CORONAVIRUS INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. provisional patent application No. 63/077,482, filed on September 11, 2020; the content of which is herein incorporated in entirety by reference.

FIELD OF TECHNOLOGY

[0002] The present disclosure generally relates to compositions and methods for prevention or treatment of Coronavirus infections, particularly SARS-CoV-2 viral infection. More specifically, the disclosure relates to recombinant, pseudotyped retroviral virus-like particles (VLPs) comprising a Coronavirus spike (S) protein or an immunogenic portion thereof on the viral envelope, and uses thereof as a vaccine against SARS-CoV-2.

BACKGROUND

[0003 ] The coronavirus disease 2019 (COVID- 19) epidemic that started in the Wuhan province of

China in December 2019 has quickly become pandemic and has spread worldwide. COVID-19 is a result of infection by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). In addition to its severe health threat, COVID-19 has profound socioeconomic consequences (1).

[0004] SARS-CoV-2 is the seventh coronavirus that has been identified so far. HCoV-NL63, HCoV-229E, HCoV-OC43 and HKU1 strains are constantly present in the human population and cause mild flu-like symptoms (2). In contrast, SARS-CoV and the Middle East respiratory syndrome (MERS)- CoV, like SARS-CoV-2, are pathogenic in humans. These viruses are responsible respectively for the SARS-CoV epidemic that originated in China in 2002, and the MERS-CoV epidemic that emerged 10 years later in the Middle East (3). Fortunately, these two viruses did not spread widely, with only 8,096 cases reported for SARS-CoV and 2,494 cases reported for MERS. No new cases of SARS-CoV have been reported since 2004, although MERS is still endemic in the Middle East (4).

[0005] The three pathogenic coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2) are zoonotic and have emerged from bats, with dromedary camels, palm civets and probably pangolins being the intermediary hosts for MERS-CoV, SARS-CoV and SARS-CoV-2, respectively (2, 5-11). Coronaviruses are single-stranded, positive-sense RNA viruses composed of four structural proteins: the spike (S) protein, the nucleocapsid protein, the envelope protein and the membrane protein (2). The S protein is about 180 kDa in size and assembles as a trimer at the virus surface. It is composed of two subunits, SI and S2, which are responsible for viral attachment and fusion to cells. MERS-CoV binds to the dipeptidyl peptidase 4 cell-surface receptor, whereas SARS-CoV and SARS-CoV-2 enter cells via the angiotensin-converting enzyme 2 (ACE2) receptor (11-17).

[0006] Efforts have been made to identify candidate neutralizing antibodies (Nabs) that could block the interaction of the SARS-CoV-2 S protein with the ACE2 receptor. Such Nabs could potentially be used therapeutically for infected patients (18). Several vaccine strategies are also being pursued for COVID- 19, with S protein being the major target (19-21). These vaccines are being produced from a wide variety of platforms, including RNA, DNA, recombinant proteins, viral vector-based or virus-like particles (VLPs), and live attenuated or inactivated vaccines (19-21).

[0007] Vaccines made from RNA, DNA and proteins are usually easier to manufacture than those that are viral derived, but it is generally accepted that vaccines made from the original virus (attenuated) or from VLPs can induce a better immune response (22). It is expected that induction of a strong immune response will be important for a COVID- 19 vaccine, as it will be desirable to induce high-titer Nabs for a long -lasting period of time.

[0008] Preliminary results obtained in animals and in humans have shown that both humoral and cellular immune responses can be obtained with different vaccine strategies. Nabs titers achieved in humans were comparable to those measured in the serum of COVID-19 convalescent individuals (21, 23-32). Recently, a study evaluating a potential DNA vaccine indicated that macaques were protected upon SARS- CoV-2 challenges 13 weeks after vaccination (33). However, only long-term studies in humans will reveal the efficacy of these vaccines.

[0009] Virus-like particles (VLPs) are produced by the assembly of viral proteins that do not contain genetic material and are thus unable to replicate. VLPs can be advantageous for their immunostimulatory activity: they are highly recognized by antigen-presenting cells and the repetitive arrangement of antigens on their surface is capable of inducing both innate and adaptive immune responses with a high level of Nabs (22). VLP-based vaccines have been successfully developed for Human Papilloma Virus, Hepatitis B, E, and A Viruses, and influenza virus (22). [0010] One major hurdle for development of a COVID- 19 vaccine is the capability of mass production to allow widespread immunization for the entire worldwide population. VLP -based vaccines have the potential for large-scale production and are therefore desirable for a COVID-19 vaccine.

[0011] There remains a need for an effective COVID- 19 vaccine capable of large-scale production in order to more efficiently and successfully protect against infection with SARS-CoV-2.

SUMMARY

[0012] It is an object of the present technology to ameliorate at least some of the deficiencies present in the prior art. Embodiments of the present technology have been developed based on the inventors’ appreciation that there is a need for improved compositions and methods for prevention and/or treatment of Coronavirus infection, including COVID-19.

[0013] In some aspects, the present disclosure relates to recombinant, pseudotyped retroviral virus-like particles (VLPs) comprising a Coronavirus spike (S) protein or an immunogenic portion thereof on the viral envelope, as well as compositions and uses thereof for inducing an immune response against Coronavirus infection. Specifically, we have engineered and characterized a new Moloney murine leukemia virus (MLV) VLP platform that has the potential for large-scale production of a COVID- 19 vaccine. VLPs and compositions provided herein can be used alone or as a boost with other vaccines.

[0014] According to a first broad aspect, the present disclosure relates to a recombinant, pseudotyped retroviral virus-like particle (VLP) comprising a Coronavirus spike (S) protein or an immunogenic portion thereof on the viral envelope, wherein the VLP is replication-defective, and wherein the VLP is capable of inducing an immune response against the Coronavirus in a subject. The Coronavirus may be, for example and without limitation, HCoV-NL63, HCoV-229E, HCoV-OC43, HKU1, SARS-CoV, MERS-CoV, or SARS-CoV-2. In a particular embodiment, the Coronavirus is SARS-CoV-2.

[0015] In some embodiments of the present technology, the coronavirus S protein incorporated into the VLP is the full-length S protein, or an immunogenic portion thereof. In certain embodiments, the S protein is truncated at the C-terminal, e.g., by about 19 amino acids. In an embodiment, the S protein has the sequence set forth in SEQ ID NO: 1 or 3. In an embodiment, the S protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 2 or 4.

[0016] Any suitable retroviral virus may be used. Lor example, the retroviral virus may be a leukemia virus or a lenti virus. [0017] In some embodiments, the immune response is a protective immune response, i.e., the immune response is sufficient to provide immunity against Coronavirus infection. In some embodiments, the immune response comprises a neutralizing antibody response. For example, the neutralizing antibody response may block the interaction of the Coronavirus S protein with a cellular receptor. In a particular embodiment, the Coronavirus is SARS-CoV-2 and the neutralizing antibody response blocks the interaction of the Coronavirus SARS-CoV-2 S protein with the ACE2 receptor.

[0018] In accordance with a second broad aspect, there is provided a composition comprising the VLP described herein and a pharmaceutically acceptable diluent, carrier, adjuvant or excipient.

[0019] In accordance with a third broad aspect, there is provided a vaccine for prevention or treatment of a Coronavirus infection in a subject, the vaccine comprising an effective amount of the VLP or the composition described herein. In some embodiments, the vaccine further comprises an adjuvant.

[0020] In some embodiments the subject is a mammal, such as without limitation a human.

[0021 ] In a fourth broad aspect, there are provided methods for preventing or treating Coronavirus infection, comprising administering to a subject the VLP, composition or vaccine described herein, such that Coronavirus infection is prevented or treated in the subject.

[0022] In another broad aspect, there are provided methods of inducing immunity against Coronavirus infection comprising administering to a subject the VLP, composition or vaccine described herein, such immunity against Coronavirus infection is induced in the subject.

