

[0068] The terms "protein", "polypeptide" and "peptide" refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, etc. are included in the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The term "amino acid" refers to either natural and / or unnatural or synthetic amino acids, including both D or L optical isomers, and amino acid analogs. The one-letter and three-letter codes for each of the natural amino acids are known to those of skill in the art.

[0069] As used herein, the term "comprising" and its cognates are used in their inclusive sense, i.e., equivalent to the term "including" and its corresponding cognates.

[0070] overview The present disclosure provides a method for the production of recombinant vesicular stomatitis virus (rVSV) in a scalable suspension cell culture production system that can produce virus titers comparable to adherent cell culture processes. In particular, the method of the present disclosure involves a specific combination of culture, infection, and harvest parameters for the surprising production of high titers of replication-incompetent rVSV in suspension cell culture. The method can be used, for example, to produce replication-incompetent rVSV with the envelope glycoprotein of any one of a variety of enveloped viruses (e.g., SARS-CoV-2 or Ebola) for use, for example, as a pseudovirus in viral antibody neutralization assays or for use in vector-based vaccines.

[0071] Methods for producing replication-incompetent recombinant VSV Embodiments of the present disclosure are directed to methods for producing replication-incompetent recombinant VSV particles that incorporate envelope glycoproteins expressed from a plasmid or other vector in packaging cells from which the rVSV particles bud.

[0072] With reference to an exemplary production schematic depicted in FIG. 1, a method for producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV) can include (a) inoculating a plurality of packaging cells (e.g., HEK293 cells) into a suspension cell culture medium (e.g., a bioreactor) on day 0; (b) transfecting the packaging cells in the suspension cell culture medium on day 1 with a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein (VSV-G is depicted in FIG. 1, however, this could be substituted with any viral envelope glycoprotein, such as, for example, the spike glycoprotein as depicted in FIG. 6). (c) on day 2, introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of 0.001-3 to infect a population of packaging cells expressing viral envelope glycoproteins, where the rVSV has been engineered not to express functional envelope glycoproteins (e.g., rVSV-ΔG), and (d) following viral replication and budding, isolating the rVSV produced from the population of packaging cells 15-65 hours post-infection, where the viral envelope glycoproteins are incorporated into the viral envelope of the isolated rVSV (i.e., where the virus buds through the membrane of the packaging cells).

[0073] The packaging cells (or host cells) used for recombinant VSV production can be any cell in which VSV replicates, for example, mammalian cells and some insect (e.g., Drosophila) cells. Immortalized cell lines or tumor cell lines can be used. Many cell lines commonly known in the art are available for use. By way of example, such cell lines include, but are not limited to, BHK (baby hamster kidney) cells, CHO (Chinese hamster ovary) cells, HeLA (human) cells, mouse L cells, Vero (monkey) cells, African green monkey kidney (AGMK) cells, ESK-4, PK-15, EMSK cells, MDCK (Madin-Darby canine kidney) cells, MDBK (Madin-Darby bovine kidney) cells, HEK293 (human) cells, and Hep-2 cells. Such cell lines are publicly available, for example, from the ATCC and other culture depositories. In some embodiments, the packaging or host cells are mammalian cells. In some cases, the packaging cell or host cell is a primate cell or a human cell. In some embodiments, the packaging cell or host cell is a human embryonic kidney (HEK) cell. In some examples, the HEK cell is a HEK293 cell, which may be selected from a HEK293F cell, a HEK293T cell, a HEK293SF cell, and a HEK293S cell. In some embodiments, the packaging cell or host cell is a HEK293F cell. In some cases, the packaging cell or host cell is a HEK293F cell that grows in suspension cell culture.

[0074] The culture medium (or production medium) used for suspension cell culture of the packaging cells or host cells may be any suitable product. The culture medium is preferably animal-derived component-free, protein-free, and chemically defined. An exemplary culture medium is the CTS TM LV-MAX TM Production medium (ThermoFisher Scientific).

[0075] The transfection of packaging cells or host cells can be achieved by any suitable method known in the art. In some embodiments, the transfection is achieved by using a transfection reagent (to promote the uptake of vectors or plasmids), such as polyethylenimine transfection reagent (e.g., PEI MAX™, Polysciences), Lipofectamine transfection reagent, or FectoVIR®-AAV transfection reagent (Polyplus, France). Alternatively, plasmid DNA uptake can also be enhanced by electroporation of cells, whereby a high-voltage current is applied for a few milliseconds across the cuvette containing the cells and DNA.

[0076] In various embodiments, transfection of the vector or plasmid into multiple packaging or host cells is performed when the cell density of the packaging cells reaches a sufficient density. In some embodiments, transfection is performed when the cell density is at least 3×10 6 In various embodiments, the cell density is at least about 1×10 6 Cells / mL, at least approximately 1.5 x 10 6 Cells / mL, at least approximately 2 x 10 6 Cells / mL, at least approximately 2.5 x 10 6 Cells / mL, at least approximately 3 x 10 6 Cells / mL, at least approximately 3.5 x 10 6 Cells / mL, at least approximately 4 x 10 6 Cells / mL, at least approximately 4.5 x 10 6 cells / mL, or at least about 5 x 10 6 In cells / mL.

[0077] In some cases, transfection of packaging or host cells is accomplished using a DNA concentration of at least 1.5 μg / mL. In various embodiments, the DNA concentration in the vector or plasmid is at least about 0.5 μg / mL, at least about 0.6 μg / mL, at least about 0.7 μg / mL, at least about 0.8 μg / mL, at least about 0.9 μg / mL, at least about 1.0 μg / mL, at least about 1.1 μg / mL, at least about 1.2 μg / mL, at least about 1.3 μg / mL, at least about 1.4 μg / mL, at least about 1.5 μg / mL, at least about 1.6 μg / mL, at least about 1.7 μg / mL, at least about 1.8 μg / mL, at least about 1.9 μg / mL, at least about 2.0 μg / mL, at least about 2.5 μg / mL, at least about 3.0 μg / mL, at least about 3.5 μg / mL, at least about 4.0 μg / mL, at least about 5.5 μg / mL, or at least about 5.0 μg / mL.

[0078] In various embodiments, transfection is accomplished through the use of a transfection reagent to DNA ratio of about 1 to about 5. In some cases, the transfection reagent to DNA ratio is about 2 to about 3. In some cases, the transfection reagent to DNA ratio is 2.5. In various embodiments, the transfection reagent to DNA ratio is about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0.

