WO2022115503A1

WO2022115503A1 - Treatment and prevention of coronavirus infection

Info

Publication number: WO2022115503A1
Application number: PCT/US2021/060679
Authority: WO
Inventors: David Estell; Scott D. Power; Quinn Q. ZHU; Xiaochun FAN; Seung-Pyo Hong; Perry Caimi; Felipe O. BENDEZU; Zhixiong Xue
Original assignee: Dupont Nutrition Biosciences Aps; Danisco Us Inc.
Priority date: 2020-11-25
Filing date: 2021-11-24
Publication date: 2022-06-02

Abstract

Provided herein, inter alia, are recombinant Yarrowia cells comprising a nucleic acid encoding a viral protein or a fragment thereof for use in the treatment and prevention of viral disease as well as methods for making and using the same.

Description

TREATMENT AND PREVENTION OF CORONAVIRUS INFECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to IIS. Provisional Patent Application No. 63/118,219, filed November 25, 2020. the disclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] Provided herein, inter aha, are compositions for the treatment and prevention of coronavirus infection as well as methods for making and using the same.

BACKGROUND

[0003] Coronaviruses (Coronaviridae) have been long recognized as one of the causa tive agents of common cold and respiratory infections in humans and a variety of respiratory illnesses in animals. However, it has only been in the 21st century that coronavirus variants have emerged as pandemic pathogens. In 2002, the SARS-CoV virus emerged and in short order demonstrated the high infectivity characteristic of modem strains. The appearance of MERS coronavirus on the Arabian Peninsula followed a similar trajectory.

[0004] Since the outbreak ofSARS (Severe Acute Respiratory' Syndrome) coronavirus 2 (SARS- CoV-2), causing coronavirus disease (CGVID-19), the world has experienced a fast spread of this highly infec tious virus leading to a global pandemic. While approximately 15-20% of the tested cases are asymptomatic, most of the affected patients have mild symptoms that includes fever (83-98% of symptomatic cases), cough (59-82% of symptomatic cases), shortness of breath (19-55% of symptomatic eases) and muscle ache (11-44% of symptomatic cases). However, in some patients, this disease will progress to a more severe state that will develop around 8 days after the infection has occurred. The main symptoms go from dyspnea to respiratory distress with 3-29% of patients needing admission to Intensive Care Units (ICUs). The disease course then iay lead to acute respiratory distress syndrome (ARDS) (17-29% of patients hospitalized), severe sepsis with shock and in some cases multiple organ dysfunction within one week. Last, it has been estimated that the global mortality rate of infected patients is approximately 5-7%.

[0005] The challenges presented by this virus are various: its high contagiousness combined with a fair share of asymptomatic carriers allow for the infection to spread rapidly and undetected amongst the population. This leads to a. fast increase of cases in ail countries infected which has put huge pressure on existing healthcare infrastructures leading to high death tolls.

[0006] Furthermore, unlike the other coronaviruses, this variant has retained its virulence even though as an RNA virus, it is expected to undergo mutation at a relatively high frequency. In addition, the lack of anima l models of the disease has hindered the ability of vaccine developers to demonstrate efficacy and the rapid spread has called for employing shortcuts to get to human trials without a wealth of animal data.

[0007] Consequently, all infected countries, except a handful of them, went into extensive lockdowns, limiting social gathering, restricting travels and forcing most businesses to limit activities to its bare minimum and in most cases to close. Such measures have triggered the “economics of stoppage” creating a global recession with devastating consequences for the global population.

[0008] Over the past few months, government, non-governmental organizations (NGOs) and private films have refocused some of their efforts to find treatments against COVID-19. The most common approaches can be clustered into three main categories: i) passive immnnity/neutralizmg antibodies, ii) vaccines and iii) drug repurposing. What is needed, therefore, are new and effective interventions that can safely and effectively prevent and/or treat COVID-19.

[0009] The subject matter disclosed herein addresses these needs and provides additional benefits as well

SUMMARY

[0010] Provided herein, inter alia , are recombinant yeast (such as Yarrowia) cells comprising a nucleic acid encoding one or more viral proteins or fragments thereof (such as a coronavirus protein or a fragment thereof, such as a spike protein) for use in the treatment and prevention of viral disease as well as methods for making and using the same.

[0011] In some aspects, provided herein is a recombinant Yarrowia cell comprising a nucleic acid encoding a coronavirus spike protein or a fragmentthereof. In some embodiments, the Yarrowia ceil is selected from the group consisting of Yarrowia Iwbuia, Yarrowia deformans , Yarrowia Hpolytica, Yarrowia porcino, Yarrowia yokushimemis , and Yarrowia parOphonii. In some embodiments of any of the embodiments disclosed herein, the spike protein or fragment thereof is expressed miracelMariy, is secreted exiracellularly, or is displayed on the surface of the Yarrowia cell. In some embodiments of any of the embodiments disclosed herein, the coronavirus spike protein or fragment thereof is derived from a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), QC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and SARS-CoV-2 vims. In some embodiments, the coronavirus is a SARS-CoV-2 virus. In some embodiments of any of the embodiments disclosed herein, the nucleic acid encoding the coronavirus spike protein receptor binding domain (RBD) comprises SEQ ID NO: l. In some embodiments of any of the embodiments disclosed herein, the coronavirus spike protein receptor binding domain (RBD) comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the coronavirus spike protein further comprises a signal peptide selected from the group consisting of signal peptides encoded by SEQ ID NO; 6, SEQ ID NQ:8, SEQ ID NO; 10, and SEQ ID NO; 12. In some embodiments of any of the embodiments disclosed herein, the recombinant cell further comprises a nucleic acid encoding one or more additional coronavirus proteins or fragment thereof. In some embodiments, the additional coronavirus protein is a coronavirus nucleocapsid phosphoprotein or fragment thereof. In some embodiments, the coronavirus nucleocapsid phosphoprotein comprises the amino acid sequence of SEQ ID NO; 27, SEQ ID NO:28, or SEQ ID NO:29. In some embodiments of any of the embodiments disclosed herein, the coronavirus spike protein and/or additional coronavims proteins further comprises a cell surface anchoring peptide selected from the group consisting of cell surface anchoring peptides encoded by SEQ ID NO; 17, SEQ ID NO; 18, SEQ ID NO: 19, and SEQ ID NO;20. [0012] In other aspects, provided herein is a. vector comprising the nucleic acid encoding a coronavirus spike protein or fragment thereof for expression in any of the recombinant cells disclosed herein. In some embodiments, the vector further comprises a promoter encoded by SEQ ID NO:3 and/or a. terminator encoded by SEQ ID NO:4

[0013] in additional aspec ts, provided herein is a composition comprising any of the recombinant cells disclosed herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the composition is lyophilized or freeze dried. In some embodiments, the composition is encapsulated or coated, In some embodiments of any of the embodiments disclosed herein, the composition is a food product, food ingredient, dietary supplement, or medicament. In some embodiments of any of the embodiments disclosed herein, at least about 100 mg composition to at least about 10 g composition of recombinant Yarrowia cells are present in the composition. In some embodiments of any of the embodiments disclosed herein, the composition is a probiotic, In some embodiments of any of the embodiments disclosed herein, the composition has been pasteurized or heat treated.

[0014] In further aspects, provided herein is a tablet, prolonged-release capsule, prolonged- release granule, powder, sachet, or gummy comprising any of the compositions disclosed herein. In some embodiments of any of the embodiments disclosed herein, the composition is formulated for intranasal administration.

[0015] In another aspect, provided herein is a mechanical inhaler comprising any of the compositions disclosed herein.

[0016] In yet further aspects, provided herein is a kit comprising any of the compositions disclosed herein, a tablet, prolonged-release capsule, prolonged-release granule, powder, sachet, or gummy comprising any of the compositions disclosed herein; or a mechanical inhaler inhaler comprising any of the compositions disclosed herein: and b) written instructions for administration to a subject.

[0017] In still further aspects, provided herein is a method of treating or preventing illness and/or symptoms associated with a coronavirus infection in a subject in need thereof, said method comprising administering a therapeutically effective dose of any of the recombinant cells disclosed herein or any of the compositions disclosed herein to the subject, in some embodiments, said illness is a respiratory illness. In some embodiments, the respiratory illness is acute respiratory distress syndrome (ARDS). In some embodiments, the respiratory illness is pneumonia. In some embodiments of any of the embodiments disclosed herein, said symptoms are one or more of shortness of breath or difficulty breathing (dyspnea), fatigue, muscle or body aches, nausea or vomiting, diarrhea, cough, fever, loss or change of sense of smell (anosmia) and loss or change of sense of taste (ageusia). In some embodiments of any of the embodiments disclosed herein, said preventing or treating is achieved by stimulation of the immune system in the subject when in contact with any of the recombinant cells disclosed herein . In some embodiments, said stimulation of the immune system is a T-cell-mediated immune response and/or a B-cell-mediaied immune response. In some embodiments of any of the embodiments disclosed herein, the corona virus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU 1 (beta coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and SARS-CoV-2 virus, hr some embodiments, the coronavirus is a SARS-CoV-2 virus. In some embodiments of any of the embodiments disclosed herein, the subject has one or more pre-existing conditions selected from the group consisting of obesity, type II diabetes, chronic lung disease or moderate to severe asthma, heart conditions, immunocompromised, chronic kidney disease and liver disease. In some embodiments of any of the embodiments disclosed herein, the sub ject is 65 years of age or older and/or is a resident in a musing home or long-term care facility or jail or prison or university'. In some embodiments of any of the embodiments disclosed herein, the subject is a human. In some embodiments of any of the embodiments disclosed herein, the subject is a non-human animal.

[0018] In other aspects, provided herein are any of the recombinant cells provided herein for use in preventing or heating illness and/or symptoms associated with coronaviruses in a subject in need thereof, In some embodiments, said illness is a respiratory illness. In some embodiments of any of the embodiments disclosed herein, the respiratory illness is acute respiratory distress syndrome (ARDS). In some embodiments, the respiratory illness is pneumonia. In some embodiments of any of the embodiments disclosed herein, said symptoms are one or more of cough, fever, shortness of breath or difficulty breathing (dyspnea), fatigue, muscle or body aches, nausea or vomiting, diarrhea, loss or change of sense of smell ( anosmia), and loss or change of sense of taste (ageusia). In some embodiments of any of the embodiments disclosed herein, said preventing or treating is achieved by stimulation of the immune system in the subject when in contact with any of the recombinant ceils disclosed herein. In some embodiments, said stimulation of the immune system is a. T-cell-mediated immune response and/or a B-celi- mediated immune response, in some embodiments of any of the embodiments disclosed herein, the coronavirus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus). HKU1 (beta coronavirus). MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and SARS-CoV-2 virus. In some embodiments, the coronavirus is a SARS-CoV-2 virus. In some embodiments of any of the embodiments disclosed herein, the subject has one or more pre-existing conditions selected from the group consisting of obesity, type II diabetes, chr ome lung disease or moderate to severe asthma, heart conditions, inummoconipromised, chronic kidney disease, liver disease. In some embodiments of airy of the embodiments disclosed herein, the subject is 65 years of age or older and/or is a resident in a nursing home or long-term care facility or jail or prison or a university.

In some embodiments of any of the embodiments disclosed herein, the subject is human.