[0023] In another broad aspect, there are provided methods of diagnosing Coronavirus infection in a subject, the method comprising the steps of: providing a sample from the subject; providing the VLP described herein; and determining whether said sample comprises antibodies or immune cells specifically binding the VLP; wherein the presence of said antibodies or immune cells in the sample indicates Coronavirus infection in the subject.

[0024] In another broad aspect, there are provided methods of detecting the presence of neutralizing antibodies against the Coronavirus in a subject, the method comprising the steps of: providing a sample from the subject; providing the VLP described herein; and determining whether said sample comprises antibodies specifically binding the VLP and/or blocking interaction of the VLP with a corresponding cellular receptor. [0025] In particular embodiments of the various aspects of the present technology, the coronavirus is SARS-CoV-2 and CO VID-19 is prevented or treated. In some embodiments, the subject is a mammal, e.g., a human.

[0026] In another broad aspect, there is provided a method for producing the VLP described herein, the method comprising the steps of: transfecting HEK293 cells with a vector for expression of MLV Gag-Pol and a vector for expression of a Coronavirus S protein or an immunogenic portion thereof, to produce 293GP-S cells; infecting the 293GP-S cells with a retroviral vector, to produce infected cells; culturing the infected cells under conditions sufficient to produce the VLP in the supernatant; and harvesting the supernatant from the infected cells, the supernatant comprising the VLP. The HEK293 cells may be transiently transfected or stably transfected with the vector for expression of the Coronavirus S protein. In some embodiments, the immunogenic portion is an S protein that is truncated at the C-terminal, such as without limitation a protein having the amino acid sequence set forth in SEQ ID NO: 3.

[0027] A VLP, a composition or a vaccine of the present technology may be administered by injection, e.g., intravenously, subcutaneously, intramuscularly, or orally. In some embodiments, a VLP, composition or vaccine of the present technology is administered in combination with a second agent for preventing or treating a coronavirus infection. The second agent may be administered concomitantly with the VLP, composition or vaccine, or they may be administered sequentially, i.e., one before the other. In some embodiments, the second agent is a second vaccine for coronavirus, e.g., a second vaccine for providing immunity against the same coronavirus, e.g., SARS-CoV-2.

[0028] Use of a VLP as described herein in the manufacture of a medicament for prevention or treatment of a coronavirus infection is also provided.

[0029] Other aspects and features of the present technology will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Further aspects and advantages of the present technology will become better understood with reference to the description in association with the following in which:

[0031] FIG. 1 shows a drawing of a c-terminal truncated version of the SARS-CoV-2 spike (AS), and a schematic representation of stable virus-like particle (VLP) producer cells that have been established by transfection of293GP cells with a plasmid encoding AS. 293GP-AS cells release VLPs and extracellular vesicles (EVs).

[0032] FIG. 2 shows expression of S protein at the surface of 293 cells. FACS analysis of 293 cells transiently transfected with plasmids encoding the Galv envelope, the full-length S protein or the AS version was conducted, as indicated. S protein was detected with an anti-S 1 antibody.

[0033] FIG. 3 shows expression of ACE2 measured by FACS analysis at the surface of 293-ACE2 cells.

[0034] FIGS. 4A-4B show transduction efficiency of different GFP pseudotyped vectors produced in transient transfections. Two days after infection of 293-ACE2 cells, titers of VSV-G-, Galv-, S- and AS-pseudotyped vectors were measured by FACS analysis (FIG. 4A) or evaluated by fluorescence microscopy (FIG. 4B). Values presented are the mean ± SD of three independent experiments. Fluorescent and bright-field pictures are displayed. The envelope pseudotype and the volume used for infection are indicated.

[0035] FIGS. 5A-5B show characterization of stable VLP producer cells. FIG. 5A shows S expression measured by FACS analysis of 293GP, 293GP-S and 293GP-AS cells with an anti-S 1 antibody. FIG. 5B shows GFP fluorescence of 293GP-Galv/GFP, 293GP-S/GFP and 293GP-AS/GFP measured by FACS analysis.

[0036] FIGS. 6A-6B show transduction efficiency of GFP pseudotyped vectors released from stable producers. FIG. 6A displays fluorescent and bright-field pictures. The envelope pseudotype and the volume used for infection are as indicated. FIG. 6B shows titers of Galv-, S- and AS-pseudotyped vectors produced from stable producers and measured by FACS analysis two days after infection. Values presented are the mean ± SD of three independent experiments.

[0037] FIG. 7 shows fusion mediated by S and AS. 293, 293GP-S and 293GP-AS were mixed with 293-ACE2 cells at a 1/10 ratio. Syncytia (arrows) were observed 24 h later.

[0038] FIGS. 8A, 8B and 8C show quantification of S and AS into VLPs. FIG. 8A shows Western blot analysis from concentrated supernatants of 293GP and 293 cells using anti-S2 and anti-p30 antibodies. Different amounts of IgG-S2 were also loaded on the gel to quantify the amount of S2 into VLPs. FIG. 8B shows differences between S and AS incorporation into VLPs. All the bands detected by the anti-S2 antibody for the quantification of S and AS were counted and normalized with the signal obtained for MLV p30. Values presented are the mean ± SD of three independent experiments analyzed twice in Western blot. FIG. 8C shows Western blot analysis of SARS CoV-2 S protein in cellular extracts. Signals for S2, S and multimeric forms of S were detected with the anti-S2 antibody. The Gag precursor pr65 was detected with the anti-p30 antibody.

[0039] FIGS. 9A-9B show incorporation of SARS-CoV-2 AS into MLV VLPs. Western blot analysis with antibodies against S2 and p30 on collected fractions separated with an iodixanol velocity gradient of 293-AS (FIG. 9A) and 293GP-AS (FIG. 9B) supernatants is shown.

[0040] FIG. 10 are Western blots showing the presence of SARS-CoV-2 AS incorporated into VLPs. Western blot analysis with antibodies directed against S2 and p30 on concentrated supernatants from stable 293GP cells expressing AS from different strains.

[0041] FIG. 11 shows a schematic representation of the fusion protein between ICAM-1 and flagellin (Flagecam) and a schematic representation of stable virus-like particle (VLP) producer cells that release VLPs and extracellular vesicles (EVs) that display at their surface AS and flagellin.

[0042] FIG. 12 shows a schematic representation of VLP-AS and VLP-AS-Flagecam. Both VLPs are made of Moloney murine leukemia (MLV) structural protein with the capsid (CA) and the matrix (MA) that are displayed, and a C-terminal truncated version of the SARS-CoV-2 spike. VLP-AS-Flagecam displays also the adjuvant flagellin.

[0043] FIG. 13 are Western blots on VLPs produced from 293GP-AS and 293GP-AS-Flagecam. VLPs and EVs were concentrated on a sucrose cushion and antibodies used were directed against S2, SI, p30 and flagellin. Different amounts of S were also loaded on the gel to quantify the amount of SI and S2 into VLPs.

DETAILED DESCRIPTION

[0044] The present technology is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the technology may be implemented, or all the features that may be added to the instant technology. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which variations and additions do not depart from the present technology. Hence, the following description is intended to illustrate some particular embodiments of the technology, and not to exhaustively specify all permutations, combinations and variations thereof.

[0045] As used herein, the singular form “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0046] The recitation herein of numerical ranges by endpoints is intended to include all numbers subsumed within that range (e.g., a recitation of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 4.32, and 5).

[0047] The term “about” is used herein explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. For example, the term “about” in the context of a given value or range refers to a value or range that is within 20%, preferably within 15%, more preferably within 10%, more preferably within 9%, more preferably within 8%, more preferably within 7%, more preferably within 6%, and more preferably within 5% of the given value or range.