[0079] In various embodiments, the vector or plasmid transfected into the packaging cell (or host cell) comprises a nucleic acid molecule encoding a viral envelope glycoprotein, which may be a heterologous glycoprotein (i.e., a glycoprotein not normally produced by VSV). In some cases, the viral envelope glycoprotein is a vesicular stomatitis virus G protein. In some cases, the viral envelope glycoprotein is a class I fusion protein. In some cases, the viral envelope glycoprotein is a class II fusion protein. In some cases, the viral envelope glycoprotein is a class III fusion protein. In some embodiments, the viral envelope glycoprotein is a viral envelope glycoprotein of a virus selected from the group consisting of flavivirus, alphavirus, togavirus, coronavirus, herpesvirus, hepadnavirus, poxvirus, paramyxovirus, rhabdovirus, bunyavirus, filovirus, retrovirus, hepatitis virus, influenza A virus, and influenza B virus.

[0080] In various embodiments, the vector or plasmid transfected into the packaging cell (or host cell) comprises a nucleic acid molecule encoding a coronavirus spike glycoprotein, e.g., a spike protein from SARS-CoV-2. In some cases, the spike glycoprotein comprises the amino acid sequence of SEQ ID NO: 1. In some cases, the spike glycoprotein comprises the amino acid sequence of SEQ ID NO: 2.

[0081] In various embodiments, the vector or plasmid transfected into the packaging cell (or host cell) comprises a nucleic acid molecule encoding an Ebola virus glycoprotein. In some cases, the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 3. In some cases, the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 4.

[0082] In any of the various embodiments of the methods contemplated herein, the suspension cell culture medium is not exchanged prior to introducing the rVSV into the suspension cell culture medium (as discussed below). Although medium exchange prior to or close to infecting the packaging cells with virus is often considered advantageous to remove residual elements from the transfection process, the inventors have surprisingly discovered that these advantages are diminished in the context of other factors (e.g., MOI and harvest timing) that allow medium exchange to be avoided, thereby increasing the scalability of the production process.

[0083] In various embodiments, infection of packaging cells (or host cells) with recombinant VSV (that does not express a functional envelope glycoprotein) is carried out by introducing the virus into suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.001 to about 5. In some cases, the MOI is about 0.001 to about 0.5. In some cases, the MOI is about 0.001 to about 1. In some cases, the MOI is about 0.005 to about 3. In some cases, the MOI is about 0.005 to about 1.5. In some cases, the MOI is about 0.001 to about 0.1. In some cases, the MOI is about 0.005 to about 0.05. In some cases, the MOI is about 0.008 to about 0.012. In some cases, the MOI is about 0.01±0.001. In some cases, the MOI is about 0.5 to about 1.5. In some cases, the MOI is about 0.8 to about 1.2. In some cases, the MOI is about 1±0.1. In various embodiments, the MOI is about 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.0225, 0.025, 0.0275, 0.03, 0.0325, 0.035, 0.0375, 0.04, 0.0425, 0.045, 0.0475, or 0.5. In some cases, the MOI is about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.05, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or 5.

[0084] In the methods discussed herein, the suspension cell culture medium is maintained at temperature during inoculation and transfection, and the temperature of the suspension cell culture medium is reduced to a lower temperature when the packaging cells are infected with the rVSV. In some cases, the temperature of the suspension cell culture is about 37°C during inoculation and transfection, and is reduced to about 32°C to about 36.5°C at or near the time of infection (e.g., within 1 hour). In some cases, the temperature is reduced to about 35.5°C. In some cases, the temperature is reduced to about 34°C. In various embodiments, the temperature of the cell culture during inoculation and transfection is 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, 37.5°C, 37.6°C, 37.7°C, 37.8°C, 37.9°C, or 38°C. In various embodiments, including those mentioned in the immediately preceding sentence, the reduced temperature may be 32° C., 32.1° C., 32.2° C., 32.3° C., 32.4° C., 32.5° C., 32.6° C., 32.7° C., 32.8° C., 32.9° C., 33° C., 33.1° C., 33.2° C., 33.3° C., 33.4° C., 33.5° C., 33.6° C., 33.7° C., 33.8° C., 33.9° C., 34 ... °C, 34.1°C, 34.2°C, 34.3°C, 34.4°C, 34.5°C, 34.6°C, 34.7°C, 34.8°C, 34.9°C, 35°C, 35.1°C, 35.2°C, 35.3°C, 35.4°C, 35.5°C, 35.6°C, 35.7°C, 35.8°C, 35.9°C, 36°C, 36.1°C, 36.2°C, 36.3°C, 36.4°C, or 36.5°C.