[0019] In yet another aspect, provided herein is airy of the compositions disclosed herein for use in preventing or treating illness and/or symptoms associated with corona viruses in a subject in need thereof. In some embodiments, said illness is a respiratory illness. In some embodiments of any of the embodiments disclosed herein, the respiratory illness is acute respiratory distress syndrome (ARDS). In some embodiments, the respiratory illness is pneumonia. In some embodiments of any of the embodiments disclosed herein, said symptoms are one or more of cough, fever, shortness of breath or difficulty breathing (dyspnea), fatigue, muscle or body aches, nausea or vomiting, diarrhea, loss or change of sense of smell (anosmia), and loss or change of sense of taste (ageusia). In some embodiments of any of the embodiments disclosed herein, said preventing or treating is achieved by stimulation of the immune system in the subject when in contact with any of the compositions disc losed herein. In some embodiments, said stimulation of the immune system is a T-cell-mediated immune response and/or a B-cell-mediated immune response. In some embodiments of any of the embodiments disclosed herein, the coronavirus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus),

OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and SARS-CoV-2 virus. In some embodiments, the coronavints is a SARS-CoV-2 virus. In some embodiments of any of the embodiments disclosed herein, the subject has one or more pre-existing conditions selected from the group consisting of obesity, type II diabetes, chronic lung disease or moderate to severe asthma, heart conditions, immunocompromised, chronic kidney disease, liver disease. In some embodiments of any of fee embodiments disclosed herein, the subject is human . In some embodiments of any of the embodiments disclosed herein, the subject is 65 years of age or older anchor is a resident in a nursing home or long-term care facility or jail or prison or a university.

[0020] In other aspects, provided herein is a use of any of the recombinant cells provided herein or any of fee compositions provided herein for preventing or treating illness and/or symptoms associated with coronaviruses in a subject in need thereof. In some embodiments of any of the embodiments disclosed herein, said illness is a respiratory illness. In some embodiments of any of the embodiments disclosed herein, the respiratory illness is acute respiratory distress syndrome (ARDS). In some embodiments of any of the embodiments disclosed herein, the respiratory illness is pneumonia. In some embodiments of any of the embodiments disclosed herein, said symptoms are one or more of cough, fever , shortness of breath or difficulty breathing (dyspnea), fatigue, muscle or body aches, nausea or vomiting, diarrhea, loss or change of sense of smell (anosmia), and loss or change of sense of taste (ageusia). In some embodiments of any of the embodiments disclosed herein, said preventing or treating is achieved by stimulation of the immune system in the subject when in contact with any of the recombinant cells disclosed herein. In some embodiments, said stimulation of the immune system is a T-cell-mediated immune response and/or a B-cell-mediated immune response. In some embodiments of any of the embodiments disclosed herein, the coronavirus is selected from fee group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and SARS-CoV-2 virus. In some embodiments, the coronavirus is SARS-CoV-2 virus. In some embodiments of any of the embodiments disclosed herein, the subject has one or more preexisting conditions selected from the group consisting of obesity, type II diabetes, chronic lung disease or moderate to severe asthma, heart conditions, immunocompromised, chronic kidney disease, liver disease. In some embodiments of any of the embodiments disclosed herein, the subject is 65 years of age or older and/or is a resident in a musing home or long-term care facility or jail or prison or a university. In some embodiments of any of the embodiments disclosed herein, the subject is human. In some embodiments of any of the embodiments disclosed herein, the subject is a non-human animal.

[0021] In another aspect, provided herein is a recombinant Yarrowia cell comprising one or more nucleic acids encoding one or more viral proteins or fragments thereof. In some embodiments, the Yarrowia cell is selected from the group consisting of Yarrowia bubula, Yarrowia deformans, Yarrowia Hpohtica, Yarrowia porcina, Yarrowia yahishimens is. and Yarrowia parophonii. In some embodiments of any of the embodiment s disclosed herein, the viral proiein(s) or fragments) thereof are expressed intracellularly, secreted extracellularly, or displayed on the surface of the Yarrowia cell. In some embodiments of any of the embodiments disclosed herein, the viral protein is derived from an influenza virus. In some embodiments, the influenza virus is influenza virus A, influenza virus B, influenza virus C, or influenza virus D. In some embodiments, the influenza virus is influenza virus A and the viral protein(s) or fragments thereof are derived from strain H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, and/or H6N1. In some embodiments of any of the embodiments disclosed herein, the viral proiein(s) are hemagglutinin and/or neuraminidase. In some embodiments of any of the embodiments disclosed herein, the viral protein(s) further comprise one or more cell surface anchoring peptide selected from the group consisting of cell surface anchoring peptides encoded by SEQ ID NO; 17, SEQ ID NO: 18, SEQ ID NO; 19, and SEQ ID NO.20.

[0022] In further aspects, provided herein is a vector comprising the nucleic acid encoding one or more viral proteins or fragment thereof for expression in any of the recombinant cells provided herein as well as a composition comprising one or more viral proteins or fragment thereof disclosed herein and/or a tablet, prolonged-release capsule, prolonged-release granule, powder, sachet, or gummy comprising one or more viral proteins or fragment thereof disclosed herein.

[0023] In another aspect, provided herein is method of treating or preventing illness and/or symptoms associated with a respiratory viral infection in a subject in need thereof, said method comprising administering a therapeutically effective dose of any of the recombinant cells disclosed herein or any of the compositions disclosed herein. [0024] In a further aspect, provided herein is a method for producing a coronavirus spike protein or fragment thereof, the method comprising culturing any of the recombinant Yarrowia cells disclosed herein in media under suitable conditions for the production of the spike protein or fragment thereof, wherein the spike protein or fragment thereof is secreted extracellularly In some embodiments, the method further comprises recovering the spike protein or fragment thereof fr om the media.

[0025] In still further aspects, provided herein is a composition comprising a recombinant coronavirus spike protein or fr agment thereof produced and extracellularly secreted by any of the recombinant Yarrowia cells disclosed herein and a pharmaceutically acceptable earner or excipient.

[0026] In another aspect, provided herein is a tab let, prolonged-release capsule, prolonged- release granule, powder, sachet, or gummy comprising a recombinant coronavirus spike protein or fragment thereof produced and extracellularly secreted by any of the recombinant Yarrowia cells disclosed herein.

[0027] In another aspect, provided herein is a mechanical inhaler comprising a recombinant coronavirus spike protein or fr agment thereof produced and extracellularly secreted by any of the recombinant Yarrowia cells disclosed herein and a pharmaceutically acceptable carrier or excipient.

[0028] In other aspects, provided herein is a method of treating or preventing illness and/or symptoms associated with a coronavirus infection in a subject in need thereof said method comprising administering a therapeutically effective dose of composition comprising a recombinant coronavirus spike protein or fr agment thereof produced and extracellularly secreted by any of the recombinant Yarrowia cells disclosed herein to the subject.

[0029] An yet another aspect, provided herein is a method for producing one or more viral proteins or fragments thereof the method comprising culturing the recombinant Yarrowia cells disclosed herein in media under suitable conditions for the production of one or more viral proteins or fragments thereof, wherein one or more viral proteins or fr agments thereof is secreted extracellularly. In some embodiments, the method further comprises recovering the one or more viral proteins or fragments thereof from the media. In some embodiments of any of the embodiments disclosed herein, the viral protein is derived from an influenza virus. In some embodiments, the influenza virus is influenza virus A, influenza virus B, influenza virus €, or influenza, virus D. In some embodiments , the influenza virus is influenza virus A and the viral protein(s) or fragments thereof are derivedfrom strain H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2. H7N2, H7N3, H10N7, H7N9, and/or H6N1. In some embodiments of any of the embodiments disclosed herein, the viral profein(s) are hemagglutinin and/or neuraminidase.

[0030] In still further aspects, provided herein is a composition comprising a recombinant viral protein or fragment thereof produced and extracellularly secreted by any of the recombinant Yarrowia cells disclosed herein and a pharmaceutically acceptable carrier or excipient. In some embodiments of any of the embodiments disclosed herein, the viral protein is derived from an influenza virus. In some embodiments, the influenza virus is influenza virus A, influenza virus B, influenza virus C, or influenza virus D. In some embodiments, the influenza vires is influenza virus A and the viral proteinis) or fragments thereof are derived fr om strain H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, and/or H6N1. In some embodiments of any of the embodiments disclosed herein, the viral protein(s) are hemagglutinin and/or neuraminidase.

[0031] In yet another aspect, provided herein is a tablet, prolonged-release capsule, prolonged- release granule, powder, sachet, or gummy comprising a composition comprising a recombinant viral protein or fragment thereof produced and extracellularly secreted by any of the recombinant Yarrawkt cells disclosed herein. In some embodiments of any of the embodiments disclosed herein, the viral protein is derived from an influenza vires. In some embodiments, the influenza vires is influenza vires A, influenza virus B, influenza vires C, or influenza vires D. In some embodiments, the influenza vires is influenza vires A and the viral protein(s) or fragments thereof are derived from strain H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7. H7N9, and/or H6NL In some embodiments of any of the embodiments disclosed herein, the viral protein(s) are hemagglutinin and/or neuraminidase.

[0032] In further aspects, provided herein is a mechanical inhaler comprising a composition comprising a recombinant viral protein or fragment thereof produced and extracellularly secreted by any of the recombinant Yarrowia cells disclo sed herein and a pharmaceutically acceptable excipient, in some embodiments of any of the embodiments disclosed herein, the viral protein is derived from an influenza virus. In some embodiments, the influenza virus is influenza virus A, influenza virus B, influenza virus C, or influenza virus D. In some embodiments, the influenza virus is influenza virus A and the viral proteinCs) or fragments thereof are derived from strain. H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, and/or H6N1. In some embodiments of any of the embodiments disclosed herein, the viral protein(s) are hemagglutinin and/or neuraminidase.

[0033] In another aspect, provided herein is a method of treating or preventing illness and/or symptoms associated with a respiratory viral infection in a subject in need thereof said method comprising administering a therapeutically effective dose of a c omposition comprising a recombinant viral protein or fragment thereof produced and exiracellular!y secreted by any of the recombinant Yarrowia cells disclosed herein and a pharmaceutically acceptable excipient. In some embodiments of any of the embodiments disclosed herein, the viral protein is derived from an influenza virus. In some embodiments, the influenza virus is influenza virus A, influenza virus B, influenza virus C, or influenza virus D. In some embodiments, the influenza virus is influenza virus A and the viral protein(s) or fragments thereof are derived from strain H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, and/or H6N1. In some embodiments of any of the embodiments disclosed herein, the viral protein(s) are hemagglutinin and/or neuraminidase.

[0034] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0035] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entiles, etc.) are referenced. Tire disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference m their entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG, 1 depicts Western blot detection of SP273S Intracellular Expression in Yarrowia. Lane 1: Molecular weight marker; Lanes 2: Wild type untransformed Yarrowia; Lanes 3 and 10 Recombinant Spike protein (1 to 674 AA) senes as positive controls (Catalog #: PI 524, BioVision ); Lane 3-9 and 11-15: Cell lysates of different Yarrowia isolates transformed with DNA fragment of pZP2-SP273S.

[0037] FIG. 2 depicts Western blot detection of SP273S Secreted Expression from culture supernatants when using LIP2 and MFa signal sequences. Lane 1 : Molecular weight marker; Lanes 2 & 3: Wild type untransformed Yarrowia control and GFP transformed Yarrowia', Lanes 4 to 9: Culture supernatant of different Yarrowia isolates transformed with DNA fragment of pL2SP273; Lanes 10 tol5: Culture supernatant of different Yarrowia isolates transformed with DNA fragment of pMfSP273.

[0038] FIG. 3 depicts Western blot detection of SP273S Secreted Expression from culture supernatants when using SUC2 and XPR2 signal sequences. Lane 1: Molecular weight marker; Lanes 2 & 3: Wild type imtransformed Yarrowia control and GFP fransfomied Yarrowia; Lanes 4 to 8: Culture supematant of different Yarrowia isolates transformed with DNA fragment of pS2SP273 ; Lane 9: Wild type untransformed Yarrowia control; Lanes 10 to 12: Culture supematant of different Yarrowia isolates transformed with DNA fragment of pX2SP273., Left side thin arrow indicates non-specific band in all strains.