[0048] The expression “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0049] As used herein, the term “Coronavirus” refers to a large group of viruses named for the crown-like spike proteins on their surface. These viruses belong to the enveloped RNA virus family Coronaviridae that animals and human share, based on the frequent mutations that lead these viruses to a rapid adaptation to one species or another. A characteristic that distinguishes these infections is a rapid spread and, often, a different pathogenicity of the viruses according to the categories, but above all to the age of the affected hosts. They consist of single -stranded positive -sense genomes, and are currently classified into four genera based on the differences in their protein sequences. Genera are Alphacoronavirus, lineage A, B (as SARS-CoV) and C Betacoronavirus (as MERS-CoV), Gammacoronavirus and Deltacoronavirus (these two latter have not been reported to cause human disease). Non-limiting examples of coronaviruses include HCoV-NL63, HCoV-229E, HCoV-OC43, HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. [0050] As used herein, the term “virus-like particle (VLP) refers to particles that closely resemble viruses, but are non-infectious because they contain no viral genetic material and are therefore unable to replicate. VLPs can be naturally occurring or can be synthesized through the individual expression of viral structural proteins, which can then self-assemble into the virus-like structure. Combinations of structural capsid proteins from different viruses can be used to create recombinant VLPs. VLPs have been successfully produced from components of a wide variety of viruses including Parvoviridae (e.g. adeno- associated virus), Retroviridae (e.g. HIV), Flaviviridae (e.g. Hepatitis C virus), Paramyxoviridae (e.g. Nipah) and bacteriophages (e.g. QP, AP205). VLPs can be produced in many cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast and plant cells. VLPs can be advantageous for their immunostimulatory activity, as generally they are highly recognized by antigen-presenting cells and the repetitive arrangement of antigens on their surface can induce both innate and adaptative immune response with a high level of neutralizing antibodies in some cases (22).

[0051] As used herein, the term “pseudotyped” refers to a virus or viral vector produced in combination with foreign viral envelope proteins. A pseudotyped VLP comprises one or more foreign viral envelope proteins in its viral envelope. Such envelope proteins can allow a pseudotyped VLP to readily enter different cell types via the corresponding host receptor. Pseudotyped VLPs generally do not carry the genetic material required to produce additional viral envelope proteins, and therefore cannot pass on the phenotypic changes to progeny viral particles. Pseudotyped VLPs are also generally replication incompetent, due to the inability to produce viral envelope proteins. Pseudotyped VLPs can be used to vaccinate animals against proteins expressed on the envelope. They can also be used for serological testing to test whether a treatment can protect host cells from infection, for example by testing in vitro or in cell culture for the presence of protective antibodies that bind and/or neutralize the foreign envelope protein.

[0052] As used herein, the term “Nab” refers to a neutralizing antibody. Neutralizing antibodies generally block the interaction of a cell-surface protein with its corresponding receptor on a host cell. In this way, a Nab may, for example, block entry of a virus into a cell expressing its corresponding receptor, thereby neutralizing the infectivity of the virus.

[0053] As used herein, the term “isolated” refers to nucleic acids or polypeptides that have been separated from their native environment, including but not limited to virus, proteins, glycoproteins, peptide derivatives or fragments or polynucleotides. For example the expression “isolated nucleic acid molecule” as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences, which naturally flank the nucleic acid (i.e. sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived.

[0054] In one embodiment, the present technology relates to recombinant, pseudotyped retroviral virus-like particles (VLPs) that are derived from a retrovirus and comprise one or more foreign viral envelope protein in the viral envelope. In certain embodiments, the VLPs are derived from a retrovirus that is not a coronavirus. In certain embodiments, the foreign viral envelope protein expressed in the viral envelope of the pseudotyped VLP is a Coronavirus spike (S) protein or an immunogenic portion thereof. The S protein may be the full-length S protein or an immunogenic portion thereof.

[0055] In an embodiment, the foreign viral envelope protein expressed in the viral envelope of the pseudotyped VLP is a SARS-CoV-2 S protein. In an embodiment, the S protein is truncated at the C- terminal, e.g., by about 19 amino acids. Non-limiting examples of S proteins and immunogenic portions thereof for use in pseudotyped VLPs in accordance with the present technology are given in Table 1.

Table 1. Exemplary sequences of coronavirus S proteins and immunogenic portions thereof. Codon-optimized nucleotide

[0056] In certain embodiments, the present technology relates to compositions of VLPs described herein for prevention or treatment of Coronavirus infections. In one embodiment, the present technology relates to a composition of a VLP described herein for prevention or treatment of SARS-CoV-2 infection.

[0057] In certain embodiments, the present technology relates to vaccines comprising the VLPs or compositions thereof described herein. In some embodiments, the present technology relates to a vaccine composition comprising a VLP described herein for prevention or treatment of a coronavirus infection, and optionally an adjuvant. In one embodiment, the present technology relates to a vaccine composition comprising a VLP described herein for prevention or treatment of SARS-CoV-2 infection, and optionally an adjuvant.

[0058] In some embodiments, the present technology relates to a VLP or composition or vaccine thereof in a form suitable for administration by injection. For example, the VLP, composition or vaccine may be administered parenterally, such as without limitation by subcutaneous, intravenous, intramuscular, intraperitoneal or intrastemal injection or using infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions).

[0059] In some embodiments, the present technology provides compositions, e.g., pharmaceutically acceptable compositions, vaccines, and the like, which include a VLP provided herein formulated together with a pharmaceutically acceptable carrier, diluent or excipient. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0060] The pharmaceutical compositions herein may include a "therapeutically effective amount" or a "prophylactically effective amount" of a VLP. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a VLP may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effect is outweighed by the therapeutically beneficial effects. A "therapeutically effective dosage" preferably inhibits a measurable parameter by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. [0061] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, e.g., immune response.

[0062] In some embodiments, the present technology provides methods for manufacturing the VLP. Such methods can include transient or stable transfection of cells with a vector expressing a Coronavirus S protein or an immunogenic portion thereof; infecting the transfected cells with a retroviral vector; and harvesting the supernatant which comprises the VLP. In an embodiment, the cells are stably transfected with the coronavirus S protein or the immunogenic portion thereof. In an embodiment, the immunogenic portion is an S protein truncated at the C-terminal, such as without limitation a protein having the sequence set forth in any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15 and 19.

[0063] In some embodiments, the present technology provides methods for preventing or treating a coronavirus infection comprising administering to a subject a VLP, composition, or vaccine described herein, such that the coronavirus infection is prevented or treated in the subject. Methods of inducing immunity against coronavirus infection in a subject, such that coronavirus infection is prevented or treated in the subject, are also provided.

[0064] In some embodiments, the present disclosure relates to the use of a composition for prevention and/or treatment of SARS-CoV-2 infection in a subject, wherein the composition comprises an effective amount of a VLP derived from a murine leukemia virus and expressing the Coronavirus S protein or an immunogenic portion thereof in the viral envelope. In some such embodiments, the virus is Moloney murine leukemia virus. In some such embodiments, the S protein has the sequence set forth in SEQ ID NO: 1 or is an immunogenic portion thereof. In some such embodiments, the S protein is truncated at the C- terminal. In some such embodiments, the S protein has the sequence set forth in SEQ ID NO: 3. In some such embodiments, the S protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16 or SEQ ID NO: 20.

[0065] In some embodiments, the present disclosure relates to the use of a vaccine for inducing immunity against SARS-CoV-2 infection in a subject, wherein the vaccine comprises an effective amount of a VLP or composition thereof, the VLP being derived from a murine leukemia virus and expressing the Coronavirus S protein in the viral envelope. In some such embodiments, the virus is Moloney murine leukemia virus. In some such embodiments, the S protein has the sequence set forth in SEQ ID NO: 1 or is an immunogenic portion thereof. In some such embodiments, the S protein is truncated at the C-terminal. In some such embodiments, the S protein has the sequence set forth in SEQ ID NO: 3. In some such embodiments, the S protein is encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 20.