[0085] In various embodiments, the infected packaging cells are cultured for a period of time before the viral particles are harvested and isolated. In some embodiments, the rVSV particles produced from the packaging cells are isolated at about 40 to about 50 hours post infection (hpi). In some cases, the rVSV particles produced from the packaging cells are isolated at about 43 hpi to about 47 hpi. In various embodiments, the rVSV particles produced from the packaging cells are isolated at about 44 hpi or about 45 hpi.In some cases, rVSV particles produced from packaging cells were 40 hpi, 40.1 hpi, 40.2 hpi, 40.3 hpi, 40.4 hpi, 40.5 hpi, 40.6 hpi, 40.7 hpi, 40.8 hpi, 40.9 hpi, 41 hpi, 41.1 hpi, 41.2 hpi, 41.3 hpi, 41.4 hpi, 41.5 hpi, 41.6 hpi, 41.7 hpi, 41.8 hpi, 41.9 hpi, 42 hpi, 42.1 hpi, 42.2 hpi, 42.3 hpi, 42.4 hpi, 42.5 hpi, 42.6 hpi, 42.7 hpi, 42.8 hpi, 42.9 hpi, 43.1 hpi, 43.2 hpi, 43.3 hpi, 43.4 hpi, 43.5 hpi, 43.6 hpi, 43.7 hpi, 43.8 hpi, 43.9 hpi, 44.1 hpi, 44.2 hpi, 44.3 hpi, 44.4 hpi, 44.5 hpi, 44.6 hpi, 44.7 hpi, 44.8 hpi, 44.9 hpi, 45.1 hpi, 45.2 hpi, 45.3 hpi, 45.4 hpi, 45.5 hpi, 45.6 hpi, 45.7 hpi, 45.8 hpi, 45.9 hpi, 46.1 hpi, 46.2 hpi, 46.3 hpi, 46.4 hpi, 46.5 hpi, 46.6 hpi 2.4hpi, 42.5hpi, 42.6hpi, 42.7hpi, 42.8hpi, 42.9hpi, 43hpi, 43.1hpi, 43.2hpi, 43.3hpi, 43.4hpi, 43.5hpi, 43.6hpi, 43.7hpi, 4 3.8hpi, 43.9hpi, 44hpi, 44.1hpi, 44.2hpi, 44.3hpi, 44.4hpi, 44.5hpi, 44.6hpi, 44.7hpi, 44.8hpi, 44.9hpi, 45hpi, 45.1hpi, 45. 2hpi, 45.3hpi, 45.4hpi, 45.5hpi, 45.6hpi, 45.7hpi, 45.8hpi, 45.9hpi, 46hpi, 46.1hpi, 46.2hpi, 46.3hpi, 46.4hpi, 46.5hpi, 46 .6hpi, 46.7hpi, 46.8hpi, 46.9hpi, 47hpi, 47.1hpi, 47.2hpi, 47.3hpi, 47.4hpi, 47.5hpi, 47.6hpi, 47.7hpi, 47.8hpi, 47.9hpi, 48 hpi, 48.1 hpi, 48.2 hpi, 48.3 hpi, 48.4 hpi, 48.5 hpi, 48.6 hpi, 48.7 hpi, 48.8 hpi, 48.9 hpi, 49 hpi, 49 hpi, 49.1 hpi, 49.2 hpi, 49.3 hpi, 49.4 hpi, 49.5 hpi, 49.6 hpi, 49.7 hpi, 49.8 hpi, 49.9 hpi, or 50 hpi, and in some cases, the rVSV particles produced from the packaging cells are isolated at about 15 hpi to about 35 hpi. In some cases, the rVSV particles produced from the packaging cells are isolated at about 20 hpi to about 30 hpi. In various embodiments, the rVSV particles produced from the packaging cells are isolated at about 24 hpi, or about 25 hpi.In some cases, rVSV particles produced from packaging cells were 20 hpi, 20.1 hpi, 20.2 hpi, 20.3 hpi, 20.4 hpi, 20.5 hpi, 20.6 hpi, 20.7 hpi, 20.8 hpi, 20.9 hpi, 21 hpi, 21.1 hpi, 21.2 hpi, 21.3 hpi, 21.4 hpi, 21.5 hpi, 21.6 hpi, 21.7 hpi, 21.8 hpi, 21.9 hpi, 22 hpi, 22.1 hpi, 22.2 hpi, 22.3 hpi, 22.4 hpi, 22.5 hpi, 22.6 hpi, 22.7 hpi, 22.8 hpi, 22.9 hpi, 23.1 hpi, 23.2 hpi, 23.3 hpi, 23.4 hpi, 23.5 hpi, 23.6 hpi, 23.7 hpi, 23.8 hpi, 23.9 hpi, 24.1 hpi, 24.2 hpi, 24.3 hpi, 24.4 hpi, 24.5 hpi, 24.6 hpi, 24.7 hpi, 24.8 hpi, 24.9 hpi, 25.1 hpi, 25.2 hpi, 25.3 hpi, 25.4 hpi, 25.5 hpi, 25.6 hpi, 25.7 hpi, 25.8 hpi, 25.9 hpi, 26.1 hpi, 26.2 hpi, 26.3 hpi, 26.4 hpi, 26.5 hpi, 26.6 hpi 2.4hpi, 22.5hpi, 22.6hpi, 22.7hpi, 22.8hpi, 22.9hpi, 23hpi, 23.1hpi, 23.2hpi, 23.3hpi, 23.4hpi, 23.5hpi, 23.6hpi, 23.7hpi, 2 3.8hpi, 23.9hpi, 24hpi, 24.1hpi, 24.2hpi, 24.3hpi, 24.4hpi, 24.5hpi, 24.6hpi, 24.7hpi, 24.8hpi, 24.9hpi, 25hpi, 25.1hpi, 25. 2hpi, 25.3hpi, 25.4hpi, 25.5hpi, 25.6hpi, 25.7hpi, 25.8hpi, 25.9hpi, 26hpi, 26.1hpi, 26.2hpi, 26.3hpi, 26.4hpi, 26.5hpi, 26 .6hpi, 26.7hpi, 26.8hpi, 26.9hpi, 27hpi, 27.1hpi, 27.2hpi, 27.3hpi, 27.4hpi, 27.5hpi, 27.6hpi, 27.7hpi, 27.8hpi, 27.9hpi, 28 hpi, 28.1 hpi, 28.2 hpi, 28.3 hpi, 28.4 hpi, 28.5 hpi, 28.6 hpi, 28.7 hpi, 28.8 hpi, 28.9 hpi, 29 hpi, 29.1 hpi, 29.2 hpi, 29.3 hpi, 29.4 hpi, 29.5 hpi, 29.6 hpi, 29.7 hpi, 29.8 hpi, 29.9 hpi, or 30 hpi, and in some cases, rVSV particles produced from the packaging cells are isolated at about 30 hpi to about 40 hpi.In some cases, rVSV particles produced from packaging cells were 30 hpi, 30.1 hpi, 30.2 hpi, 30.3 hpi, 30.4 hpi, 30.5 hpi, 30.6 hpi, 30.7 hpi, 30.8 hpi, 30.9 hpi, 31 hpi, 31.1 hpi, 31.2 hpi, 31.3 hpi, 31.4 hpi, 31.5 hpi, 31.6 hpi, 31.7 hpi, 31.8 hpi, 31.9 hpi, 32 hpi, 32.1 hpi, 32.2 hpi, 32.3 hpi, 32.4 hpi, 32.5 hpi, 32.6 hpi, 32.7 hpi, 32.8 hpi, 32.9 ...1 hpi, 32.2 hpi, 32.3 hpi, 32.4 hpi, 32.5 hpi hpi, 32.3hpi, 32.4hpi, 32.5hpi, 32.6hpi, 32.7hpi, 32.8hpi, 32.9hpi, 33hpi, 33.1hpi, 33.2hpi, 33.3hpi, 33.4hpi, 33.5h pi, 33.6hpi, 33.7hpi, 33.8hpi, 33.9hpi, 34hpi, 34.1hpi, 34.2hpi, 34.3hpi, 34.4hpi, 34.5hpi, 34.6hpi, 34.7hpi, 34.8hpi , 34.9hpi, 35hpi, 35.1hpi, 35.2hpi, 35.3hpi, 35.4hpi, 35.5hpi, 35.6hpi, 35.7hpi, 35.8hpi, 35.9hpi, 36hpi, 36.1hpi, 36 .2hpi, 36.3hpi, 36.4hpi, 36.5hpi, 36.6hpi, 36.7hpi, 36.8hpi, 36.9hpi, 37hpi, 37.1hpi, 37.2hpi, 37.3hpi, 37.4hpi, 37.5 hpi, 37.6hpi, 37.7hpi, 37.8hpi, 37.9hpi, 38hpi, 38.1hpi, 38.2hpi, 38.3hpi, 38.4hpi, 38.5hpi, 38.6hpi, 38.7hpi, 38.8hpi, 38.9hpi, 39hpi, 39.1hpi, 39.2hpi, 39.3hpi, 39.4hpi, 39.5hpi, 39.6hpi, 39.7hpi, 39.8hpi, 39.9hpi, or 40hpi.