[0039] FIG. 4A and FIG, 4B depict Western blot detection of SP273S Displayed Expression from cultures when using PIRl (FIG. 4A) and CWP1 (FIG. 4B) anchors. FIG. 4A: Lane 1; Intracellular expressed SP273S, Lane 2: Molecular weight marker; Lanes 3: Wild type imtransformed Yarrowia control; Lanes 4 and 5 : Cell lysates of different Yarrowia isolates fransfomied with DNA fragment of pX2SP273PR. FIG. 4B: Lane 1 : Molecular weight marker; Lane 2: Intracellular expressed SP273S, Lanes 3; Wild type untransformed Yarrowia control; Lanes 4: Yarrowia transformed with DNA fragment of pX2SP273W.

[0040] FIG. 5 depicts immunofluorescence microscopy of Yarrowia cells with surface displayed SP273 with CWP1 or PIRl anchor as well as wild type control. [0041] FIG. 6 depicts Western blot detection for Yarrowia transformant expressing the SP273 protein intracellularly, at high level. Lane M: Molecular weight marker; Lane 1-5: Cell lysates of different Yarrowia isolates transformed with DNA fragment of pZP2-SP273AU.

[0042] FIG. 7 depicts a FACS analysis histogram of Yarrowia cells wiidtype control and SP273 displayed with two different anchor protein domains, CWP1 and PIR1, SP045 and SP046 strains respectively. On X-axis is the signal of fluorescence and Y-axis is the relative abundance of cells.

DETAILED DESCRIPTION

[0043] Mucosal IgA is broadly cross-reactive against microbiota and helps to maintain microbiota homeostasis within the host. Without being bound to theory, by ingesting or inhaling microorganisms that have been engineered to express homologous epitopes with respiratory viruses, such as coronaviruses, including SARS-CoV-2, surface polypeptides (mainly S protein), it could be possible to induce cross-reactive IgA antibodies that could treat or reduce the risk of infection by cross-reacting with viral (e.g. SARS-CoV-2 or other coronavirus virions) at mucosal surfaces. Mucosal IgA antibodies also function in transporting viral particles from host side of the epithelium to microbiota side via polymeric Ig receptor expressed in epithelial cells.

[0044] Accordingly, in order to overcome the challenges presented by the current outbreak of SARS-CoV-2 and other coronavirus and/or influenza virus caused/associated respiratory illness or disease, the inventors believe that a key to a successful outcome for preventing or treating the population against coronavirus associated severe respiratory syndrome disease in general and COVID-19 specifically will be to provide a product that would: (i) enable the immune system to (a) secrete antibodies against respiratory viruses (e.g., SARS-CoV-2), (b) stimulate pre-existing cross-reactive T cells (e.g. SARS-CoV-2 cross-reactive T cells), and (3) develop novel cross- reactive T cells (e.g. SARS-CoV-2 cross-reactive T cells) using microbes engineered to express one or more viral (e.g. SARS-CoV-2) antigens (for example, S protein or fragment thereof) and/or (ii) activate the immune system to stimulate anti-viral immunity before infection with respiratory viruses (e.g. SARS-CoV2). The present inventors believe that this approach can be used alone and/or in addition to other approaches being considered currently by other research groups, like vaccines or monoclonal antibody therapies. I. Definitions

[0045] As used herein, “recombinant” or “non-naturaT refers to an organism, microorganism (such as Yarrmvia), cell, nucleic acid molecule, or vector that has at least one engineered genetic alteration or has been modified by the introduction of a heterologous nucleic acid molecule, or refers to a cell that has been altered such that the expression of a heterologous or endogenous nucleic acid molecule or gene can be controlled. Recombinant also refers to a cell that is derived from a non-natural cell or is progeny of a non-natur al cell having one or more such modifications. Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins (such as the S protein or a fragment thereof), or other nucleic acid molecule additions, deletions, substitutions or other functional alteration of a cell's genetic material. For example, recombinant cells may express genes or other nucleic acid molecules that are not found in identical or homologous form within a native (wild-type) cell (e.g. , a fusion or chimeric protein), or may provide an altered expression pattern of endogenous genes, such as being over-expressed, under-expressed, minimally expressed, or not expressed at all.

[0046] The term “coronavirus infection” or “CoV infection,” as used herein, refers to infection with a coronavirus such as SARS-CoV-2, MERS-CoV, or SARS-CoV. The term includes coronavirus respiratory tract infections, often in the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastro-intestinal symptoms such as diarrhea, organ failure (kidney failure and renal dysfunction), septic shock, and death in severe eases.

[0047] The term “CoV-S”, also called “S” or “S protein” refers to the spike protein of a coronavirus, and can refer to specific S proteins such as SARS-CoV-2-S, MERS-CoV S, and SARS-CoV S. The SARS-CoV -2- Spike protein is a 1273 amino acid type I membrane glycoprotein which assembles into trimers that constitute the spikes or peplomers on the surface of the enveloped coronavirus particle. The protein has two essentia l functions, host receptor binding and membrane fusion, which are attributed to the N-terminal (S1) and C- terminal (S2) halves of the S protein. CoV-S binds to its cognate receptor via a receptor binding domain (RBD) present in the SI subunit. In some embodiments, the amino acid sequence of the SARS- CoV-2 spike protein for use in the recombinant Yarrowia cells disclosed herein is the amino acid sequence of the S protein RBD. In oilier embodiments, the amino acid sequence of the SAE.S- CoV-2 spike protein for use in the recombinant Yarrowia cells disclosed herein is exemplified by flie amino acid sequence of SEQ ID NO: 2. The term “CoV-S” includes protein valiants of CoV spike protein isolated from different CoV isolates, recombinant CoV spike protein or a fragment thereof as well as “escape variants” that have evolved resistance to antibody binding and neutralization. The term also encompasses CoV spike protein or a fragment thereof coupled to, for example, a signal sequence or an anchoring protein.

[0048] The term “virus” includes any virus whose infection in the body of a subject is treatable or preventable by administration of any of the recombinant Yarrowia cells comprising a nucleic acid encoding a viral surface antigen (e.g. a coronavirus spike protein or a fragment thereof or an influenza virus antigen) disclosed herein (e.g., wherein infectivity of the virus is at least partially dependent on CoV-S). In an embodiment of the invention, a “virus” is any vires that expresses spike protein (e.g., CoV-S). The term “virus” also includes a CoV-S -dependent respiratory virus which is a virus that infects the respiratory tissue of a subject (e.g. , upper and/or lower respiratory tract, trachea, bronchi, lungs) and is treatable or preventable by administration of an anti-CoV-S antibody or antigen-binding fragment thereof. For example, in an embodiment of the invention, vires includes coronavirus, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). SARS-CoV (severe acute respiratory syndrome coronavirus), and MERS-CoV (Middle East respiratory' syndrome (MERS) coronavirus). Coronaviruses can include the genera oa alphacoronavruses , betaeorona viruses, gammacorona viruses, and deltacoronaviruses. In some embodiments, the recombinant Yarrowia cells comprising a nucleic acid encoding a coronavirus spike protein or a fragment thereof disclosed herein can treat or prevent infection with an alphacoronavirus, a betacoronavirus, a gammacoronavirus, and/or a deltacoronavirus. In certain embodiments, this treatment and/or prevention can be specific for a particular genus of coronavirus or for a particular subgroup of a genus. “Viral infection” refers to the invasion and multiplication of a vires in the body of a subject.

[0049] The term “gene” refers to a nucleic acid molecule that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' noncoding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, hut that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that lias been introduced into the genome by a transformation procedure.

[0050] The term “coding sequence” refers to a nucleotide sequence which codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5^' non-coding seequences), within, or downstream (3^* non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, RNA processing site, effector binding sites, and stem-loop structures.

[0051] The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid molecule so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence, i.e., the coding sequence is under the transcriptional control of the promoter. Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0052] The terms “regulatory sequence” or “control sequence” are used interchangeably herein and refer to a segment of a nucleotide sequence which is capable of increasing or decreasing expression of specific genes within an organism. Examples of regulatory sequences include, but are not limited to, promoters, signal sequence, operators and the like. As noted above, regulatory sequences can be operably linked in sense or antisense orientation to the coding sequence/gene of interest.

[0053] “Promoter” or “promoter sequences” refer a regulatory sequence feat is involved in binding RNA polymerase to initiate transcription of a gene. The promoter may be an inducible promoter or a constitutive promoter. A preferred promoter used in fee invention is the FBAINm Promoter (SEQ 11) NO:3), [0054] The “3' non-coding sequences” refer to DNA sequences located downstream of a coding sequence and include sequences encoding regulatory signals capable of affecting niRNA processing or gene expression, such as termination of transcription. A preferred terminator used in the invention is the Pex20 Terminator (SEQ ID NO:4).

[0055] As used herein, "microorganism" or “microbe” refers to a bacterium, a. fungus, a virus, a protozoan, and other microbes or microscopic organisms, in some embodiments, the term "microorganism" or “microbe” refers to a fungus such as a yeast, for example, a yeast of the genus Yarrowia ( e.g Y. Hpolyticd).

[0056] The term “CFU” as used herein means “colony forming units” and is a measure of viable cells in which a colony represents an aggregate of cells derived from a single progenitor cell.

[0057] For purposes of this disclosure, a “biologically pure strain” means a strain containing no other microbial strains in quantities sufficient to interfere with replication of the strain or to be detectable by normal microbial techniques.

[0058] The term "isolated” means a substance in a form or environment that does not occur in nature and does not reflect the extent to which an isolate has been purified, but indicates isolation or separation from a native form or native environment. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any cell (such as a host cell), enzyme, engineered enzyme, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature: (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated.

[0059] The term “percent identity” or “percent sequence identity” is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the number of matching nucleotides or amino acids between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W._ ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I { Griffin, A. M., and Griffin, H. G_, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heitije, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Deverenx, J., eds.) Stockton Press, NY (1991). Methods to determine identity and similarity are codified in publicly available computer programs. Percent identity may be determined using standard techniques known hi the ait. Useful algorithms include the BLAST algorithms (See, Altschul et al., JMol Biol 215:403-410, 1990; and Karim and Altschul, Proc Natl Acad Sci USA , 90:5873-5787,

1993). The BLAST program uses several search parameters, most of which are set to the default values. The NCBI BLAST algorithm finds the most relevant sequences in terms of biological similarity but is not recommended for query sequences of less than 20 residues (Altschul et al., Nucleic Acids Res , 25:3389-3402, 1997; and Schaffer et al, Nucleic Acids Res, 29:2994-3005, 2001). Exemplary default BLAST parameters for a nucleic acid sequence searches include: Neighboring words threshold = 11; E- value cutoff = 10; Scoring Matrix = MUC.3.1 (match = 1, mismatch = -3); Gap Opening = 5; and Gap Extension = 2. Exemplary default BLAST parameters for amino acid sequence searches include: Word size = 3; E-value cutoff = 10; Scoring Matrix = BLOSUM62; Gap Opening = 11; and Gap extension = 1.

[0060] As used herein with regard to nucleotide or ammo acid residue positions, "'corresponding to” or “corresponds to” or “correspond to” or “corresponds” refers to (i) a nucleotide or an amino acid residue at an enumerated position in a nucleic acid or a protein or peptide; or (ii) a nucleic acid or an ammo acid residue that is analogous, homologous, or equivalent to an enumerated residue in a nucleic acid or a protein or peptide. As used herein, “corresponding region” generally refers to an analogous position hi a related protein or a reference protein.