Pharmaceutical Compositions and Methods

[0066] There are provided herein compositions and methods for the prevention or treatment of Coronavirus infection in a subject comprising virus-like particles (VLPs) expressing a Coronavirus spike (S) protein or an immunogenic portion thereof. Compositions and methods for inducing an immune response to a Coronavirus, e.g., SARS-CoV-2, are also provided. Methods provided herein comprise administration of a VLP as described herein to a subject in an amount effective to induce an immune response against Coronavirus, thereby reducing, eliminating, preventing, or treating disease caused by Coronavirus infection. Compositions and methods are also provided for the generation of antibodies for use in passive immunization against Coronavirus infection.

[0067] Coronavirus may be any coronavirus for which immunization, prevention or treatment is desired, including without limitation SARS-CoV, MERS-CoV, or SARS-CoV-2. In some embodiments, compositions and methods for immunization against SARS-CoV-2 are provided. In some embodiments, methods for prevention and/or treatment of COVID- 19 are provided.

[0068] The term “subject” as used herein refers to a subject in need of prevention or treatment for a Coronavirus infection. A subject may be a vertebrate, such as a mammal, e.g., a human, a non-human primate, a rabbit, a rat, a mouse, a cow, a horse, a goat, or another animal. Animals include all vertebrates, e.g., mammals and non-mammals, such as mice, sheep, dogs, cows, avian species, ducks, geese, pigs, chickens, amphibians, and reptiles. In an embodiment, a subject is a human.

[0069] "Treating" or "treatment" refers to either (i) the prevention of infection or reinfection, e.g., prophylaxis, or (ii) the reduction or elimination of symptoms of the disease of interest, e.g., therapy. "Treating" or "treatment" can refer to the administration of a composition comprising a VLP or composition as described herein, or to the administration of antibodies raised against a VLP of the present disclosure. Treating a subject with a VLP or composition thereof can prevent or reduce the risk of infection and/or recurrence and/or induce an immune response to a Coronavirus.

[0070] Treatment can be prophylactic (e.g., to prevent or delay the onset of the disease, to prevent the manifestation of clinical or subclinical symptoms thereof, or to prevent recurrence of the disease) or therapeutic (e.g., suppression or alleviation of symptoms after the manifestation of the disease). "Preventing" or "prevention" refers to prophylactic administration or vaccination with a VLP or a composition thereof in a subject who has not been infected or who is symptom -free after Coronavirus infection

[0071] As used herein, the term "immune response" refers to the response of immune system cells to external or internal stimuli (e.g., antigens, cell surface receptors, cytokines, chemokines, and other cells) producing biochemical changes in the immune cells that result in immune cell migration, killing of target cells, phagocytosis, production of antibodies, production of soluble effectors of the immune response, and the like. An “immunogenic” molecule is one that is capable of producing an immune response in a subject after administration.

[0072] “Active immunization” refers to the process of administering an antigen (e.g., an immunogenic molecule) to a subject in order to induce an immune response. In contrast, “passive immunization” refers to the administration of active humoral immunity, usually in the form of pre-made antibodies, to a subject. Passive immunization is a form of short-term immunization that can be achieved by the administration of an antibody or an antigen-binding fragment thereof. Antibodies can be administered in several possible forms, for example as human or animal blood plasma or serum, as pooled animal or human immunoglobulin, as high-titer animal or human antibodies from immunized subjects or from donors recovering from a disease, as polyclonal antibodies, or as monoclonal antibodies. Typically, immunity derived from passive immunization provides immediate protection or treatment but may last for only a short period of time.

[0073] In some embodiments, there are provided compositions and methods for active immunization against Coronavirus infection. Compositions and methods are provided for inducing an immune response to a coronavirus in a subject, comprising administering to the subject a VLP, and optionally an adjuvant, in an amount effective to induce an immune response in the subject. In one embodiment, there is provided a composition comprising an effective immunizing amount of a VLP of the present disclosure and an adjuvant, wherein the composition is effective to prevent or treat coronavirus infection in a subject in need thereof. In an embodiment, the VLP comprises the full-length spike (S) protein of a coronavirus, as described herein. In another embodiment, the VLP comprises an immunogenic portion of the S protein of a coronavirus, e.g., a C-terminal truncated S protein, e.g., the S protein with 19 amino acids deleted in the cytoplasmic tail. In an embodiment, an adjuvant is not required, i.e., compositions and methods are provided for inducing an immune response to coronavirus in a subject, comprising administering to the subject a VLP described herein and a pharmaceutically acceptable carrier, excipient, or diluent, in an amount effective to induce an immune response in the subject.

[0074] Adjuvants generally increase the specificity and/or the level of immune response. An adjuvant may thus reduce the quantity of antigen necessary to induce an immune response, and/or the frequency of injection necessary in order to generate a sufficient immune response to benefit the subject. Any compound or compounds that act to increase an immune response to an antigen and are suitable for use in a subject (e.g., pharmaceutically-acceptable) may be used as an adjuvant in compositions, vaccines, and methods of the present technology. An adjuvant may be any suitable molecule known to increase the response of the immune system.

[0075] In other embodiments, there are provided compositions and methods for passive immunization comprising a neutralizing antibody or an antigen-binding fragment thereof specific for a coronavirus S protein and prepared using the VLPs, compositions or methods provided herein. As used herein, the term "antibody" refers to any immunoglobulin or intact molecule as well as to fragments thereof that bind to a specific antigen or epitope. Such antibodies include, but are not limited to polyclonal, monoclonal, chimeric, humanized, single chain, Fab, Fab', F(ab')2, F(ab)' fragments, and/or F(v) portions of the whole antibody and variants thereof. All isotypes are encompassed by this term, including IgA, IgD, IgE, IgG, and IgM.

[0076] As used herein, the term "antibody fragment" refers to a functionally equivalent fragment or portion of antibody, i.e., to an incomplete or isolated portion of the full sequence of an antibody which retains the antigen binding capacity (e.g., specificity, affinity, and/or selectivity) of the parent antibody. Non-limiting examples of antigen-binding portions include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; (vi) an isolated complementarity determining region (CDR); and (vii) a single chain Fv (scFv), which consists of the two domains of the Fv fragment, VL and VH. Other non-limiting examples of antibody fragments are Fab' fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0077] An intact "antibody" comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHi, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0078] As used herein, the term "monoclonal antibody" or “mAb” refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. A "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions (if present) derived from human germline immunoglobulin sequences. In one aspect, human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. A "humanized antibody" refers to at least one antibody molecule in which the amino acid sequence in the non-antigen binding regions and/or the antigen-binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding properties. Humanized antibodies are typically antibody molecules from non-human species having one or more CDRs from the non-human species and a framework region from a human immunoglobulin molecule. The term “chimeric antibody” refers to an antibody in which different portions are derived from different animal species, e.g., an antibody having a variable region derived from a murine mAb and a human immunoglobulin constant region.

[0079] As used herein, the term "antigen" refers to a substance that prompts the generation of antibodies and can cause an immune response. The terms “antigen” and "immunogen" are used interchangeably herein, although, in the strict sense, immunogens are substances that elicit a response from the immune system, whereas antigens are defined as substances that bind to specific antibodies. An antigen or fragment thereof can be a molecule (i.e., an epitope) that makes contact with a particular antibody.

[0080] The terms "specific for" or "specifically binding" are used interchangeably to refer to the interaction between an antibody and its corresponding antigen. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigen or epitope). In order for binding to be specific, it should involve antibody binding of the epitope(s) of interest and not background antigens, i.e., no more than a small amount of cross reactivity with other antigens. Antibodies, or antigen-binding fragments, variants or derivatives thereof of the present disclosure can also be described or specified in terms of their binding affinity to an antigen. The affinity of an antibody for an antigen can be determined experimentally using methods known in the art. The term "high affinity" for an antibody typically refers to an equilibrium association constant (Kaff) of at least about 1 x 10 ⁷ liters/mole, or at least about 1 x 10 ⁸ liters/mole, or at least about 1 x 10 ⁹ liters/mole, or at least about 1 x 1O ¹⁰ liters/mole, or at least about 1 x 10 ¹¹ liters/mole, or at least about 1 x 10 ¹² liters/mole, or at least about 1 x 10 ¹³ liters/mole, or at least about 1 x 10 ¹⁴ liters/mole or greater. KD, the equilibrium dissociation constant, can also be used to describe antibody affinity and is the inverse of Kaff.