[0086] VSV is generally secreted from the host or packaging cells into the culture medium. Thus, isolating the VSV product may include collection from the cell culture fluid. In some cases, the rVSV produced by the cell line may be isolated, for example, using an affinity matrix. Briefly, the method of isolating rVSV may include adding VSV to an affinity matrix to produce bound VSV, washing the bound VSV, and eluting the VSV from the affinity matrix. The present disclosure encompasses modified VSV that includes a non-naturally occurring fusion protein on the outer surface of the virus. The non-natural protein may be a fusion protein that includes an affinity tag and a viral envelope glycoprotein, or may be derived from the packaging cell. The packaging cell line may be engineered to express one or more affinity tags on its plasma membrane, which will be acquired by the virus as it buds through the membrane. One example of an affinity tag is the use of histidine residues that bind to an immobilized nickel column. Affinity tags also include antibodies. Other protocols for affinity purification may be used as known in the art, including, but not limited to, batch processing, a solution of virus and affinity matrix, pelleting the VSV binding matrix by centrifugation, and isolating the virus. Alternatively, VSV can be collected and purified from culture supernatant, and the supernatant can be clarified to remove cellular debris. One method of isolating and concentrating the virus is to pass the supernatant through a tangential flow membrane concentration. The volume of the harvest can be further reduced by pelleting through a glycerol cushion and concentration on cesium chloride or on a sucrose step gradient or other form of gradient.

[0087] In various embodiments, the suspension cell culture production method of the present disclosure comprises producing at least 1×10 6 The suspension cell culture production method of the present disclosure produces rVSV at an infectious titer of at least 1×10 plaque forming units (pfu) / mL. 7In some cases, the suspension cell culture production methods disclosed herein produce rVSV at an infectious titer of at least 1×10 pfu / mL. 8 In some cases, the suspension cell culture production method of the present disclosure produces rVSV at an infectious titer of at least 5×10 pfu / mL. 8 In some cases, the suspension cell culture production methods disclosed herein produce rVSV at an infectious titer of at least 1×10 pfu / mL. 9 In various embodiments, the suspension cell culture production method of the present disclosure produces rVSV at an infectious titer of 1×10 6 pfu / mL, 2 x 10 6 pfu / mL, 3 × 10 6 pfu / mL, 4 × 10 6 pfu / mL, 5 × 10 6 pfu / mL, 6 × 10 6 pfu / mL, 7 × 10 6 pfu / mL, 8 × 10 6 pfu / mL, 9 × 10 6 pfu / mL, 1 × 10 7 pfu / mL, 2 x 10 7 pfu / mL, 3 × 10 7 pfu / mL, 4 × 10 7 pfu / mL, 5 × 10 7 pfu / mL, 6 × 10 7 pfu / mL, 7 × 10 7 pfu / mL, 8 × 10 7 pfu / mL, 9 × 10 7 pfu / mL, 1 × 10 8 pfu / mL, 2 x 10 8 pfu / mL, 3 × 10 8 pfu / mL, 4 × 10 8 pfu / mL, 5 × 10 8 pfu / mL, 6 × 10 8 pfu / mL, 7 × 10 8 pfu / mL, 8 × 10 8 pfu / mL, 9 × 10 8 pfu / mL, 1 × 10 9 pfu / mL, 2 x 10 9 pfu / mL, 3 x 10 9 pfu / mL, 4 × 10 9 pfu / mL, 5 × 109 pfu / mL, 6 × 10 9 pfu / mL, 7 × 10 9 pfu / mL, 8 × 10 9 pfu / mL, or 9 x 10 9 with an infectious titer of pfu / mL or at least 1 x 10 6 pfu / mL, 2 x 10 6 pfu / mL, 3 × 10 6 pfu / mL, 4 × 10 6 pfu / mL, 5 × 10 6 pfu / mL, 6 × 10 6 pfu / mL, 7 × 10 6 pfu / mL, 8 × 10 6 pfu / mL, 9 × 10 6 pfu / mL, 1 × 10 7 pfu / mL, 2 x 10 7 pfu / mL, 3 × 10 7 pfu / mL, 4 × 10 7 pfu / mL, 5 × 10 7 pfu / mL, 6 × 10 7 pfu / mL, 7 × 10 7 pfu / mL, 8 × 10 7 pfu / mL, 9 × 10 7 pfu / mL, 1 × 10 8 pfu / mL, 2 x 10 8 pfu / mL, 3 × 10 8 pfu / mL, 4 × 10 8 pfu / mL, 5 × 10 8 pfu / mL, 6 × 10 8 pfu / mL, 7 × 10 8 pfu / mL, 8 × 10 8 pfu / mL, 9 × 10 8 pfu / mL, 1 × 10 9 pfu / mL, 2 x 10 9 pfu / mL, 3 × 10 9 pfu / mL, 4 × 10 9 pfu / mL, 5 × 10 9 pfu / mL, 6 × 10 9 pfu / mL, 7 × 10 9 pfu / mL, 8 × 10 9 pfu / mL, or 9 x 10 9Produce rVSV with an infectious titer of pfu / mL.

[0088] In various embodiments, the suspension cell culture production methods of the present disclosure produce rVSV at an infectious titer that is at least comparable to the titer produced by an adherent cell culture control (producing the same rVSV). In some cases, the suspension cell culture production methods of the present disclosure produce rVSV at an infectious titer that is at least 1.5-fold, at least 2-fold, at least 2.5-fold, or at least 3-fold greater than the titer produced by an adherent cell culture control (producing the same rVSV).

[0089] Vesicular stomatitis virus (VSV) VSV, a member of the Rhabdoviridae family, is a negative-stranded virus that replicates in the cytoplasm of infected cells, does not undergo genetic recombination or reassortment, has no known transformation potential, and does not integrate any part of its genome into the host. VSV contains an approximately 11 kilobase genome that encodes five proteins called nucleocapsid (N), polymerase proteins (L) and (P), surface glycoprotein (G), and peripheral matrix protein (M). The genome is tightly wrapped in the nucleocapsid (N) protein and also contains polymerase proteins (L) and (P). After infection of cells, the polymerase protein initiates the transcription of five subgenomic viral mRNAs from the negative-sense genome that code for viral proteins. The polymerase protein also participates in the replication of the full-length viral genome that is packaged into progeny virions. The matrix (M) protein binds to the RNA genome / nucleocapsid core (RNP) and also binds to the glycosylated (G) protein, which extends from the outer surface of the array of spike-like projections and is involved in binding to cell surface receptors and initiating the infection process. The recombinant VSV can be identical to the VSV discussed above, except for the deletion of the gene encoding the glycosylated (G) protein, or the mutation of the gene encoding the glycosylated (G) protein, such that a non-functional protein is produced, as discussed herein. The recombinant VSV used for infection does not encode a functional G glycoprotein, but contains the G glycoprotein in its viral envelope.