[0061] As used herein, “prevent,” "preventing,” “prevention” and grammatical var iations thereof refers to a method of partially or completely delaying or precluding the onset or recurrence of a disorder or condition (such as a respiratory condition, for example, COVID-19) and/or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a disorder or condition or reducing an subject's risk of acquiring or reacquiring a disorder or condition or one or more of its attendant symptoms.

[0062] As used herein, the term “reducing” in relation to a particular trait, characteristic, feature, biological process, or phenomena refers to a decrease in the particular trait, characteristic, feature, biological process, or phenomena. The trait, characteristic, feature, biological process, or phenomena can be decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater than 100%.

[0063] As used herein, the term “subject” refers to a mammal (e.g., rat, mouse, cat, dog, cow, pig, sheep, horse, goat, rabbit, mink), preferably a human, for example, in need of prevention and/or treatment of a disease or disorder such as viral infection or cancer. The subject may have a viral infection, e.g. , an influenza infection, or be predisposed to developing an infection. Subjects predisposed to developing au infection, or subjects who may be at elevated risk for contracting an infection (e.g. , of coronavirus or influenza virus), include subjects with compromised immune systems because of autoimmune disease, subjects receiving immunosuppressive therapy (for example, following organ transplant), subjects afflicted with human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS), subjects with forms of anemia that deplete or destroy white blood cells, subjects receiving radiation or chemotherapy, or subjects afflicted with an inflammatory disorder. Additionally, subjects of very' young (e.g., 5 year s of age or younger) or old age (e.g., 65 years of age or older) are at increased risk. Moreover, a subject may be at risk of contracting a viral infection due to proximity to an outbreak of the disease, e.g. subject resides in a densely-populated city or in close proximity to subjects having confirmed or suspected infections of a virus, or in prison or other penal institution, or in a university, or choice of employment, e.g. hospital worker, pharmaceutical researcher, traveler to infected area, or frequent flier.

[0064] “Treat” or '‘treating” or “administer” or “administering” means to administer a recombinant Yarrowia cell comprising a nucleic acid encoding a coronavirus spike protein or a fragment thereof (such as by feeding or by gavage) to a subject having one or more signs or symptoms of a disease or infection, e.g. , viral infection, for which the recombinant Yarrowia cell is effective when administered to file subject at an effective or therapeutically effective amount or dose.

[0065] An “effective” or ‘therapeutically effective” dose of a recombinant Yarrowia cell, Yarrawia cell lysate, or fractionated Yarrowia cell lysate, e.g. , a recombinant Yarrawia cell comprising a nucleic acid encoding a corona virus spike protein or a fragment thereof, for treating or preventing a viral infection (such as a coronavirus infection, such as infection by SAKS CoV- 2), refers to fire amount of recombinant Yarrawia cell, Yarrowia cell lysate, or fractionated Yarrowia cell lysate sufficient to alleviate one or more signs and/or symptoms of the infection in fire treated subject, whether by preventing the manifestation of one or more signs and/or symptoms of the infection, by inducing the regression or elimination of such signs and/or symptoms, or by inhibiting the progression of such signs and/or symptoms.

[0066] Certain ranges are presented herein with numerical values being preceded by the term ''about.'' The term "about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number can be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. For example, in connection with a numerical value, the term “about” refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in context.

[0067] As used herein, the singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise.

[0068] As used herein, “optional” or “optionally” means that the subsequently circumstance or limitation on scope does or does not occur, and that the description includes instances where the circumstance or limitation on scope occurs and instances where it does not. For example, a composition that optionally contains additional exogenous enzymes means that the enzymes can be present or not present in the composition.

[0069] It is further noted that the claims may be chaffed to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” Limitation.

[0070] It is also noted that the term “consisting essentially of,” as used herein refers to a. composition wherein the components) after the term is in the presence of other known components) in a. total amount that is less than 30% by weight of the total composition and do not contribute to or interferes with the actions or activities of the components).

[0071] It is further noted that the term '’comprising,” as used herein, means including, but not limited to, the component(s) after the term “comprising.” The components) after the term “comprising” are required or mandatory, but the composition comprising the components) can further include other non-mandatory or optional component(s).

[0072] It is also noted that the term “consisting of,” as used herein, means including, and limited to, the components) after the term "consisting of.” The components) after the term “consisting of” are therefore required or mandatory, and no other components) are present in the composition.

[0073] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include event higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0074] Unless defined otherwise herein, all technica l and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains,

[0075] Other definitions of terms may appear throughout the specification.

II. Compositions ft. Spike Protein Polypeptides [0076] Corona virus virions axe spherical with diameters of approximately 125 mu. The most prominent: feature of coronaviruses is the club-shaped spike projections emanating from the surface of the virion. These spikes are a defining feature of the virion and give them the appearance of a. solar· corona, prompting the name, coronaviruses. Within the envelope of the virion is the nucleocapsid. Coronaviruses have helically symmetrical inicleocapsids, which is uncommon among positive-sense RNA viruses, but far more common for negative-sens» RNA viruses. SARS-CoV-2, MERS-CoV, and SARS-CoV belong to the Coronaviridae family. The initial attachment of the virion to the host cell is initiated by interactions between the S protein and its receptor. The sites of receptor binding domains (RBD) within the SI region of a coronavirus S protein vary depending on the virus, with some having the RBD at the C-tenninus of SI. The S-protein/receptor interaction is the primary determinant for a coronavirus to infect a host species and also governs the tissue tropism of the virus. Many coronaviruses utilize peptidases as their cellular receptor. Following receptor binding, the virus must next gain access to the host cell cytosol. This is generally accomplished by acid-dependent proteolytic cleavage of S protein by a eathepsin, TMPRRS2 or another protease, followed by fusion of the viral and cellular membranes.

[0077] Provided herein are recombinant Yarrowia cells expressing a nucleic acid encoding a. coronavirus spike protein or a fragment thereof. The nucleic acid encoding the spike protein can encode the entire spike protein (for example, the nucleic acid encoded by SEQ ID NO:25 or a nucleic acid sharing at least about 80% sequence identity to SEQ ID NO: 25, such as at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2S), encoding the polypeptide encoded by SEQ ID NO:26 or a polypeptide sharing at least about 70% sequence identity to SEQ ID NO:26, such as at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,

94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 26) or a fragment thereof (for example, a domain, such as the RBD domain). The corona virus spike protein or fragment thereof can be derived from any coronavirus such as, without limitation, 229E (alpha coronavirus), NL63 (alpha coronavirus), GC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and/or SARS-CoV- 2 virus. ΎΊ [0078] In some embodiments, the spike protein fragment expressed by the recombinant Yarrawia can correspond to the nucleotide sequence enco ding a segment or portion of a 2019- nCoV spike protein, suck as residues 319 to 591 of the 2019-nCoV spike protein (PBD ID: 6VSB), which can, in other embodiments, be farther codon optimized for expression in a yeast cell (such as a Yarrawia cell, for example, Y Hpolytica). In some embodiments, the polynucleotide for expression in the recombinant yeast cell comprises the polynucleotide encoded by SEQ ID NO: 1 (or a nucleic acid sharing at least about 80% sequence identity to SEQ ID NO:l, such as at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:l) which produces a 273 amino acid residue translation product encoded by SEQ ID NO:2 (or a polypeptide sharing at least about 70% sequence identity to SEQ ID NO:2, such as at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2).

[0079] In some embodiments, the spike protein or fragment thereof is fused to a signal peptide to facilitate secretion. The signal peptide can be any signal peptide suitable for facilitating secretion of a recombinant protein from a yeast cells ( such as a Yarrowia cell, such as, without limitation, the PIP2, XPR2, MFa, or SUC2 signal peptides). In other embodiments, the fused signal peptide/spike protein or fragment thereof is encoded by the amino acid sequence of any one of SEQ ID Nos: 13, 14, 15, or 16 (or a polypeptide sharing at least about 70% sequence identity to SEQ ID Nos: 13, 14, 15, or 16, such as at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID Nos: 13, 14, 15, or 16).

[0080] In further embodiments, the spike protein or fragment thereof is also (i.e. in addition to the signal peptide) fused to an anchor protein (for example, a protein comprising a cell surface- adhering domain anchor transmembrane domain and an extracellular region for conjugation with a recombinant protein) to facilitate display on the surface of a yeast cell (e.g. a Yarrowia cell). The anchor protein can be any anchor protein suitable for facilitating display of a recombinant protein on the surface of a Yarrowia cell (such as, without limitation, the CWPl, CWP3, CWP6, or PIR1 anchor proteins). In other embodiments, the fused signal peptide/spike protein or fragment thereo£½achor protein is encoded by the amino acid sequence of any one of SEQ ID NOs:21, 22, 23, or 24 (or a polypeptide sharing at least about 70% sequence identity' to SEQ ID NOs:21, 22, 23, or 24, such as at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:21, 22, 23, or 24).

B. Other viral proteins

[0081] In additional embodiments, other viral proteins can be expressed in or on recombinant Yarrowia cells (intracellularly, on the surface of, or extracellularly secreted), such as viral proteins derived from an influenza virus. There are four types of influenza virus, termed influenza viruses A, B, C, and D. Aquatic bir ds are the primary source of Influenza A virus (IAV), which is also widespread in various mammals, including humans and pigs. Influenza B virus (IBV) and Influenza C virus (ICV) primarily infect humans, and Influenza D virus (IDV) is found in cattle and pigs. LAV and IBV circulate in humans and cause seasonal epidemics, and ICV causes a mild infection, primarily in children. IDV can infect humans but is not known to cause illness. In humans, influenza viruses are primarily transmitted through respiratory droplets produced from coughing and sneezing. Transmission through aerosols and intermediate objects and surfaces contaminated by the virus also occur.

[0082] As of 2019, 18 H subtypes and 11 N subtypes have been identified. Most potential combinations have been reported in birds, but H17-18 and NiO-11 have only been found in bats. Only H subtypes HI -3 and N subtypes N1-2 are known to have circulated in humans, the current IAV subtypes in circulation being H1N1 and H3N2. Accordingly, in some embodiments, where the influenza virus is influenza virus A, the viral protein(s) or fragment thereof expressed in or on recombinant Yarrowia cells (iiitracellularly, on the surface of, or extracelluJarly secreted) are derived from strain H1N1 H2N2, H3N2, H5NT H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, and/or H6N1.

C. Vectors [0083] Also provided herein are recombinant vectors for expressing a viral protein (such as a coronavirus spike protein or fragment thereof (including the fragment encoded by SEQ ED NO:I)) in Yarrowia (e.g., Y. lipo!ytica) which contains one or more expression control sequences operably linked and controlling expression of the spike protein or fragment thereof.

As used herein, the term “operably linked” means functionally associated with an expression control sequence (e.g., a promoter or a terminator) of a DNA sequence, wherein the expression of the DNA sequence is regulated by an expression control sequence by functional binding.

[0084] As used herein, the term “promoter” means a DNA sequence capable of regulating the expression of a gene coding sequence or functional RNA. Promoters suitable for use in the yeast Yarrawia include, without limitation, promoters for metallothioneln, 3 -phosphogly cerate kinase (Hess et al, J Adv. Enzyme Reg. 7: 149 (1968) and Holland et ah, Biochem. 17: 4900, (1978)), glucuronidase, glutamate dehydrogenase, enolase. glyceraldeliyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglucose isomerase or the the FBAINrn promoter (Hong et ah. 2012, Yeast, 2012; 29:59- 72; such as the FBAINrn encoded by SEQ ID NO: 3). In some embodiments, multiple copies of an expression cassette can be used to increase expression either intracellularly or for surface display.

[0085] The recombinant vector of the present invention may further comprise a transcription terminator for termination of transcription such as, without limitation, an HSP150 terminator, a

TRP1 terminator, an ADHI terminator, a GAP terminator, an MFl terminator, an XPR terminator, a Pex20 terminator, or other functional terminator.