[0081] VLPs described herein are typically combined with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition. Pharmaceutically acceptable carriers can include a physiologically acceptable compound that acts to, e.g., stabilize, or increase or decrease the absorption or clearance rate of a pharmaceutical composition. Physiologically acceptable compounds can include, e.g., carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of glycopeptides, or excipients or other stabilizers and/or buffers. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, e.g., phenol and ascorbic acid. Detergents can also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. Pharmaceutically acceptable carriers and formulations are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. ("Remington's"). One skilled in the art would appreciate that the choice of a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the route of administration of the VLP or composition of the present disclosure, and on its particular physio-chemical characteristics.

[0082] Compositions and vaccines of the present technology may be administered by any suitable means, for example, orally, such as in the form of pills, tablets, capsules, granules or powders; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular, intraperitoneal or intrastemal injection or using infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions); nasally, such as by inhalation spray, aerosol, mist, or nebulizer; topically, such as in the form of a cream, ointment, salve, powder, or gel; transdermally, such as in the form of a patch; transmucosally; or rectally, such as in the form of suppositories. The present compositions may also be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.

[0083] It is often advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic or immunogenic effect in association with the required pharmaceutical carrier.

[0084] In an embodiment, a composition or vaccine is prepared as an injectable, either as a liquid solution or suspension, or as a solid form which is suitable for solution or suspension in a liquid vehicle prior to injection. In another embodiment, a composition or vaccine is prepared in solid form, emulsified or encapsulated in a liposome vehicle or other particulate carrier used for sustained delivery. For example, a vaccine can be in the form of an oil emulsion, a water in oil emulsion, a water-in-oil-in-water emulsion, a site-specific emulsion, a long-residence emulsion, a sticky emulsion, a microemulsion, a nanoemulsion, a liposome, a microparticle, a microsphere, a nanosphere, or a nanoparticle. A vaccine may include a swellable polymer such as a hydrogel, a resorbable polymer such as collagen, or certain polyacids or polyesters such as those used to make resorbable sutures, that allow for sustained release of a vaccine.

[0085] In some embodiments, compositions provided herein include one or more additional therapeutic or prophylactic agents for coronavirus infection. For example, a composition may contain a second agent for preventing or treating coronavirus infection. Examples of such second agents include, without limitation, antiviral agents.

[0086] In alternative embodiments, compositions of the present technology may be employed alone, or in combination with other suitable agents useful in the prevention or treatment of coronavirus infection. In some embodiments compositions of the present technology are administered concomitantly with a second composition comprising a second therapeutic or prophylactic agent for coronavirus infection. In some embodiments compositions of the present technology are administered concomitantly with a second vaccine against coronavirus infection, e.g., a second vaccine against the same coronavirus. In some such embodiments, the coronavirus is SARS-CoV-2.

[0087] As used herein, a "therapeutically effective amount" or "an effective amount" refers to an amount of a composition, vaccine, antigen, or antibody that is sufficient to prevent or treat coronavirus infection, to alleviate (e.g., mitigate, decrease, reduce) at least one of the symptoms associated with coronavirus infection, and/or to induce an immune response to a coronavirus, such that benefit to the subject is provided. The effective amount of a composition, vaccine, antigen, or antibody may be determined by one of ordinary skill in the art. Examples of dosage amounts for an adult human include, without limitation, from about 0.1 to 500 mg/kg of body weight of antigen or antibody per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 5 times per day, or weekly, or biweekly. [0088] In some embodiments, an effective amount of a composition comprising a VLP contains about 0.05 to about 1500 pg, about 10 to about 1000 pg, about 30 to about 500 pg, or about 40 to about 300 pg, or any integer between those values. For example, a VLP may be administered to a subject at a dose of about 0.1 pg to about 200 mg, e.g., from about 0.1 pg to about 5 pg, from about 5 pg to about 10 pg, from about 10 pg to about 25 pg, from about 25 pg to about 50 pg, from about 50 pg to about 100 pg, from about 100 pg to about 500 pg, from about 500 pg to about 1 mg, or from about 1 mg to about 2 mg, with optional boosters given at, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, two months, three months, 6 months and/or a year later.

[0089] A composition, vaccine, antigen or antibody may also be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid). For prophylactic purposes, the amount of peptide in each dose is selected as an amount which induces an immunoprotective response without significant adverse side effects in a typical vaccine. Following an initial vaccination, subjects may receive one or several booster immunisations adequately spaced.

[0090] It will be understood that the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion and clearance, drug combinations, and severity of the particular condition.

Diagnostic Methods

[0091] There are provided methods of diagnosing coronavirus infection based on detecting the presence of a coronavirus in a subject using a VLP as described herein. Known methods of detecting viral antigens in a sample from a subject may be used to detect the presence of an S protein binding agent (such as an antibody, neutralizing antibody, T-cell, and the like) that binds specifically to coronavirus S protein, using the VLP as a reagent, the presence of molecules that bind the coronavirus S protein incorporated in the VLP being indicative of the presence of a coronavirus infection in the subject.

[0092] A sample may be, for example, a saliva sample, a blood sample, or a urine sample. Antigens may be detected using a variety of common techniques in the art, such as without limitation detection using an enzyme immunoassay or a cellular assay.

[0093] In one embodiment, there is provided use of the VLP or composition thereof as described herein for detecting the presence of a coronavirus, e.g., SARS-CoV-2, in a subject. In another embodiment, there is provided a method of detecting the presence of a coronavirus, e.g., SARS-CoV-2 in a subject comprising: obtaining a sample from the subject, and assaying the sample for the presence of antibodies that bind specifically to the VLP, to determine the presence of the coronavirus S-protein in the subject, wherein the presence of antibodies specific for the S-protein indicates presence of coronavirus in the subject. In another embodiment, there is provided use of the VLP or composition thereof as described herein for detecting the presence of neutralizing coronavirus antibodies in a subject, using the VLP as a reagent in a cellular assay to detect neutralizing antibodies in a sample from the subject.

Kits

[0094] Also within this disclosure are kits comprising an effective amount of a VLP or a composition or a vaccine thereof. A kit can include one or more other elements including: instructions for use; other reagents, e.g., a detectable label, a therapeutic agent, etc; devices or other materials for preparing the VLP or the composition or the vaccine for administration; pharmaceutically acceptable carriers; adjuvants; and devices or other materials for administration to a subject.

[0095] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. A kit will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.

[0096] Also provided are kits for diagnosing coronavirus infection in a subject comprising VLP reagents for detecting the presence of one or more VLP-binding agent in a sample from the subject, such as an antibody, neutralizing antibody, T-cell, and the like. Instructions for use or for carrying out the diagnostic methods described herein may also be provided in a kit, as well as additional reagents, solvents, buffers, etc., required for carrying out the methods described herein.

[0097] The technology described herein is not meant to be limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It should also be understood that terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

EXAMPLES [0098] The examples below are given to illustrate the practice of various embodiments of the present disclosure. They are not intended to limit or define the entire scope of this disclosure. It should be appreciated that the disclosure is not limited to the particular embodiments described and illustrated herein but includes all modifications and variations falling within the scope of the disclosure as defined in the appended embodiments.

Example 1: Engineering and Characterization of a MLV VLP vaccine.

[0099] A new MLV-derived VLP platform that could be used for the production of a COVID-19 vaccine was established and characterized.

[00100] The SARS CoV-2 S protein migrates to the cell surface. To develop a Moloney murine leukemia virus (MLV)-derived virus-like particle (VLP) COVID-19 vaccine, we first confirmed that the SARS CoV-2 Spike (S) protein migrates to the cell surface, which is required for production of an MLV- derived VLP COVID-19 vaccine.