[0090] After attachment of VSV via the (G) protein to a receptor on the host surface, the virus penetrates the host and uncoats, releasing RNP particles. The polymerase protein carried with the virus binds to the 3' end of the genome and sequentially synthesizes individual mRNAs encoding N, P, M, and L, followed by the negative-sense progeny genome. The newly synthesized N, P, and L proteins associate in the cytoplasm and form an RNP core that binds to a region of the plasma membrane enriched in both M protein and glycoproteins encoded by transfected plasmids. Viral particles arise, followed by budding or release of progeny viruses.

[0091] Any of a variety of VSV strains can be utilized in the methods of the present disclosure. VSV strains include Indiana, New Jersey, Piry, Colorado, Kokkal, Chandipura, and San Juan. The complete nucleotide sequence and predicted protein sequence of the VSV genome are known and available as Genbank VSVCG, accession number J02428; NCBI Seq ID 335873, and published in Rose and Schubert, 1987, in The Viruses: The Rhabdoviruses, Plenum Press, NY. pp. 129-166. The VSV New Jersey strain is available from the American Type Culture Collection (ATCC) and has the ATCC accession number VR-159. The VSV Indiana strain is available from the ATCC and has the ATCC accession number VR-1421.

[0092] Exemplary Production Methods In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a suspension cell culture medium with a plurality of HEK293 cells selected from HEK293F cells, HEK293T cells, HEK293SF cells, or HEK293S cells, wherein the temperature of the suspension cell culture medium is about 36.6° C. to about 37.4° C.; (b) transfecting the HEK293 cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a viral envelope glycoprotein. (c) reducing the temperature of the suspension cell culture medium to about 32.5°C to about 35.5°C and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.005 to about 0.015 to infect a population of HEK293 cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express functional envelope glycoprotein, and (d) isolating the rVSV produced from the population of packaging cells 40 to 48 hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0093] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a suspension cell culture medium with a plurality of HEK293 cells selected from HEK293F cells, HEK293T cells, HEK293SF cells, or HEK293S cells, wherein the temperature of the suspension cell culture medium is about 36.8° C. to about 37.2° C.; (b) transfecting the HEK293 cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a viral envelope glycoprotein. (c) reducing the temperature of the suspension cell culture medium to about 33°C to about 35°C and introducing rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.008 to about 0.012 to infect a population of HEK293 cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express a functional envelope glycoprotein, and (d) isolating the rVSV produced from the population of packaging cells 42 to 46 hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0094] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a suspension cell culture medium with a plurality of HEK293 cells selected from HEK293F cells, HEK293T cells, HEK293SF cells, or HEK293S cells, wherein the temperature of the suspension cell culture medium is 37° C.±0.1° C.; (b) transfecting the HEK293 cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a viral envelope glycoprotein. (c) reducing the temperature of the suspension cell culture medium to about 34°C ± 0.1°C and introducing rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.01 to infect the population of HEK293 cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express functional envelope glycoprotein, and (d) isolating the rVSV produced from the population of packaging cells 43-45 hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0095] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the suspension cell culture medium has a temperature of about 36.6° C. to about 37.4° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, the viral envelope glycoprotein being selected from the group consisting of a flavivirus, an alphavirus, a togavirus, a coronavirus, a herpesvirus, a hepadnavirus, a poxvirus, a paramyxovirus, a rhabdovirus, and the like. (c) reducing the temperature of the suspension cell culture medium to about 32.5° C. to about 35.5° C. and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.005 to about 0.015 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, wherein the rVSV has been engineered not to express a functional envelope glycoprotein; and (d) isolating the produced rVSV from the population of packaging cells 40 to 48 hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0096] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the suspension cell culture medium has a temperature of about 36.8° C. to about 37.2° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, the viral envelope glycoprotein being selected from the group consisting of a flavivirus, an alphavirus, a togavirus, a coronavirus, a herpesvirus, a hepadnavirus, a poxvirus, a paramyxovirus, a rabdovirus, a rabdoi ... (c) reducing the temperature of the suspension cell culture medium to about 33° C. to about 35° C. and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.008 to about 0.012 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, wherein the rVSV has been engineered not to express a functional envelope glycoprotein; and (d) isolating the produced rVSV from the population of packaging cells 42 to 46 hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0097] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the temperature of the suspension cell culture medium is 37° C.±0.1° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, the viral envelope glycoprotein being selected from the group consisting of a flavivirus, an alphavirus, a togavirus, a coronavirus, a herpesvirus, a hepadnavirus, a poxvirus, a paramyxovirus, a rabies virus ... (c) reducing the temperature of the suspension cell culture medium to about 34° C.±0.1° C. and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.01 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, wherein the rVSV has been engineered not to express a functional envelope glycoprotein; and (d) isolating the produced rVSV from the population of packaging cells 43-45 hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0098] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the suspension cell culture medium has a temperature of about 36.6° C. to about 37.4° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, wherein the viral envelope glycoprotein optionally comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. (c) reducing the temperature of the suspension cell culture medium to about 32.5°C to about 35.5°C and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.005 to about 0.015 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express a functional envelope glycoprotein, and (d) isolating the produced rVSV from the population of packaging cells 40 to 48 hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0099] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the temperature of the suspension cell culture medium is about 36.8° C. to about 37.2° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, wherein the viral envelope glycoprotein optionally comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. (c) reducing the temperature of the suspension cell culture medium to about 33° C. to about 35° C. and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.008 to about 0.012 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express a functional envelope glycoprotein, and (d) isolating the produced rVSV from the population of packaging cells 42 to 46 hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0100] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the temperature of the suspension cell culture medium is 37° C.±0.1° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, wherein the viral envelope glycoprotein optionally comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. (c) reducing the temperature of the suspension cell culture medium to about 34°C ± 0.1°C and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.01 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express a functional envelope glycoprotein, and (d) isolating the produced rVSV from the population of packaging cells 43-45 hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0101] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the suspension cell culture medium has a temperature of about 36.6° C. to about 37.4° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, wherein the viral envelope glycoprotein is optionally selected from the group consisting of SEQ ID NO:3 or (c) reducing the temperature of the suspension cell culture medium to about 32.5°C to about 35.5°C and introducing rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.005 to about 0.015 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, wherein the rVSV has been engineered not to express functional envelope glycoprotein, and (d) isolating the produced rVSV from the population of packaging cells 40 to 48 hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0102] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the suspension cell culture medium has a temperature of about 36.8° C. to about 37.2° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, wherein the viral envelope glycoprotein is optionally selected from the group consisting of SEQ ID NO: 3 or SEQ ID NO: 4. or an Ebola virus glycoprotein comprising the amino acid sequence of SEQ ID NO:4; (c) reducing the temperature of the suspension cell culture medium to about 33° C. to about 35° C. and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.008 to about 0.012 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express functional envelope glycoprotein; and (d) isolating the produced rVSV from the population of packaging cells 42 to 46 hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0103] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the temperature of the suspension cell culture medium is 37° C.