[0086] The recombinant vector of the present invention includes a selection marker for facilitating selection of the transformant. The selectable marker is, for example, a gene capable of complementing an antibiotic resistance gene, or a yeast strain having a metabolic deficiency characteristic, for example, tryptophan or a histidine-deficient mutant strain. In some embodiments, the selectable marker comprises URA3, LEU2, HIS3, TRP1, HIS4, ARG4, or an antibiotic resistance gene.

[0087] The recombinant vector of the present invention can include a replication origin capable of maintaining the vector in T lipolytiea. For example, suitable replication origins include 2 m or ARS1. The vector of the present invention may contain a yeast centromeric sequence (CEN) at the origin of replication for stabilization of the vector.

[0088] Since the manipulation of the vector may he more efficient in a bacterial strain, lirerecombinant vector of the present invention can also include a replication origin replicable in a bacterial cell, such as, but not limited to, ColEl, Ori, or oriT.

[0089] In some embodiments, the recombinant vectors for use in expression of a coronavirus spike protein or fragment thereof in a Yarrowia cell can be constructed to facilitate intracellular, secreted, or surface display of the spike protein or fr agment thereof.

[0090] When constructed for intracellular expression, the vector can include an expression cassette comprising a promoter (such as the FBAINm promoter), the nucleic acid to be expressed (such as the nucleic acid encoded by SEQ ID NO:l), and a terminator (such as the Pex20 terminator. In some embodiments, the functional components of the vector constructed for intracellular expression of a coronavirus spike protein or fragment thereof in a Yarrowia cell correspond to those shown in Table 1.

[0091] When constructed for secreted expression, the vector can additionally include a signal peptide. A “signal peptide” as used herein refers to an amino-terminal extension on a protein to be secreted. Nearly all secreted proteins use an ammo-terminal protein extension which plays a crucial role in the targeting to, and translocation of, precursor proteins across the membrane and which is generally proteoiytical!y removed by a signal peptidase during or immediately following membrane transfer. Any signal peptide capable of facilitating the secretion of a coronavirus spike protein or fragment thereof from a. Yarrowia cell can be employed including, without limitation, the LIP2 signal peptide (for example the nucleic acid sequence of SEQ ID NO: 5 encoding the polypeptide of SEQ ID NO:6), the XPR2 signal peptide (for example the nucleic acid sequence of SEQ ID NO:7 encoding the polypeptide of SEQ ID NG:8), the MFa signal peptide (for example the nucleic acid sequence of SEQ ID NO:9 encoding the polypeptide of SEQ ID NO:10), or the SUC2 signal peptide (for example the nucleic acid sequence of SEQ ID NO:ll encoding the polypeptide of SEQ ID NO:12). In some embodiments, the functional components of the vector constructed for secreted expression of a coronavirus spike protein or fragment thereof fr om a. Yarrowia cell correspond to those shown in Table 2.

[0092] When constructed for displayed expression on the surface of a Yarrowia ceil, the vector can additionally include an anchor protein. The term “anchor protein” as used herein, refers to a polypeptide or protein of which a portion is found outside the cell wall or membrane or the outer surface of an expression system which can be used to tether a protein of interest to the outer surface of a cell. Any anchor protein capable of facilitating the surface display of a coronavirus spike protein or fragment thereof on the surface of a Yarrowia cell can be employed including, without limitation, the CWP1 anchor protein or functional fragment thereof (for example, the polypeptide ofSEQ ID NO:17) or a functional fragment or portion thereof, the CWP3 anchor protein (for example, the polypeptide ofSEQ ID NO: 18) or a functional fragment or portion thereof the CWP6 signal peptide (for example, the polypeptide of SEQ ID NO: 19) or a functional fragment or portion thereof, or the PIRl anchor protein (for example, the polypeptide ofSEQ ID NO:20) or a functional fragment or portion thereof, In some embodiments, the functional components of the vector constructed for displayed expression of a coronavirus spike protein or fragment thereof from on the surface of a Yarrowia cell correspond to those shown in Table 3.

D. Recombinant Yarrowia cells

[0093] Provided herein are recombinant Yarrowia cells comprising a nucleic acid encoding a coronavirus spike protein or a fragment thereof. Yarrowia is a fungal genus in the family

Dipodascaceae. Any Yarrowia cell suitable for expression of recombinant proteins can be used, including those from Yarrowia bubula, Yarrowia deformans , Yarrowia Iipolytica, Yarrowia porcina, Yarrowia yakushimensis, and Yarrowia parophonii.

[0094] In some embodiments, the recombinant Yairowia cell is a recombinant Yarrowia lipolytica cell. Y. iipolytica is a dimorphic alkane-assimilating yeast, classified as GRAS based on its association with several food-related processes (Zinjarde, 2014, Food Ckem. 152, 1—10). This yeast can use diverse carbon sources, including glucose (but not sucrose), alcohols, acetate, and hydrophobic substrates such as alkanes, fatty acids, and oils (Barth and Gaillardin, 1997, FEMS Microbiol. Rev. 19, 219-237). The glyeosylation pattern of Y. Iipolytica is closer to the mammalian high-mannose type of glycosylation Sian that of S. cerevisiae (Song et al, 2007. Appl. Environ. Microbiol. 73, 4446 - 4454; Moon et al., 2013 , J Microbiol 51, 506-514; Oh el a!., 2016, J. Microbiol 51 , 506-514). Further, recombinant. Y. lipojytica has been used as an oral vaccine to prevent nervous necrosis virus in cultured marine fish (Lira et al., 2017, 1. Microbiol., 55(8), 655-664).

[0095] Constructs or vectors comprising the nucleic aeids(s) of interest (such as a nucleic acid encoding a spike protein or fragment thereof) may be introduced into a host cell such as Yarrowia by any standard technique. These techniques include transformation (e.g. , lithium acetate transformation (Methods in Enzymology, 194:186-187 (1991))), protoplast fusion, holistic impact, electroporation, microinjection, or any other method that introduces the nucleic acids(s) of interest into the host cell. More preferred herein for Yarrowia lipolytica are Integra lion techniques based on linearized fragments of DNA, as described in U.S. Pal. No. 4,880,741 and No. 5,071,764 and Chen, D. C. et al. {Appl Microbiol Biotechnol., 48(2):232-235 (1997)).

[0096] For convenience, a host cell (such as a Yarrowia host cell) that has been manipulated by any method to take up a nucleic acid sequence {e.g., an expression cassette or a vector comprising an expression cassette) is referred to herein as "'transformed”, “transformant” or “recombinant”. The transformed host will have at least one copy of the expression cassette and may have two or more, depending upon whether the expression cassette is integrated into the genome or is present on an extrachromosomal element having multiple copy numbers. The transformed host cell can be identified by various selection techniques, as described in U.S. Pat. No. 7,238,482 and U.S. Pat. No. 7,259,255.

[0097] Preferred selection methods for use herein are resistance to kanamyeiix, hygromycin and the amino glycoside G418, as well as ability to grow on media lacking uracil, leucine, lysine, tryptophan or histidine. In alternate embodiments, 5-fluoroorotic acid (5-fluorouracil-0- carboxylic acid monohydrate; “5-FOA”) is used for selection of yeast Ura- mutants. The compound is toxic to yeast cells that possess a functioning URA3 gene encoding orotidine 5'- monopkosphate decarboxylase (OMP decarboxylase) (Bartel, P. L. and Fields, S., Yeast 2- Hybrid System, Oxford University New York, v. 7, pp 109-147, 1997).

[0098] More specifically, one can first knockout the native Ura3 gene to produce a strain having a lira- phenotype, wherein selection occurs based on 5-FOA resistance. Then, a cluster of multiple chimeric genes and a new Ura3 gene could be integrated into a different locus of the YafTowia genome to thereby produce a new strain having a Ura+· phenotype. Subsequent integration would produce anew Ura3- strain (again identified using 5-FOA selection), when the introduced Ura3 gene is knocked out. Thus, the Ura3 gene, in combination with 5-FOA selection, can be used as a selection marker in multiple rounds of transformation and thereby readily permit genetic modifications to be integrated into the Yarrowia genome in a facile manner.

[0099] The transformed Yarrowia host cell is grown under conditions that optimize expression of recombinant genes (e.g., a gene encoding a spike protein or fragment thereof). In general, media conditions may be optimized by modifying the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the amount of different mineral ions, the oxygen level, growth temperature, pH, etc. Yarrowia lipolytiea are generally grown in a complex media such as yeast extract-peptone-dextrose broth (YPD) or a defined minimal media that lacks a component necessary for growth and thereby forces selection of the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCQ Laboratories, Detroit, Midi.)).

[0100] Fermentation media for the methods and host cells described herein must contain a suitable carbon source, such as are taught in U.S. Pat. No. 7,238,482. Although it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing sources, preferred carbon sources are sugars, glycerol and/or fatty acids. In some embodiments, the carbon source is glucose and/or fatty acids containing between 10-22 carbons.

[0101] Nitrogen may be supplied from an inorganic (e.g. , (NH₄)₂SO₄) or organic (e.g., urea or glutamate) source. In addition to appropriate carbon and nitrogen sources, the fermentation media must also contain suitable minerals, salts, cofactors, buffers, vitamins and other components known to those skilled in the art.

[0102] Preferred growth media for the methods and host cells described herein are common commercially prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growth media may also be used and the appropriate medium for growth of Yarrowia lipolytiea will be known by one skilled in the art of microbiology or fermentation science, A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 is preferred as the range for the initial growth conditions. The fermentation may be conducted under aerobic or anaerobic conditions, wherein microaerobic conditions are preferred.

E. Pharmaceutical and Other Compositions

[0103] While it is possible to administer any of the recombinant Yarrowia cell comprising a nucleic acid encoding a viral protein or fragment thereof (e.g. a coronavirus spike protein or a fragment thereof) thereof alone according to the present invention (i.e., without any support, diluent or excipient, for example, where the cells are lyophilized or freeze dried), recombinant Yairowia cells are typically administered on or in a support as part of a product, in particular as a component or at least as one of the components of a composition such as a dietary supplement, a nutritional supplement, a food product or a pharmaceutical acceptable composition or formulation. These produc ts typically contain additional components well known to those skilled in the art.

[0104] The recombinant Yarrowia cell-containing compositions as described in the present application are used in or for preventing or treating illnesses and/or symptoms associated with coronavirus infection or other viral infection (e.g. influenza virus) in a subject in need thereof.

[0105] Additionally, where the recombinant Yarrowia cell has been designed to secret the expressed viral protein (e.g., an influenza viral protein or fragment thereof or coronavirus spike protein or fragment thereof), the secreted protein can be isolated and formulated into a pharmaceutical or other composition for administration to a subject.

[0106] Further, where the recombinant Yarrowia cell has been designed to express the viral protein intracell ularly (e.g., an influenza viral protein or fragment thereof or coronavirus spike protein or fragment thereof), the Yarrowia cell can be lysed and the lysate can be formulated into a pharmaceutical or other composition for administration to a subject. Alternatively, the viral protein can be extracted or otherwise isolated from the recombinant Yarrowia cell lysate.

[0107] The coronavirus can be one of the coronaviruses selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and SARS-CoV-2 virus, or any other coronavirus belonging to the family Coronavirda or any new variants of these viruses. In particular, the coronavirus is SARS-CoV-2 virus.

1. Food Product

[0108] In one embodiment, recombinant Yarrowia cells comprising a nucleic acid encoding a viral protein or fragment thereof [e.g. a coronavirus spike protein or a fragment thereof) or an isolated viral protein or fragment thereof produced and secreted by a recombinant Yarrowia cell are used according to the invention in a food product, such as a food supplement, a medical food, a drink or a powder based on milk. Here, the term "food" is used in a broad sense and covers food for humans as well as food for animals (ie. a feed). In a preferred aspect, the food is for human consumption.