[00101] Stable VLP producing cell lines have been established with several spike variants for producing VLP vaccines against SAR-CoV-2. The amino acid differences among the spike variants are shown in Table 2 below.

Table 2: Amino acid differences between SARS-CoV-2 spikes used for VLP production

V2: South- African variant (Beta variant; B. 1.351). Pl and P2: Brazilian variant (Gamma variant). PldeltaCS: Pl with a mutation to inactivate cleavage site (S1/S2). Delta variant: B.1.617.2.

[00102] Since coronaviruses assemble at the ER-Golgi intermediate compartment (2), we first verified that the S protein would migrate efficiently to the cell surface. A full-length optimized version of the S gene (“S”), as well as a version with the last 57 nucleotides deleted from the 3’ end (“AS”), were cloned in an expression vector. We note that an endoplasmic reticulum (ER) retention signal is present in the cytoplasmic tail of the S protein of coronaviruses, and it has been reported that a 19-amino acid deletion at the C-terminal of SARS-CoV S protein (FIG. 1) increases the production of MLV or vesicular stomatitis (VSV) infectious particles (34-40). After transfection in 293 cells, both S versions were detected at a very similar intensity at the cell surface (FIG. 2). These results indicated that S protein was able to migrate efficiently to the cell surface, and that in these experimental conditions, the localization of S protein at the cell surface was not impaired by its ER retention signal.

[00103] Inefficient transient production of infectious recombinant MLV viruses pseudotyped with the SARS-CoV-2 S protein. The production of VLP pseudotyped with S or AS (VLP-S) was next assessed by generating green fluorescent protein (GFP) recombinant viruses produced in transient transfections. Titers were measured by FACS analysis on 293-ACE2 cells, a cell line generated by stable transfection that is 61% positive for ACE2 (FIG. 3). Titers of 3.2 x 10 ⁷ lU/ml and 1.5 x 10 ⁶ lU/ml were obtained for VSV-G- and Galv-pseudtotyped viruses, although titers of S and AS-pseudotyped viruses were below the detection limit of 10 ⁴ lU/ml (FIG. 4A). Only a few GFP cells could be observed by fluorescence microscopy after infection with the AS-pseudotyped virus and none were observed for cells infected with the S-pseudotyped vector (FIG. 4B). These results indicated that the transient production system was inefficient for generating VLP-S, even with AS.

[00104] AS-pseudotyped MLV recombinant viral particles are efficiently released from stable producer cells. We have shown previously that stable retrovirus packaging cell lines can generate Galv- pseudotyped vectors with at least 10-fold higher titers as compared to transient transfection productions (41). We therefore tested whether S or AS stably expressed in 293GP cells (293 cells that express MLV Gag-Pol) could be a better system to produce VLP-S.

[00105] Stable populations of 293GP cells expressing S and AS were generated by transfection. In these cells, S and AS were able to localize at the cell surface at even higher levels than what was found in transient transfection (FIG. 5A). A GFP retroviral vector was then introduced in these cells by infection (FIG. 5B), and titers of GFP viruses released by these new producers were measured after infecting 293- ACE2 cells. Only a few GFP positive cells could be detected by fluorescence microscopy after infection of 293-ACE2 cells with the S-pseudotyped vector, but a very high percentage of fluorescent cells was observed after infection with the AS-pseudotyped virus. A high number of GFP positive cells was seen with the Galv virus diluted 10-times as compared to the other two vectors (FIG. 6A). Titers of 1.6 x 10 ⁷ IU/ml and 10 ⁵ lU/ml were measured for the Galv and AS-pseudotyped viruses, respectively, and as expected, the S-pseudotyped vector titer was below the detection limit of 10 ⁴ lU/ml (FIG. 6B). These results showed that the production of recombinant viral particles was robust from stable producers expressing AS and inefficient with the full-length version of SARS-CoV-2 S protein.

[00106] Deletion of the 19 amino acid cytoplasmic tail of S does not enhance its fusogenicity. As producer cells express the same amount of S and AS at the cell surface, one possible explanation for the high transduction efficiency of AS-pseudotyped vectors could be due to an increased fusogenicity. The fusion of S and AS was thus assessed in a syncytia formation assay by mixing 293GP cells expressing S or AS with 293-ACE2 cells. The number and the size of syncytia evaluated one day after mixing were very similar between S and AS mixtures, and there were none with the control 293 cells (FIG. 7). These results showed that the deletion of the 19 amino acids in the cytoplasmic tail of S protein did not have a significant effect on its fusogenicity.

[00107] High amounts of SARS-CoV-2 AS protein are incorporated into MLV VLPs released from stable producer cells. For a VLP-derived SARS-CoV-2 vaccine to be a viable option, sufficient amounts of S protein must be incorporated at the surface of the released particles. Western blots were performed with an anti-S2 antibody to evaluate the quantity of S protein incorporated into VLPs produced in transient transfections and from stable producers. Two bands were detected around 90 KDa that are most likely two glycosylated forms of S2. The uncleaved S protein migrated at around 180 kDa, and two other bands above 250 kDa were also detected in the AS samples that that had more intense signals. These bands could be dimeric and trimeric forms of S, as has been suggested (17). The amount of S2 detected at the surface of VLPs produced in transient transfections or released from stable producers was much higher with the truncated version of S (AS) than with the full-length version (FIG. 8A). MLV viral particles produced in transient transfection or from stable producers were detected with an antibody against p30. A 4-fold and a 15 -fold difference was found with the transient and the stable production systems, respectively (FIG. 8B), although there was less than a 1.5-fold difference between S and AS in cellular extracts (FIG. 8C). More AS was also released as compared to the full-length protein in the supernatants of stably transfected 293 cells, however the amount of AS detected was 4-to-5 times lower than the one released from the 293GP- AS. The amount of S2 equivalent present in the supernatant of 293GP-AS cells was high and evaluated at 1.25 pg/ml using the IgG-S2 standard (FIG. 8A). These results indicated that the incorporation of S protein into MLV VLPs was especially efficient from stable producers with the truncated version of S (AS).

[00108] SARS-CoV-2 AS protein is preferentially incorporated into MLV VLPs versus extracellular vesicles. As stable transfected 293 cells were capable of releasing S or AS, we further characterized the supernatants of the 293GP-AS. We used an iodixanol velocity gradient to discriminate VLPs from extracellular vesicles (EVs) as this technique has been used in the past to successfully separate human immunodeficency viruses (HIV) from EVs (42, 43). Western blots with anti-S2 and anti-p30 antibodies were performed on the collected gradient fractions of 293-AS and 293GP-AS supernatants. S2 was detected in the top fractions from the 293-AS supernatant but there was none in the last 3 bottom fractions (FIG. 9A). A similar detection pattern was observed in the top fractions from the supernatant of 293GP-AS, but the majority of S2 came from the 2 bottom fractions in which a band for the uncleaved S protein was also detected. The p30 signal was present in these two fractions, indicating that the majority of AS released from the 293GP-AS cells was incorporated into VLPs (FIG. 9B).

[00109] Conclusions. We have established and characterized a new MLV-derived VLP platform that could be used for the production of a COVID-19 vaccine. The efficient pseudotyping of MLV particles with S protein is a prerequisite to establish a robust VLP platform. We showed that S protein could be detected at the cell surface at a similar level as the AS protein in transient transfected cells as well as in stable producers (FIG. 2 and FIG. 4A). These results indicate that S protein can bypass its natural localization and efficiently migrates at the cell surface when it is overexpressed. Despite similar amounts of S and AS at the cellular membrane, the truncated version was more efficiently incorporated into MLV viral particles. A 4- and a 15 -fold difference were obtained with VLPs produced in transient transfection experiments and from stable producers, respectively (FIG. 8B). A hypothesis which has been proposed for SARS-COV and SARS-CoV-2 is that the 19 amino-acid deletion in the cytoplasmic tail of S facilitates pseudotyping by decreasing steric interference with retroviral matrix proteins (47, 48, 50); our results invalidate this hypothesis since more AS was also found in the supernatant of 293 transfected cells that did not express MLV Gag-Pol (FIG. 8A). Parental and 293GP cells released EVs that can incorporate AS more efficiently than S (FIG. 9).