±0.1° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, wherein the viral envelope glycoprotein is optionally selected from the group consisting of SEQ ID NO:3. or an Ebola virus glycoprotein comprising the amino acid sequence of SEQ ID NO:4; (c) reducing the temperature of the suspension cell culture medium to about 34°C ± 0.1°C and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.01 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express functional envelope glycoprotein; and (d) isolating the produced rVSV from the population of packaging cells 43-45 hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0104] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a suspension cell culture medium with a plurality of HEK293 cells selected from HEK293F cells, HEK293T cells, HEK293SF cells, or HEK293S cells, wherein the temperature of the suspension cell culture medium is about 36.6° C. to about 37.4° C.; (b) transfecting the HEK293 cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a viral envelope glycoprotein. (c) reducing the temperature of the suspension cell culture medium to about 32.5°C to about 35.5°C and introducing rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.5 to about 1.5 to infect a population of HEK293 cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express a functional envelope glycoprotein, and (d) isolating the rVSV produced from the population of packaging cells 20 to 28 hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0105] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a suspension cell culture medium with a plurality of HEK293 cells selected from HEK293F cells, HEK293T cells, HEK293SF cells, or HEK293S cells, wherein the temperature of the suspension cell culture medium is about 36.8° C. to about 37.2° C.; (b) transfecting the HEK293 cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a viral envelope glycoprotein. (c) reducing the temperature of the suspension cell culture medium to about 33° C. to about 35° C. and introducing rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.8 to about 1.2 to infect the population of HEK293 cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express functional envelope glycoprotein, and (d) isolating the rVSV produced from the population of packaging cells 22 to 26 hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0106] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a suspension cell culture medium with a plurality of HEK293 cells selected from HEK293F cells, HEK293T cells, HEK293SF cells, or HEK293S cells, wherein the temperature of the suspension cell culture medium is 37° C.±0.1° C.; (b) transfecting the HEK293 cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a viral envelope glycoprotein. (c) reducing the temperature of the suspension cell culture medium to about 34°C ± 0.1°C and introducing rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) of about 1 to infect a population of HEK293 cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express a functional envelope glycoprotein, and (d) isolating the rVSV produced from the population of packaging cells 23-25 ​​hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0107] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the suspension cell culture medium has a temperature of about 36.6° C. to about 37.4° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, the viral envelope glycoprotein being selected from the group consisting of a flavivirus, an alphavirus, a togavirus, a coronavirus, a herpesvirus, a hepadnavirus, a poxvirus, a paramyxovirus, a rabdovirus, a rabdoi ... (c) reducing the temperature of the suspension cell culture medium to about 32.5° C. to about 35.5° C. and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.5 to about 1.5 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, wherein the rVSV has been engineered not to express a functional envelope glycoprotein; and (d) isolating the produced rVSV from the population of packaging cells 20 to 28 hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0108] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the suspension cell culture medium has a temperature of about 36.8° C. to about 37.2° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, the viral envelope glycoprotein being selected from the group consisting of a flavivirus, an alphavirus, a togavirus, a coronavirus, a herpesvirus, a hepadnavirus, a poxvirus, a paramyxovirus, a rhabdovirus, a rab ... (c) reducing the temperature of the suspension cell culture medium to about 33° C. to about 35° C. and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.8 to about 1.2 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, wherein the rVSV has been engineered not to express a functional envelope glycoprotein; and (d) isolating the produced rVSV from the population of packaging cells 22 to 26 hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0109] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the temperature of the suspension cell culture medium is 37° C.±0.1° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, the viral envelope glycoprotein being selected from the group consisting of a flavivirus, an alphavirus, a togavirus, a coronavirus, a herpesvirus, a hepadnavirus, a poxvirus, a paramyxovirus, a rabvirus, a rabies virus ... (c) reducing the temperature of the suspension cell culture medium to about 34° C.±0.1° C. and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 1 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, wherein the rVSV has been engineered not to express a functional envelope glycoprotein; and (d) isolating the produced rVSV from the population of packaging cells 23-25 ​​hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0110] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the suspension cell culture medium has a temperature of about 36.6° C. to about 37.4° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, wherein the viral envelope glycoprotein optionally comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. (c) reducing the temperature of the suspension cell culture medium to about 32.5°C to about 35.5°C and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.5 to about 1.5 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express a functional envelope glycoprotein, and (d) isolating the produced rVSV from the population of packaging cells 20 to 28 hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0111] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the suspension cell culture medium has a temperature of about 36.8° C. to about 37.2° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, wherein the viral envelope glycoprotein optionally has the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. (c) reducing the temperature of the suspension cell culture medium to about 33° C. to about 35° C. and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.8 to about 1.2 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express functional envelope glycoprotein, and (d) isolating the produced rVSV from the population of packaging cells 22 to 26 hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0112] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the temperature of the suspension cell culture medium is 37° C.±0.1° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, wherein the viral envelope glycoprotein optionally comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. (c) reducing the temperature of the suspension cell culture medium to about 34°C ± 0.1°C and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 1 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express a functional envelope glycoprotein, and (d) isolating the produced rVSV from the population of packaging cells 23-25 ​​hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0113] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the suspension cell culture medium has a temperature of about 36.6° C. to about 37.4° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, wherein the viral envelope glycoprotein is optionally selected from the group consisting of SEQ ID NO: 3 or SEQ ID NO: 4. or an Ebola virus glycoprotein comprising the amino acid sequence of SEQ ID NO:4; (c) reducing the temperature of the suspension cell culture medium to about 32.5°C to about 35.5°C and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.5 to about 1.5 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, wherein the rVSV has been engineered not to express functional envelope glycoprotein; and (d) isolating the produced rVSV from the population of packaging cells 20 to 28 hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0114] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the suspension cell culture medium has a temperature of about 36.8° C. to about 37.2° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, wherein the viral envelope glycoprotein optionally comprises a nucleic acid molecule selected from the group consisting of SEQ ID NO: (c) reducing the temperature of the suspension cell culture medium to about 33° C. to about 35° C. and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 0.8 to about 1.2 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, wherein the rVSV has been engineered not to express functional envelope glycoprotein; and (d) isolating the produced rVSV from the population of packaging cells 22 to 26 hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

[0115] In one embodiment, the disclosure provides a method of producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), the method comprising: (a) inoculating a plurality of HEK293F cells into a suspension cell culture medium, wherein the temperature of the suspension cell culture medium is 37° C.±0.1° C.; (b) transfecting the HEK293F cells in the suspension cell culture medium with a transfection reagent (e.g., a polyethylenimine transfection reagent) and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein, wherein the viral envelope glycoprotein is optionally selected from the group consisting of SEQ ID NO: (c) reducing the temperature of the suspension cell culture medium to about 34°C ± 0.1°C and introducing the rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) rate of about 1 to infect a population of HEK293F cells expressing the viral envelope glycoprotein, where the rVSV has been engineered not to express functional envelope glycoprotein, and (d) isolating the produced rVSV from the population of packaging cells 23-25 ​​hours post-infection, where the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV. EXAMPLES

[0116] The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventor regards as his invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure.