[0109] The food may be in the form of a solution or as a solid, depending on the use and/or the mode of application and-'or the mode of administration.

[0110] When used as, or in the preparation of, a food, such as functional food, the recombinant Yarrowia cells of the present invention may be used in conjunction with one or more of: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient.

[0111] By way of example, the recombinant Yarrowia cells of the present invention can be used as an ingredient to soft drinks, a fruit juice or a beverage comprising whey protein, health teas, cocoa drinks, milk drinks and lactic acid bacteria drinks, yoghurt and drinking yoghurt, cheese, ice cream, water ices and desserts, confectionery, biscuits cakes and cake mixes, snack foods, balanced foods and drinks, fruit fillings, care glaze, chocolate bakery filling, cheese cake flavoured filling, fruit flavoured cake filling, cake and doughnut icing, instant bakery filling creams, fillings for cookies, ready-to-use bakery filling, reduced calorie filling, adult nutritional beverage, vegetable milk, acidified soy/juice beverage, aseptic/retorted chocolate drink, bar mixes, beverage powders, calcium fortified soy/plain and chocolate milk, or calcium fortified coffee beverage.

[0112] Advantageously, where the product is a food product, the recombinant Yarrowia cells should remain effective through the normal "sell-by” or "expiration" date dining which the food product is offered for sale by the retailer. Preferably, the effective time should extend past such dates until the end of the normal freshness period when food spoilage becomes apparent. The desired lengths of time and normal shelf life will vary from foodstuff to foodstuff and those of ordinary skill in the art will recognize that shelf-life times will vary upon the type of foodstuff, the size of the foodstuff, storage temperatures, processing conditions, packaging material and packaging equipment age.

2. Food ingredients

[0113] Compositions comprising recombinant Yarrowia cells comprising a nucleic acid encoding a viral protein or fragment thereof (e.g. a coronavirus spike protein or a fragment thereof) or an isolated viral protein or fragment thereof produced and secreted by a recombinant Yarrowia cell as disclosed herein may take the form of a food ingredient and/or feed ingredient.

[0114] As used herein the term “food ingredient'' or "feed ingredient" includes a composition which is or can be added to functional foods or foodstuffs as a nutritional and/or health supplement for humans and animals,

[0115] The food ingr edient may be in the form of a liquid, suspension, or solid, depending on the use and/or the mode of application and/or the mode of administration.

3. Dietary Supplements

[0116] Any of the recombinant Yatrowia cell-containing compositions or recombinant

Yarrawia cell-secreted protein compositions disclosed herein may take the form of dietary supplements or may themselves be used in combination with dietary supplements, also referred to herein as food supplements.

[0117] The term “dietary supplement” as used herein refers to a product intended for ingestion that contains a "dietary ingredient" intended to add nutritional value or health benefits to (supplement) the diet. A "dietary ingredient" may include (but is not limited to) one, or any combination, of the following substances: bacteria, yeasts, a probiotic (e.g. probiotic bacteria), a vitamin, a mineral, a herb or other botanical, an amino acid, a dietary substance for use by people to supplement the diet by increasing the total dietary intake, a concentrate, metabolite, constituent, or extract,

[0118] Dietary supplements may be found in many forms such as, without limitation, tablets, capsules (such as prolonged release capsules or enterically-coated capsules), soft gels, gel caps, liquids, sachets, gummies, or powders. Some dietary supplements can help ensure an adequate dietary intake of essential nutrients; others may help prevent or treat diseases (such as a respiratory infection, for example, COVID-19).

4. Medical Food

[0119] Any of the recombinant Yarrowia cell-containing compositions or recombinant Yarrowia cell-secreted protein compositions disclosed herein may take the form of medical foods.

[0120] By “medical food” it is meant a food which is formulated to be consumed or administered with or without the supervision of a physician and which is intended for a specific dietary management or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation. In other embodiments, a medical food is a a food which is formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary' management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation.

5. Pharmaceutical composition

[0121] The recombinant Yarrowia cells comprising a nucleic acid encoding a viral protein or fragment thereof (e.g. coronavirus spike protein or a fragment thereof) or recombinantly produced viral proteins or fragments thereof produced and secreted by recombinant Yarrowia cells disclosed herein may be used as - or in the preparation of - a pharmaceutical composition or formulation. As used herein, the term "pharmaceutical" is used in a broad sense - and covers pharmaceuticals for humans as well as pharmaceuticals for animals (i.e. veterinary applications).

[0122] In one embodiment, the pharmaceutical acceptable composition is a medicament.

[0123] The pharmaceutical composition can be for therapeutic purposes - which may be curative or palliative or preventative in nature. In some embodiments, the pharmaceutical composition functions as a vaccine for preventing or immunization against a viral (e.g. coronavirus infection, such as infection by SARS CoV-2). The term '’vaccine,” as used herein, refers to a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism and is often made from weakened or killed forms of the microbe, its toxins or one of its surface proteins (such as a coronavirus spike protein or fragment thereof). “Immunization,” as used herein means, but is not limited to, the induction of immunity and may include, for example, “active immunization” (which is the stimulation of the immune system to confer protection against a disease or disorder,for example, by administration of a vaccine, toxoid, or microbe). In some instances, the terms vaccination and immunization may be used interchangeably.

[0124] In a one embodiment of the present invention, the medicament is formulated with one or more pharmaceutically acceptable excipients for oral administration. In another embodiment, the medicament is formulated with one or more pharmaceutically acceptable excipients for nasal administration, such as via a mechanical inhaler.

[0125] A pharmaceutically acceptable composition or support may be, for example, a formulation or support in the form of creams, foams, gels, lotions, nasal sprays, aerosols, liquids/suspensions, and ointments of compressed tablets, tablets, capsules, ointments, suppositories or drinkable or inhalable solutions.

[0126] When used as - or in the preparation of - a pharmaceutical, the composition of the present invention may be used in conjunction with one or more of: a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, a pharmaceutically acceptable adjuvant, a pharmaceutically active ingredient.

[0127] Yeasts in general have been shown to be adjuvants in oral administration. Yarrowia, in particular, has been shown to drive the EL- 12/Tlil /interferon gamma path as well as to induce IL-27 leading to CDS cytotoxoc T-lymphocyte synthesis/activation. It also appears to induce IL- 17 production by the Th17 cell subset, normally involved in innate immunity of the gut epithelium including wall integrity. Therefore, in a particular embodiment, the adjuvant is a yeast and, more particularly, Yarrowia.

[0128] The pharmaceutical may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.

[0129] The recombinant Yarrowia cells disclosed herein may be used as pharmaceutical ingredients. Here, the composition may be the sole active component, or it may be at least one of a number (i.e·. 2 or more) of active components. The pharmaceutical ingredient may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.

[0130] The recombinant Yarrowia cells disclosed herein may be used according to the present invention in any suitable dosage form - whether when alone or when present in a combination with other components or ingredients. Likewise, combinations comprising the recombinant Yarrowia cells of the present invention and other components and/or ingredients (i.e. ingredients - such as food ingredients, functional food ingredients or pharmaceutical ingredients) may be used in any suitable form.

[0131 ] The recombinant Yarrowia cells disclosed herein may be used according to the present invention in the form of solid or liquid preparations or alternatives thereof Examples of solid preparations include, but are not limited to tablets, capsules, dusts, granules and powders which may be wettable, spray-dried or freeze-dried. Examples of liquid preparations include, but are not limited to, aqueous, organic or aqueous-organic· solutions, suspensions and emulsions. Suitable examples of fonns include one or more of: tablets, pills, capsules, ovules, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or contro!led-release applications.

[0132] By way of example, if the recombinant Yarrowia cells disclosed herein are used in a tablet form - such for use as a functional ingredient - the tablets may also contain one or more of: excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine; disintegrants such as starch (such as, without limitation, com, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates; granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0133] Examples of nutritionally acceptable carriers for use in preparing the dosage forms include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid 30 monoglycerides and diglycerides, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like, in some non-limiting embodiments, excipients for the dosage forms include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.

[0134] For aqueous suspensions and/or elixirs, the yeasts of the present invention may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, propylene glycol and glycerin, and combinations thereof. The dosage forms may also include gelatin capsules: fiber capsules, fiber tablets etc.; or even fiber beverages.

[0135] In one aspect, the recombinant Yarrowia cells disclosed herein may be administered in an aerosol, for example by way of a nasal spray or mechanical inhalation device, for instance for administration to the respiratory tract, such as the nasal mucosa or the lungs.

[0136] In a further embodiment, the recombinant Yarrowia cells disclosed herein may be formulated with one or more cryoprotectants. Suitable cryoprotectants include, without limitation, one or more of propyl gallate, sodium caseinate, sodium citrate, sodium glutamate, cysteine, ascorbic acid, maltodextrin, sucrose, trehalose, and/or sorbitol.

III. Methods

A. Treatment and/or prevention of illness associated with viral infection

[0137] To produce antigen specific reactions and novel antibody production, the recombinant Yarrowia cells expressing a viral protein or fragment thereof (e..g. coronavirus spike protein or fragment thereof) or isolated viral protein produced and extracellularly secreted by a recombinant Yarrowia cell should induce T-cell dependent reactions in the gut and/or mucosa, if consumed as powder or aerosolized spray, these antibodies may also be produced in the mucosa of respir atory tract.

[0138] Antigen specific T-cell dependent reactions are driven in secondary lymphoid tissues (lymph nodes/Peyer’s patches) by (i) antigen presenting cells that ingest probiotics and display cross-reactive peptides on MHC-II/ MHC-I molecules on their surface, (ii) CD4 T- cells recognizing the peptide

complexes, (iii) antigen specific CDS T-cells activated by CD4

T-cells, and (iv) B-cells activated by B-cell receptor binding the pathogen and displaying the peptide in MHC-P and (v) CD4 T-cells activated by the peptide from the same bacterium by antigen presenting cell.

[0139] T-eell dependent IgA production is driven by TGF-b (IgA class switching), and expansion/ differentiation of B-cells by IL-5, IL-6, IL-10, and IL-2I - secreted by CD4 T- cells. Tins is basically a tolerogenic Treg/ barrier protecting Th17 response to microbiota that is induced by dendritic ceils secreting IL-6, IL-10, IL-23, TGF-β.

[0140] Without being bound to theory, it is believed that ingestion or inhalation of any of the recombinant Yarrowia cells disclosed herein can induce IgA responses in the intestine and/or nasal mucosa. It has been shown that some plasma B- cells (Ah producing cells) tr avel to other mucosal sites (-10%), for example, the respiratory tract (-1-2%), to add to existing plasma cell/IgA pool.

[0141] CDS memory cells are important in eradication of virus infected cells and driving anti-viral immunity. It has been shown that healthy humans have memory CDS T-cells against commensal microbes. Further it was shown that the microbes harboring a cross-reactive peptide to tumor neoantigen was able to influence tumor reactive CDS T-cells (Bessell et al.. Commensal bacteria stimulate antitumor responses via T cell cross-reactivity; JCI Insight ; 2020;5(8)), suggesting that microbes could also influence antigen specific memory CDS T-eell pool against pathogens. CDS responses are typically initiated by IL-12TFN-γ and Till type dendritic/CD4 T- cells. Thus, microbes engineered to express a viral antigen (such as the coronavires spike protein or a fragment thereof) could influence pre-existing coronavirus cross-reactive memory CDS T cells, but also memory CD 1 T- cells.