[00110] EVs released from 293GP-AS contained less than 5% total AS protein, and they would not need to be removed from vaccine preparations. Indeed, EVs could also act as immunogens, similarly to VLPs.

[00111] Titers of recombinant GFP retroviruses released from stable producers were at least 1000- fold higher with AS compared to S, despite a 15-fold difference in the amount of the two proteins incorporated at the surface of VLPs (FIG. 5A and FIG. 8B). Since we did not find major differences in fusogenicity between S and AS in a syncytia formation assay (FIG. 7), our results suggest that recombinant viruses become fully infectious when a certain threshold of S protein is incorporated at their surface. [00112] Recombinant GFP or luciferase pseudotyped retroviral viruses are commonly used to measure the activity of Nabs present in serum of infected or vaccinated people (48-50). These reagents are convenient, as unlike SARS-CoV-2 they can be manipulated in a BSL-2 laboratory. The robust production system with the 293GP-AS cell line could be highly valuable to evaluate the presence of Nabs in large cohorts.

[00113] Based on the results of a nanoparticle vaccine containing S at 5 and 25 pg that triggered a high level of Nabs in people (24), our results suggest that a vaccine derived from the VLP platform described here could be efficient with similar or less amounts of S per dose. The yield of VLPs produced from the 293GP-AS cells could be increased if a high producer clone is selected instead of a bulk population, and if cells are cultured in bioreactors in fed-batch or perfusion modes. Mutations of the furin cleavage site located between SI and S2, and the D614G variant that is now more prevalent in the infected population could also increase the amount of S protein incorporated into VLPs (50, 55).

[00114] VLPs are known to present the antigen in a protein format that seems more potent for vaccination than the protein alone. Indeed, MLV VLPs displaying the human cytomegalovirus glycoprotein B antigen could trigger 10-times more Nabs in mice than the protein alone using the same amount of antigen (56). VLP-S could also be used as a boost for other types of vaccines, like measle virus- and adenovirusbased recombinant vectors. Such combinations were highly potent for triggering Nabs against hepatitis C proteins in mice and macaques (57).

[00115] In sum, we have shown that a virus like particle (VLP) derived from Moloney murine leukemia virus (MLV) could be engineered to become a candidate SARS-CoV-2 vaccine for mass production. Firstly, we showed that a codon optimized version of S protein expressed in cells could migrate efficiently to the cell membrane, and that infectious recombinant GFP retroviruses were successfully produced, most efficiently with a 19 amino acid C-terminal deleted version of S (AS) stably expressed in 293 cells expressing MLV Gag -Pol (293GP). Incorporation of AS was 15 -times more efficient into VLPs than the full-length version. The amount of AS incorporated into VLPs released from producer cells was robust, and around 1.25 pg/ml S2 equivalent (S is comprised of SI and S2). Thus, VLPs produced with this platform have the potential to produce a pan-coronavirus vaccine that could be used alone or as a boost with other COVID-19 vaccines being developed, as well as potential for large-scale manufacturing.

[00116] Due to their viral nature, VLPs are considered having intrinsic adjuvant properties (58). However, additional adjuvants could be incorporated into VLPs by genetic engineering to increase the potency of vaccines (59). This strategy would be advantageous as it would not increase the cost of manufacturing or goods.

Example 2: Engineering and Characterization of a MLV VLP vaccine incorporating Flagellin fused to ICAM-1.

[00117] We have tested the efficiency of incorporation of flagellin fused to ICAM-1 in the VLP- AS vaccine, the construction of which is shown in FIG. 11 and FIG. 12. This molecule could be strong adjuvants to enhance immunogenicity, efficacy in weak responders, faster immune response, immune memory, dose sparing, and broader protection (59).

[00118] It has long been known that ICAM-1 is incorporated at the surface of HIV particles or lentiviral vectors (60). We have shown that ICAM-1 is also efficiently incorporated at the surface of MLV particles, and that it can be used to present heterologous proteins.

[00119] Flagellin is a protein from Salmonella that is a strong agonist of TLR5 present on antigen- presenting cells (APCs) and T cells. The binding of flagellin induces the maturation of APCs and a Thl response that leads to a stronger humoral and cellular immunity (61). The efficacy of flagellin VLPs has already been demonstrated for rabies and influenza (62, 63).

[00120] Fusion constructs (Flagecam) have been generated and cloned in the pMD2iHygro eukaryotic expression vector (this vector allows the selection of stable producers with hygromycin). Then, the incorporation into VLPs of Flagecam has been assessed by western blot from VLPs produced in stable producer cells.

[00121] The presence of S released in the supernatant of VLP producer cells was analyzed by Western blot after 100-fold concentrated/purified by ultracentrifugation through a 20% sucrose cushion for 2 h at 100,000 g at 4°C. The pellets were resuspended in phosphate-buffered saline (PBS). Samples of 20 pl were incubated 5 min at 95°C in loading buffer containing 1% SDS and 2.5% p-mercaptoethanol, and run on a 10% SDS-polyacrylamide gel (4% stacking), followed by transfer onto nitrocellulose membranes (GE Healthcare). Immunoblotting was performed with a rabbit polyclonal antibody anti-S2 (1:5000 dilution, SinoBiological, Beijing, China), a mouse monoclonal antibody anti-Sl (1:2500 dilution, R&D Systems, Minnesota, USA), a rat monoclonal antibody anti-MLV p30 produced from the hybridoma R187 (1:700 dilution; American Type Culture Collection, Manassas, VA) and a mouse anti-Flagellin (1: 1000 dilution; Invivogen, San Diego, CA). Blots were then incubated with secondary antibodies IRDylight680 goat anti-rat IgG (1 : 10,000; Invitrogen), IRDye 800CW goat anti-rabbit IgG ( 1 : 10,000; Li-Cor Biosciences, Lincoln, NE), Alexa Fluor 680 goat anti-mouse IgG (1 : 10,000; Molecular Probes, Oregon, USA) or IR Dye 800CW goat anti-mouse IgG (1: 10,000; Li-Cor Biosciences) and analyzed with the Odyssey Infrared Imaging System (Li-Cor Biosciences). The results of the Western analysis are presented in FIG. 13 which shows VLPs produced from 293GP-AS and 293GP-AS-Flagecam.

Materials and Methods for Examples 1 and 2.

[00122] Plasmids. The expression plasmid pMD2ACE2iPuro ^r containing the human angiotensinconverting enzyme (ACE2) cDNA used to generate ACE2 positive cells was constructed as follows: the ACE2 Pmel cDNA fragment obtained from the plasmid hACE2 (Addgene; #1786) was cloned in pMD2iPuro ^r opened in EcoRV.

[00123] In some embodiments the SARS-CoV-2 S gene from the Wuhan-Hu- 1 isolate (GenBank: MN908947.3) was codon optimized (Genscript, Township, NJ) and cloned into pMD2iPuro ^r in EcoRl ^/ Xhol . A shorter version with a 19-codon deletion at the C-terminal (AS) was also constructed similarly. In some other embodiments, the SARS-CoV-2 S variant gene encoding for a D614G change in the amino acid sequence could be codon optimized (Genscript, Township, NJ) and could be cloned into pMD2iPuro ^r in EcoRl/XhoI (see SEQ ID NOs: 5-8).

[00124] The expression vectors pMD2.GalviPuro ^r and pMD2.G plasmids that encode the Galv and VSV-G envelopes, and the retroviral vector plasmid containing the GFP gene under the control of the 5’ long terminal repeat sequence were as described previously (64).