[0117] Example 1: Production of high-titer replication-incompetent recombinant vesicular stomatitis virus (rVSV) using suspension HEK293 cells in chemically defined cell culture medium A replication-incompetent form of rVSV lacking the glycoprotein gene was produced at high titers using a suspension cell line, using the production scheme depicted in Figure 1. A specific harvest timing was determined to eliminate the need for medium exchange prior to infection, reduce the adverse effects of residual transfection reagent on subsequent steps, and thus significantly improve the scalability of the process.

[0118] The materials included: -Cell line: Gibco CTS virus production cells (HEK293F-derived suspension cell line) -Cell culture medium: Gibco CTS LV-MAX production medium - Plasmid: pMD2.G -Transfection reagent: PEI MAX (40kDa) - Stock virus for infection: VSV ΔG:Fluc / GFP

[0119] On day 0, virus-producing cells were seeded at 1.7 E6 / mL in LV-MAX production medium in 125 mL shake flasks. The shake flasks were incubated at 37°C with 8% CO2 and a rocker speed of 125 rpm. A comparable experiment was performed with a temperature set point of 37°C, pH 7.0±0.3, dissolved oxygen level of 30%, and 22 W / m as shown in Table 1 below and in Figure 2. 3 The experiments were carried out in a 250 mL bioreactor (Ambr® 250 system, Sartorius AG) with a working volume of 200 mL using an agitation power input of 100 rpm, an air sparge rate of 0.01 volumes of air per liquid volume per minute (VVM), and an air overlay of 0.02 VVM.

[0120] [Table 1]

[0121] On day 1, cells were grown to a target cell density of 3.0 E6 / mL. pMD2.G plasmid was diluted in LV-MAX production medium to reach 2.5% of the cell culture volume after transfection with a final DNA concentration of 1.5ug / mL. PEI MAX was diluted in LV-MAX production medium to reach 2.5% of the cell culture volume after transfection with a PEI MAX to plasmid ratio of 2.5:1. Transfection mixtures were prepared by adding diluted PEI MAX to diluted plasmid. Transfection mixtures were gently swirled and then incubated at room temperature for 10 minutes before being added to shake flasks.

[0122] On day 2, cells were counted to determine viable cell density. VSV stock virus was thawed on ice. Stock virus was diluted 1000-fold in cold LV-MAX production medium, and the diluted stock virus was spiked directly into cell cultures using a multiplicity of infection (MOI) of 0.01. Immediately after infection, the incubator or bioreactor temperature was reduced to 34°C.

[0123] For production in the bioreactor, glucose was fed daily up to 5 g / L from day 2 onwards when residual glucose dropped to <2.5 g / L.

[0124] On day 4, approximately 48 hours post-infection (hpi), the crude cell culture was centrifuged at 3000×g for 15 min at 4° C. to clear and remove cellular debris, and the purified VSV material was stored at −80° C. until further use.

[0125] The process discussed above resulted in infectious titers of approximately 5E8-1E9 pfu / mL, which is comparable to results achieved in adherent cell production systems. Both shake flask and bioreactor experiments yielded comparable titers.

[0126] Example 2: Evaluation of different transfection, infection, culture and harvest parameters for viral titers during production of rVSV in suspension cell culture Shake flask experiments were performed as discussed in Example 1, with variation of parameters to assess the effect on virus titer. Multiplicity of infection (MOI) rate, amount of transfection DNA, transfection reagent, cell density at transfection, and virus dilution and infection media were evaluated in the first set of experiments, as shown in Figure 3. Of the various parameters tested, the MOI rate was identified as having the most significant effect on virus titer at 24, 33, and 48 hours post-infection (see the three columns on the right side of Figure 3).

[0127] A second set of shake flask experiments was also performed as described in Example 1, using the various parameters described in Table 2 below. Factors evaluated included choice of transfection reagent (PEI MAX or FectoVIR-AAV), whether or not a medium change was performed prior to infection, MOI rates ranging from 0.001 to 0.01, temperature shifts after infection (from an original temperature of 37°C) ranging from 34°C to 37°C, and harvest timings ranging from 24 hpi to 96 hpi (24, 36, 48, 72, and 96 hpi were evaluated). The resulting virus titers corresponding to each of these 15 runs are shown in Figure 4 as a function of transfection reagent (PEI MAX or FectoVIR), temperature shift, and harvest time (hpi). Figures 5A and 5B show the interaction profile of each of the various parameters relative to one another as a function of virus titers (pfu / mL) ranging from 2.78E8 to 4.89E8. As seen in Figures 5A and 5B, pre-infection medium change and a higher MOI (0.01) were preferred at 24 hpi, but the effect on viral titers became less significant at subsequent harvest time points. Based on the interaction profiles illustrated in Figures 5A and 5B, pre-infection medium change was deemed unnecessary and a harvest time of 44.4 hpi was preferred when combined with an MOI rate of 0.01, PEI MAX transfection reagent, and a temperature shift from 37 °C to 34 °C.

[0128] [Table 2]

[0129] Example 3: Production of high-titer replication-incompetent pseudotyped recombinant vesicular stomatitis virus (rVSV) using suspension HEK293 cells in chemically defined cell culture medium A pseudotyped, replication-incompetent form of rVSV lacking the glycoprotein gene was produced at high titers using a suspension cell line using the production scheme depicted in Figure 7. Production followed that detailed in Example 1, except that the multiplicity of infection (MOI) was increased from 0.01 to 1 and the harvest time was shortened from approximately 48 hours post infection (hpi) to approximately 24 hpi.

[0130] The process resulted in infectious titers more than three times higher than those produced from a comparable adherent cell production system (making the same rVSV). Titers were measured using a luciferase assay in Vero cells, and the results are shown in Figure 7. Briefly, for titer assessment of pseudotyped viruses, Vero cells were infected with pseudotyped viruses at a certain dilution ratio, and luciferase signals were read 24 hours post-infection. Titers are then reported as the number of RLU / mL (RLU, relative light units).

[0131] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims.