[0142] Without being bound to theory, microbes engineered to express viral antigens could also influence existing plasma cell activation and IgA pool. If there is existing cross-reactive IgA against SARS-CoV-2, such microbes could support total IgA production by T-eell independent mechanisms. These engineered microbes could further induce total IgA production (antigen independent) in the intestine, and potentially may have an influence in extra-intestinal IgA production as well. For example, short chain fatty acids and induction of TGF-b (IgA class- switching), IL-6, retinoic acid, BAFF and APRIL from epithelia drive the IgA production.)

[0143] Probiotic supplementation against cold and flu has been studied clinically for over 20 years. Several meta-analyses of clinical studies have shown that probiotics in general could be effective in reducing the risk and duration of the respiratory tract infectious. However, there are strain specific differences between probiotics on immune stimulation and beneficial effects against: respiratory tract infections. Research indicates that probiotics function by “training” innate immune responses, i.e.. function by priming the immune system before the viral infection. Studies have shown increased expression of interferons and innate immune cytokines prior to reducing the viral load or the risk of infectious by roughly 20%.

[0144] Although all viruses have different pathogenesis and life cycle, they still induce similar anti-viral immune responses - characterized by NK -cell, ILC1, cytotoxic T lymphocyte. Till responses, and IgG antibody production as well as production of interferons alpha, beta, gamma, and lambda, activation of the inflainmasome and Thl associated cytokines, such as IL- 12, and IP-10. Thus, stimulation of the innate immune system against SAE.S-CoV-2 could also be effective also against other coronaviruses.

[0145] Accordingly, provided herein are methods for treating or preventing illness and/or symptoms associated with a viral infection (e.g. coronavirus infection) in a subject in need thereof, said method comprising administering a recombinant Yarrowia cell comprising a nucleic acid encoding a viral (e.g. coronavirus spike protein or a fragment thereof) or a composition (such as a nutritional or pharmaceutical composition comprising the same) to the subject, In other embodiments, provided herein are methods for treating or preventing illness and/or symptoms associated with a viral infection (e.g. coronavirus infection) in a subject in need thereof, said method comprising administering a viral protein produced and extracellularly secreted by a recombinant Yarrowia cell comprising a nucleic acid encoding a viral (e.g. coronavirus spike protein or a fragment thereof) or a composition (such as a nutritional or pharmaceutical composition comprising the same) to the subject.

[0146] In one embodiment, the present invention relates to any coronavirus belonging to the family Coronaviridae. The coronavirus are enveloped viruses with a positive-sense single- stranded RNA genome and a nucleoeapsid of helical symmetry. The genome size of coronaviruses ranges from approximately 26 to 32 kilobases, one of the largest among RNA viruses. They have characteristic club-shaped spikes that project from their surface. Coronaviruses are a group of related RNA viruses and these include 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and SARS-CoY-2 virus. In a particular embodiment of the present invention, the coronaviras is SARS-CoV-2 virus.

[0147] Coronaviruses can cause various illnesses or diseases in mammals and birds. In humans, these viruses can cause respiratory tract infections that ^"can range from mild to lethal. Mild illnesses include some cases of the common cold while more lethal virus can cause severe acute respiratory syndrome (SARS) (SARS-CoY), Middle East respiratory syndrome (MERS) (MERS-CoV), and acute respiratory distress syndrome (ARDS) in the case of COVID-19 (SARS-CoV-2). In many patients, the respiratory distress is followed by severe sepsis with shock and in some cases multiple organ dysfunction within one week, resulting in a mortality rate of infected patients of approximately 2-7%.

[0148] in one embodiment, the illness caused by coronaviruses is a respiratory illness. More particularly, the respiratory illness is acute respiratory distress syndrome (ARDS). In another embodiment, the respiratory illness is pneumonia.

[0149] According to the World Health Organization (WHO), the most common symptoms of

COVID-19 are fever, dry cough and tiredness. Less common symptoms include aches and pains, sore throat, diarrhea, conjunctivitis, headache, newly acquired loss of taste or smell, a skin rash, or discoloration of fingers or toes (a.k.a. “covid toes”). Serious symptoms include difficulty breathing or shortness of breath, chest pain or pressure and loss of speech or movement.

[0150] In another embodiment, the prevention and/or treatment of the illness and/or symptoms associated with coronavimses is achieved by stimulation of the immune system in the subject when in contac t with one or more of the recombinant Yarrowia cells disc losed herein.

[0151] Some people are at high risk from coronavirus (clinically extremely vulnerable).

These include people who have had an organ transplant; people who are having chemotherapy or antibody treatment for cancer, including immunotherapy; people who are having an intense course of radiotherapy (radical radiotherapy) for lung cancer; people who are having targeted cancer treatments that can affect the immune system (such as protein kinase inhibitors or PARP inhibitors); people who have blood or bone marrow cancer (such as leukemia, lymphoma or myeloma); people who have had a recent bone marrow or stem cell transplant, or are still taking immunosuppressant medicine; people who have a severe lung condition (such as cystic fibrosis, severe asthma or severe COPD); people who have a medical condition that means they have a very high risk of getting infections, such as SOD or sickle cell; people who are taking medicine that makes them modi more likely to get infections, such as high doses of steroids or immunosuppressant medicine; and people who have a serious heart condition and are pregnant.

[0152] Some people are at moderate risk from coronavirus (clinically vulnerable). These include people who are 70 years of age or older; people who have a lung condition that’s not severe (such as asthma, COPD, emphysema or bronchitis); people who have heart disease (such as heart failure); people who have diabetes (type I or type II diabetes); people who have chronic kidney disease; people who have liver disease (such as hepatitis): people who have a condition affecting the brain or nerves (such as Parkinson’s disease, motor neuron disease, multiple sclerosis or cerebral palsy); people who have a condition that means they have a high risk of getting infections; people who are taking medicine that can affect the immune system (such as low doses of steroids); people who are obese or very obese (a BMI of 30-40 or above); people who are pregnant. Other factors that can affect risk include being male, living in a care home (such as a nursing home or long-term care facility), being an inmate in a prison or jail, or having a type A blood serotype.

[0153] hr one embodiment, the present invention relates to a subject who has one or more pre-existing conditions selected from the group consisting of obesity, type II diabetes, chronic lung disease or moderate to severe asthma, heart conditions, immunocompromised, chronic kidney disease and liver disease. In a further embodiment, the present invention relates to a subject who is 65 years of age or older and/or is a resident in a nursing home or long-term care facility or jail or prison.

[0154] The present invention encompasses propkylactically administering any of the recombinant Yarrowia cells disclosed herein to a subject who is at risk of viral infection so as to prevent such infection . “Prevent” or “preventing” means to administer a recombinant Yarrowia cells expressing a coronavirus spike protein or fragment thereof to a subject to inhibit the manifestation of a disease or infection (e.g., viral infection) in the body of a subject, for which the recombinant Yarrowia cell is effective when administered to the subject at an effective or therapeutically effective amount or dose.

[0155] In an embodiment of the invention, a sign or symptom of a viral infection in a subject is survival or proliferation of virus in the body of the subject, e.g. , as determined by viral titer assay (e.g. , coronavirus propagation in embryonated chicken eggs or coronavirus spike protein assay). Other signs and symptoms of viral infection are discussed herein.

[0156] The present invention provides a method for treating or preventing viral infection (e.g,, coronavirus infection) or for inducing the regression or elimination or inhibiting the progression of at least one sign or symptom of viral infection such as: fever or feeling feverish/chills; cough; sore throat; ninny or stuffy nose; sneezing; muscle or body aches; headaches; fatigue (tiredness); vomiting; diarrhea; respiratory tract infection; chest discomfort; shortness of breath; bronchitis; and/or pneumonia, which sign or symptom is secondary to viral infection, m a subject in need thereof (e.g., a human), by administering a therapeutically effective amount of any of the recombinant Yarrowia cells disclosed herein to the subject.

[0157] In other embodiments, the virus is an influenza virus, such as is influenza virus A, influenza virus B, influenza virus C, or influenza virus D. In a typical year, 5-15% of the population contracts influenza. There are 3-5 million severe cases annually, with up to 650,000 respiratory-related deaths globally each year. Deaths most commonly occur in high risk groups, including young children, the elderly, and people with chronic health conditions. In temperate regions of the world, the number of influenza cases peaks during winter, whereas hi the tropics influenza can occur year-round. Symptoms range from mild to severe and often include fever, runny nose, sore throat, muscle pain, headache, coughing, and fatigue. These symptoms typically begin 1-2 days and less typically 3-4 days after exposure to the virus and last for about 2-8 days. Diarrhea and vomiting can occur, particularly in children. Influenza may progress to pneumonia, which can be caused by the virus or by a subsequent bacterial infection. Other complications of infection include acute respiratory distress syndrome, meningitis, encephalitis, and worsening of pre-existing health problems such as asthma and cardiovascular disease.

[0158] The dose amount may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. In an embodiment of the invention, an effective or therapeutically effective dose of the recombinant Yarrowia cell of the present invention, for treating or preventing viral infection, e.g., in an adult human subject, is about 0.01 to about 200 mg/kg, e.g., up to about 150 mg/kg. In an embodiment of the invention, the dosage is up to about 100 mg to about 10 g (e.g., about 0.1 g, 0.2 g, 0.3 g, 0.4 g,

0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 1.5 g, 2 g, 2.5 g, 3 g, 3.5 g, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g, inclusive of all values falling in between these dosages). Depending on the severity of the infection, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the recombinant Yarrawia ceil of the present invention can be administered at an initial dose, followed by one or more secondary doses, In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of recombinant Yarrawia cell in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks,

B. Combination therapies

[0159] In an embodiment of the present disclosure, a recombinant Yairowia cell, e.g, a recombinant Yarrawia cell comprising a nucleic acid encoding a coronavirus spike protein or a fragment thereof is administered in association with one or more further therapeutic agents.

[0160] A further therapeutic agent includes, but is not limited to: an anti-inflammatory agent, an antimalarial agent, an antiviral agent, an antibody or antigen-binding fragment thereof that specifically binds TMPRSS2, and an antibody or antigen-binding fragment ther eof that specifically binds to CoV-S.

[0161] In some embodiments, an antimaiarial agent is chloroquine or hydroxychloroquine. In some embodiments, an anti-inflammatory agent is an antibody such as sarilumab , tocilizumab, or grmsrimnab or a steroid such as, without limitation, dexamethasone. In some embodiments, an antiviral agent is remdesivir (GS-5734; sold under the brand name VEKLURY^®), molnupiravir (MK-44S2, E3DD-28G1) and PF-07321332; sold under the brand name PAXLOV1D™ in combination with ritonavir). In some embodiments, the further therapeutic agent is an antibody or antigen-binding fragment that specifically binds TMPRSS2, such as H1H7017N, as described in International Patent Pub. No. WO2019/147831, incorporated by reference herein.

IV. Kits

[0162] Further provided are kits comprising one or more components that include, but are not limited to, a recombinant Yarrawia cell, e,g. , a recombinant Yairowia cell comprising a nucleic acid encoding a coronavirus spike protein or a fragment thereof, in association with one or more additional components including, but not limited to, a further therapeutic agent, as discussed herein. The recombinant Yarrowia cell and/or the further therapeutic agent can be formulated as a single composition or separately in two or more compositions, e.g., with a pharmaceutically acceptable earlier, in a pliarmaceutical composition.

[0163] in one embodiment of the invention, the kit includes a recombinant Yarrowia cell, e.g., a recombinant Yarrowia cell comprising a nucleic acid encoding a coronavirus spike protein or a fragment thereof, or a pharmaceutical composition thereof in one container (e.g., in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g., in a sterile glass or plastic vial). In some embodiments, the recombinant Yarrowia cells are lyophilized or freeze dried and are optionally further be formulated with one or more cryoprotectants. Suitable cryoprotectants include, without limitation, one or more of propyl gallate. sodium caseinate, sodium citrate, sodium glutamate, cysteine, ascorbic acid, maltodextrin, sucrose, trehalose, and/or sorbitol.