[00125] Cell Lines. 293GP, 293 cells (ATCC, CRL-11268), and their derivatives expressing the ACE2 receptor (293-ACE2), S (293GP-S and 293-S), DeltaS (293GP-AS and 293-AS) and the Galv envelope (293GP-Galv) were cultured with Dulbecco’s modified Eagle’s medium (DMEM; Wisent, Canada) supplemented with 10% fetal calf serum (Life Technologies, Grand Island, NY) and antibiotics (Wisent). Bulk populations of 293-ACE2, 293-S, 293-AS, 293GP-S, 293GP-AS and 293GP-Galv were established by transfection using the calcium phosphate procedure. Briefly, subconfluent 293 or 293GP cells plated in 10-cm dishes were transfected with 20 pg of the pMD2 plasmids expressing ACE2, S, AS or Galv. Two days later, cells were selected in puromycin for aperiod of 10 days (0.5 pg/ml). Bulk populations of 293GP-S/GFP, 293GP-AS/GFP and 293GP-Galv/GFP were generated by infections of the parental cells with a GFP vector pseudotyped with VSV-G produced in transient transfections. The 3 derived cell lines were at least 86% GFP positive (FIG. 4B). [00126] Virus Productions and Infections. The production of GFP recombinant retroviruses was generated by transient transfection of 293 cells. One day prior to transfection, 3 x 10 ⁶ cells were plated in 60-mm dishes. 293 cells were transfected for 4-hours by the calcium phosphate procedure with 1 pg of envelope expression plasmids (pMD2.G, pMD2.GalviPuro ^r , pMD2.SiPuro ^r or pMD2.ASiPuro ^r ), 4 pg of Gag-Pol expression plasmid (pMD2GPiZeo ^r ) and 5 pg of RetroVec plasmid. Two days later, 2.5 ml of viral supernatants were harvested and frozen at -80°C. Recombinant viruses from stable 293GP-S/GFP, 293GP- AS/GFP and 293GP-Galv/GFP cells were also produced similarly in 60-mm dishes.

[00127] Transduction efficiency of GFP vectors was determined by scoring fluorescent-positive target cells. 293-ACE2 cells were inoculated at a density of 2 x 10 ⁵ cells per well in 24-well plates. The next day, the medium from each well was replaced with different volume of viral supernatants containing 8 pg/ml polybrene. Two days later, cells were trypsinized and analyzed by flow cytometry. Vector titers were calculated using the following formula: (N x P) x 2/(V x D). where N= Cell number at the day of infection; P= percentage of fluorescent-positive cells determined by flow cytometry; V is the viral volume applied; and D is the virus dilution factor. Titers were calculated when the percentage of fluorescentpositive cells was comprised between 2 to 20%. Alternatively, GFP positive cells were assessed under a fluorescent microscope. The 3 x 3 mosaic images of GFP and transmitted light were acquired with a Nikon TI-E inverted microscope with a PlanApo VC 20x 0.75 NA objective using a Hamamatsu Orca-ER CCD camera. Acquisition and stitching were performed with the Nikon NIS Elements 5.02 software program. The fluorescence intensity of infected cells displayed in FIG. 3B were scanned using the Fiji software.

[00128] Syncytia formation assay. 293-ACE2 cells were mixed with 293, 293GP-S and 293GP- AS at a 9/1 ratio and plated at 4 x 10 ⁵ cells/well in a 24-well plate. Fusion activity was analyzed 24 hours (h) later by phase contrast under the same microscope used for measuring the transduction efficiency.

[00129] Protein Analysis. The presence of S at the surface of 293 cells was assessed in transient transfections. Subconfluent cells in 6-well plates were transfected for 4 hours with 5 pg of S or AS plasmids by the calcium phosphate procedure, and 24 hours later, the media was replaced with serum-free media (SFM) BalanCD HEK293 (Fujifilm Irvine Scientific, Santa Ana, CA). The next day, cells were detached without trypsin by gently pipetting up and down the medium on top of the cells. A human chimeric anti-Sl antibody (Genscript; 1:200 dilution) followed by an Alexa647-conjugated goat anti-human IgG (Jackson Laboratories; 1:400) were successively incubated with cells for labelling. The dye fixable viability stain 450 (BD Biosciences, San Jose, CA, USA) was used to exclude dead cells. The presence of S was then analyzed by flow cytometry with a BD FACS Aria II (BD Biosciences). Cells transfected with a Galv expression plasmid were used as a control. The presence of stably expressed S at the cell surface of 293GP- S and 293GP-AS were also similarly analyzed by flow cytometry.

[00130] ACE2 at the surface of 293-ACE2 cells was also analyzed by FACS. Detached cells were labelled with a mouse anti-ACE2 antibody (R&D Systems, Minneapolis, MN1/200) followed by an Alexa488 goat anti-mouse (1: 1,000; Invitrogen, Carlsbad, CA).

[00131] The presence of S released in the supernatant of transiently transfected 293GP cells was analyzed by Western blot. Subconfluent cells plated in 60 mm were transfected for 4 h with 5 pg of envelope expression plasmids and 5 pg of the GFP retroviral plasmid. One day later, the media was replaced with 2.5 ml of SFM that was then harvested the following day. Supernatants were concentrated 10-fold with a 30 kDa Amicon centrifugal unit (Millipore Sigma, Oakville, Canada) and were stored at -80°C until use. The GFP fluorescence evaluated under a microscope at the time of harvest was very similar among the different transfected plates.

[00132] Supernatants from confluent 293GP-S, 293GP-AS, 293-S and 293-AS cells were also harvested and concentrated from 60-mm dishes.

[00133] Concentrated samples of 20 pl were incubated 5 min at 95°C in loading buffer containing 1% SDS and 2.5% p-mercaptoethanol, and run on a 10% SDS -polyacrylamide gel (4% stacking) followed by transfer on nitrocellulose membranes (GE Healthcare). Immunoblotting was performed with a rabbit polyclonal antibody anti-S2 (1:400 dilution, SinoBiological, Beijing, China) and a rat monoclonal antibody anti-MLV p30 produced from the hybridoma R187 (1:2,000 dilution; American Type Culture Collection, Manassas, VA). Blots were then incubated with secondary antibodies IRDylight680 goat anti-rat IgG (1: 10,000; Invitrogen) and IRDye 800CW anti -rabbit IgG (1: 10,000; Li-Cor Biosciences, Lincoln, NE) and analyzed with the Odyssey Infrared Imaging System (Li-Cor Biosciences). Serial dilutions of known amounts of S2 terminally Fc IgG tagged (BioVendor, Bmo, Czech Republic) were used for quantification.

[00134] Cell pellets of 1 x 10 ⁶ cells were resuspended in 100 pl RIPA lysis buffer containing a protease inhibitor cocktail (Roche). Samples were centrifuged for 5 min to remove cell debris and stored at -20°C until use for Western blot analysis.

[00135] Velocity Gradient. Supernatants from 293GP-AS and 293-AS cells were harvested from confluent 150-mm dishes for two consecutive days in SFM. The supernatant was filtered with .45 pm and concentrated 60-fold by ultracentrifugation for 45 min at 100,000 x g in a AH629 rotor. Pellets containing virions and EVs were resuspended in 1ml PBS containing a protease cocktail inhibitor (Roche) during 2 h at 4°C. The resuspended vesicles were layered onto a 6-18% Optiprep™ (Stemcell Technologies, Vancouver, Canada) 11 steps discontinuous velocity gradient and centrifuged for 90 min at 176,000 x g in a SW40Ti rotor as previously described (42, 43). Fractions of approximately 800 pl were collected from the bottom after punching the wall of a tube with a gauge needle, and 20 pl of each sample were analyzed by Western blot.

INCORPORATION BY REFERENCE

[00136] All references cited in this specification, and their references, are incorporated by reference herein in their entirety where appropriate for teachings of additional or alternative details, features, and/or technical background.

EQUIVALENTS

[00137] While the disclosure has been particularly shown and described with reference to particular embodiments, it will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following embodiments.

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