Claims

1. 1. A method for producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), comprising: (a) inoculating a suspension cell culture medium with a plurality of packaging cells; (b) transfecting the packaging cells in the suspension cell culture medium with a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein; (c) infecting the population of packaging cells expressing the viral envelope glycoprotein by introducing rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) of 0.001 to 3, wherein the rVSV has been engineered so that it does not express a functional envelope glycoprotein; and (d) isolating the rVSV produced from the population of packaging cells 15 to 65 hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

2. The MOI is: (a) is 0.001 to 1.0, or (b) is between 0.005 and 0.05; or (c) is 0.008 to 0.012, or (d) is about 0.01, or (e) is between 0.5 and 1.5, or (f) is 0.8 to 1.2, or (g) about 1. The method of claim 1 .

3. The method described in claim 1 or 2, wherein the rVSV produced from the packaging cells is isolated 40 to 50 hours after infection or 43 to 47 hours after infection.

4. The method described in claim 1 or 2, wherein the rVSV produced from the packaging cells is isolated 20 to 30 hours after infection or 23 to 25 hours after infection.

5. The packaging cell comprising: (a) is a mammalian cell, or (b) is a primate cell; or (c) is a human cell, or (d) human embryonic kidney (HEK) cells, optionally wherein the HEK cells are HEK293 cells, and further optionally wherein the HEK293 cells are HEK293F cells, HEK293T cells, HEK293SF cells, or HEK293S cells.

6. The method described in claim 1 or 2, wherein the suspension cell culture medium is a serum-free and protein-free medium. (a) the isolated rVSV is produced at an infectious titer of: (i) at least 1 x 10 6 plaque-forming units (pfu) / mL; or (ii) at least 1 x 10 pfu / mL; or (iii) at least 1 x 10 pfu / mL; or (iv) at least 5 x 10 pfu / mL; or (v) at least 1 x 10 pfu / mL, and / or (b) the isolated rVSV is produced at an infectious titer at least comparable to the titer produced by an adherent cell culture control producing the same rVSV, and optionally, the isolated rVSV is produced at an infectious titer of: (i) at least two-fold higher than the titer produced by the adherent cell culture control producing the same rVSV; or (ii) the titer is at least three times higher than the titer produced by the adherent cell culture control producing the same rVSV.

8. The viral envelope glycoprotein of claim 7, wherein: (a) is a vesicular stomatitis virus G protein, or (b) is a Class I fusion protein; or (c) is a Class II fusion protein; or (d) is a Class III fusion protein; or (e) a coronavirus spike protein, optionally wherein said coronavirus spike protein is a spike protein of SARS-CoV-2, and further optionally wherein said coronavirus spike protein comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2; or (f) an Ebola virus glycoprotein, optionally wherein the Ebola virus glycoprotein comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; or (g) the viral envelope glycoprotein of a virus selected from the group consisting of flavivirus, alphavirus, togavirus, coronavirus, herpesvirus, hepadnavirus, poxvirus, paramyxovirus, rhabdovirus, bunyavirus, filovirus, retrovirus, hepatitis virus, influenza A virus, and influenza B virus. (a) transfecting the packaging cells comprises the use of a polyethylenimine transfection reagent, or the use of a Lipofectamine transfection reagent or a FectoVIR-AAV-transfection reagent; and / or (b) the cell culture medium contains a cell density of at least 3 x 10 packaging cells / mL when the packaging cells are transfected; and / or (c) transfecting the packaging cells comprises using a DNA concentration of at least 1.5 μg / mL; and / or (d) the method of claim 1 or 2, wherein transfecting the packaging cells comprises using a transfection reagent with a DNA ratio of about 2 to about 3, and optionally transfecting the packaging cells comprises using a transfection reagent with a DNA ratio of about 2.

5.

10. The suspension cell culture medium is maintained at a temperature during inoculation and transfection, and the temperature of the suspension cell culture medium is reduced to a lower temperature upon introduction of rVSV into the suspension cell culture medium, and optionally, the temperature is: (i) at about 37°C; or (ii) about 32°C to about 36.5°C; or (iii) is about 35.5°C; or (iv) the temperature is about 34°C.

11. The method described in claim 1 or 2, wherein the suspension cell culture medium is not replaced before introducing rVSV into the suspension cell culture medium.

12. A method for producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), comprising: (a) inoculating a suspension cell culture medium with a plurality of HEK293 cells, wherein the suspension cell culture medium is at a temperature of about 37°C; (b) transfecting the HEK293 cells in the suspension cell culture medium with a polyethylenimine transfection reagent and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein; (c) reducing the temperature of the suspension cell culture medium to about 34°C and introducing rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) of about 0.01 to infect the population of HEK293 cells expressing the viral envelope glycoprotein, wherein the rVSV has been engineered so that it does not express a functional envelope glycoprotein; (d) isolating the rVSV produced from the population of packaging cells 40 to 48 hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

13. A method for producing a replication-incompetent recombinant vesicular stomatitis virus (rVSV), comprising: (a) inoculating a suspension cell culture medium with a plurality of HEK293 cells, wherein the suspension cell culture medium is at a temperature of about 37°C; (b) transfecting the HEK293 cells in the suspension cell culture medium with a polyethylenimine transfection reagent and a plasmid comprising a nucleic acid molecule encoding a viral envelope glycoprotein to produce a population of packaging cells expressing the viral envelope glycoprotein; (c) reducing the temperature of the suspension cell culture medium to about 34°C and introducing rVSV into the suspension cell culture medium at a multiplicity of infection (MOI) of about 1 to infect the population of HEK293 cells expressing the viral envelope glycoprotein, wherein the rVSV has been engineered so that it does not express a functional envelope glycoprotein; (d) isolating the rVSV produced from the population of packaging cells 20 to 28 hours post-infection, wherein the viral envelope glycoprotein is incorporated into the viral envelope of the isolated rVSV.

14. The viral envelope glycoprotein of claim 1, wherein: (a) a class I fusion protein, a class II fusion protein, or a class III fusion protein; or (b) a coronavirus spike protein, optionally wherein the coronavirus is SARS-CoV-2; or (c) is an Ebola virus glycoprotein; or 14. The method of claim 12 or 13, wherein (d) the viral envelope glycoprotein is a viral envelope glycoprotein of a virus selected from the group consisting of a flavivirus, an alphavirus, a togavirus, a coronavirus, a herpesvirus, a hepadnavirus, a poxvirus, a paramyxovirus, a rhabdovirus, a bunyvirus, a filovirus, a retrovirus, a hepatitis virus, an influenza A virus, and an influenza B virus. (a) the cell culture medium contains a cell density of at least 3 x 10 6 HEK293 cells / mL when the HEK293 cells are transfected; and / or (b) transfecting the HEK293 cells comprises using a DNA concentration of at least 1.5 μg / mL; and / or (c) transfecting the HEK293 cells comprises using a transfection reagent to DNA ratio of about 2.5; and / or (d) the HEK293 cells are HEK293F cells, HEK293T cells, HEK293SF cells, or HEK293S cells, and optionally the HEK293 cells are HEK293F cells.

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