[0164] In another embodiment, the kit comprises a combination of the invention, including a recombinant Yarrowia cell, e.g. , a recombinant Yarrowia cell comprising a nucleic acid encoding a coronavirus spike protein or a fragment thereof, or pharmaceutical composition thereof in combination with one or more further therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.

[0165] If the kit includes a pharmaceutical composition for intranasal administration to a subject, the kit can include a device (e.g.. a mechanical inhaler) for performing such administration For example, the kit can include one or more mechanical inhalers or other devices as discussed above containing the recombinant Yarrowia cell, e.g. , a recombinant Yarrowia cell comprising a nucleic acid encoding a coronavirus spike protein or a fragment thereof.

[0166] The kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.

[0167] The invention can be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting.

EXAMPLES

Example 1 : Materials and Methods

[0168] Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by: Sambrook, J., Fritsch, E. F. and Maniatis,

T. Molecular Cloning: A Laboratory Manual·, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (Maniatis); T. J. Silkavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc, and Wiley- Inierscience (1987).

[0169] Materials and methods suitable for the maintenance and growth of yeast and bacterial cultures are well known in the art. Techniques suitable for use in the following Examples may be found as set out m. Manual of Methods for General Bacteriology (Piiiilipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, Eds), American Society for Microbiology: Washington, D.C. (1994)); or by Thomas D. Brock in Biotechnology*: A Textbook of Industrial Microbiology, 2nd ed., Sinauer Associates: Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used lor the growth and maintenance of cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), Biolabs (Cambridge, MA.), DIFCO Laboratories-Fisher Scientific (Detroit, Mich). GIBCO-Thermal Fisher Scientific (Gaithersburg, MD) or Sigma Chemical Company (St. Louis, MO). E. colt TOPIO and E. coli XL 1 -Blue competent cells were obtained from Invitrogen-Thennal Fisher Scientific (Carlsbad, CA). All E. coli strains were typically grown at 37° C on Luria Bertani (LB) plates.

[0170] Oligonucleotides were synthesized by Sigma-Genosys (Spring, TX). DNA fragments were synthesized and cloned by GeneArt- Gene Synthesis-Tliemio Fisher Scientific (Pleasanton, CA). DNA sequences and plasmid maps were generated by Nil vector software (Thermo Fisher Scientific). Comparisons of genetic sequences were accomplished using DNASTAR software (DNASTAR Inc., Madison, WI.

[0171] Cultivation of Yarrowia lipolyficat T lipolytica strain ATCC #20362 was purchased from the American Type Culture Collection (Rockville, Md.). Y. Ipafytica strain Y2224 is a spontaneous uracil auxoircph derivative of strain ATCC# 20362. Y Upolytica strains were usually grown at 30° C on YPD, or Basic Minimal Media (MM) or Minimal Media + Uracil or 2XSD media, as appropriate (YPD medium (per liter): 10 g of yeast extract [Difco], 20 g of Baeto peptone [Difco]; and 20 g of glucose; Minimal Media (MM) (per liter): 20 g glucose; 1.7 g yeast nitrogen base without ammo acids; 1 g praline, pH 6.1; Minims! Media + Uracil (MMIJ) (per liter): Prepare MM media as above and add 0.1 g uracil and 0.1 g uridine; 2X SD media (per liter): 13.4 g yeast nitrogen base with ammonium sulfate, 40 g Glucose, 139ml 1M NaiHPCkand 161ml 1M NaH₂PO₄).

[0172] Transformation of T lipolytica was performed according to the method of Chen, D.

C. et al. ( Appl Microbiol Biotechnol. 48(2):232-235 (1997)), unless otherwise noted.

Briefly, Yarrowia was streaked onto a YPD plate and grown at 30° C for approximately 18 hr. Several large loopfuls of cells were scraped from the plate and resuspended in 1 mL of transformation buffer containing: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M Li acetate, pH 6.0; and 0.125 mL of 2 M DTT. Then, approximately 500 ng of linearized plasmid DNA was incubated in 100 mΐ of resuspended cells and maintained at 39° C for 1 hr with vortex mixing at 15 min intervals. The cells were plated onto selection media plates and maintained at 30° C for 2 to 3 days

[0173] Protein Extraction, SDS-PAGE and Western Blot Analyses: The equivalent of 1 mL of Y, lipolytica culture at an OD600 of 1.0 was collected by centrifugation for 1 min and resuspended in 50 μL of sample buffer plus 0.1 g of glass beads, and cells were broken immediately by two cycles of boiling for 3 min and vortexing for 30 s. After 1 min centrifugation, collect the supernatant as the protein extract. The proteins extracted as described above were separated by 4-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), then all the proteins were electro-transferred onto a polyvinylidene difluoride (PVDF) membrane using a Bio-Rad Trans-Blot system. A 5% skim milk powder was used to block the membranes in PBST buffer (IX Phosphate-Buffered Saline, 0,1% Tween® 20 Detergent, MiUipore Sigma) for 60 min at room temperature. The blocking solution was removed and replaced with a solution of blocking buffer containing antibody ct-Spike SI (Cat: 40591-T62; Sino Biological Inc.), rabbit polyclonal antibody solution (1:1000 dilution in 10 mL blocking solution), and blot was incubated for at least 1 lit. The blot was washed for 5 minutes in TBST, 5 times. The blot was then incubated with goat anti-rabbit-HRP antibody (Cat: SSA004; Sino Biological, Inc.) in blocking solution at a 1:5000 antibody dilution for i hour, and then washed 5 times with PBST, for 5 minutes each time. Finally, about lOmL of the Novex chromogenic substrate 3,3',5,5'-tetramethylbenzidme (TMB) (Envitrogen cat# WP20004) (Envitrogen cat# WP20G04) was added until protein band intensity reached desired intensity. The membrane development reaction was stopped by washing the membrane in purified water for 2 min, which was repeated twice.

[0174] Fluorescence-activated cell sorting (FACS) analyses: Yarrowia cells were grown in buffered YPD (1% yeast extract, 1% peptone, 2% glucose, 0.1M phosphate buffer, pH 6.8) for 72 hours. About 10⁸ of Yarrowia cells were collected by centrifugation at 14,000rpm for 1 minute. Cell pellets were washed with iOOuL sterile water. Cells were resuspended with 100uL of MP5DP buffer (1% sodium citrate, pH7.0, 1M sorbitol, lOOmM DTT, 1% of protease inhibitor cocktail (catalog # of P8215-1ML. Sigma) and incubated at 32°C for 30 minutes. Cells were collected by centrifugation at 14,000ipm for 1 minute. After washing the cell pellets with 600uL 1M sorbitol twice, the cells were resuspended in IOOuL of PBS AS buffer (PBS buffer with 0.5% of BSA and 0.5M of sorbitol). Then 20uL cells were incubated with 100 uL of first antibody 40591-T62 (Cat: 40591-T62; Sino Biological hie.) with 1:200 dilution in PBSAS buffer for 1 hour with gentle shaking at 400 rpm. Cells were collected by centrifugation at 14000 rpm for 1 min, cell pellets were wash twice with 600 uL of PBSAS buffer, and then incubated with 100 uL of Goat anti-Rabbit IgG (H+L) Secondary Antibody A- i 1008 (Ca t# A-l 1008, Iuvitrogen), with 1 :300 dilution in PBSAS buffer, in dark for 1 hour with gentle shaking at 400 rpm. Cells were analyzed on NovoCyte Quanteon Flow Cytometer (Agilent, CA) for fluorescence.

[0175] Immunofluorescence microscopy assay: Yarrowia cells were grown in buffered YPD (i% yeast extract, 1% peptone, 2% glucose, 0.1M phosphate buffer, pH 6.8) for 72 hours. About 10^s of Yarrowia cells were collected by centrifugation at 14,000rpm for 1 minute. Cell pellets were washed with lOOuL sterile water. Cells were resuspended with lOOuL of MP5DP buffer (1% sodium citrate, pH7.5, 1M sorbitol, 100mM DTT, 1% of protease inhibitor cocktail (catalog # of P8215-1ML, Sigma). Cells were incubated at 32°C for 30 minutes. Cells were pelleted by centrifugation at 14,000rpm for 1 minute. Cell pellets were washed with 600uL 1M sorbitol twice. Cells were resuspended in lOOuL of PBS AS buffer (PBS buffer with 0.5% of BSA and G.5M of sorbitol). Then 20uL cells were incubated wife 100 uL of first antibody 40591- T62 (Cat: 40591-T62; Sino Biological Inc.) with 1:200 dilution hi PBSAS buffer for 1 hour with gentle shaking at 400 rpm. Cells were collected by centrifugation at 14000 ipm for 1 minute, cell pellets were wash twice with 600 μL of PBSAS buffer, and then incubated wife 100 uL of Goat anti-Rabbit IgG (H÷L) Secondary Antibody A-l 100S (Cat# A- 11008, Invitrogen). with 1:300 dilution in PBSAS buffer, in dark for 1 hour with gentle shaking at 400 rpm. Cells were pelleted by centrifugation at 14,000 rpm for 1 minute. Cells were washed twice wife 600uL of PBSAS buffer, then resuspended in lOuL PBSAS buffer. Image of cells were obtained with a fluorescence microscope (Leica DSM500) with objective 400X magnification, the excitation arid emission wavelengths were 425-465 mn and 485-533 mn.

Example 2: Preparation of a 2019-nCoV spike protein RBD SP273 Intracellular Expression

Cassette

[0176] The coding region corresponding to residues 319 to 591 (273 amino acids) of tire 2019-nCoV spike glycoprotein (PBD ID: 6VSB), designated here as SP273S, was codon optimized for expression in Y. lipolytica and a polynucleotide was synthesized to generate the artificial gene “SP273S^" (SEQ ID NO: 1), The 273-residue translation product SP273S protein is shown as SEQ ID NO:2.

[0177] A SP273S intracellular expression cassette was generated using the FBAINm promoter (Hong et al., 2012, Yeast; DOI: 10.1002/year.1917; GenBank accession #: XP_504407; SEQ ID NO: 3) and the Pex20 terminator (GenBank accession #; AAC23564; SEQ ID NO: 4) to control gene expression. Tire FBAINm promoter, the synthetic SPC273S coding region and the PEX20 terminator were operably linked to generate an over expression cassete:

FB AINmPro: : SP273 S : : Pex20ter . Expression vector pZP2-SP273P (Table 1) contains the SP273S expression cassette, Ura3 selection marker (GenBank accession #: CAC32856), and two arms of 810 bp 5’ region and 655 bp 3 ’ region of the POX2 locus (GenBank Accession #: KAB8282254). The nucleotide sequence of the FBAINm Promoter is SEQ IB NO:3. The nucleotide sequence of the Pex20 temiinator is SEQ ID NO 4.

Table 1: Functional components in plasmid pZP2-SP273S for intracellular expression

Example 3: Intracellular Expression of SP273S in G. lipolytica

[0178] Y UpofyHca SP273S transformants were generated by transforming strain Y2224 with the DNA fragment containing the SP273S expression cassette and URA3 selection marker that was sandwiched with POX25’ and 3 amis of plasmid pZP2-SP273S (Table 1). Since G. lipolytica strain ATCC#20362, the parent of strain Y2224, prefers non-homologous end joining mechanism to integrate the DNA fragment into its genome, several transformants were analyzed for the SP273S expression.

[0179] SDS PAGE coupled with Western Blot analyses were performed to detect the intracellular expression of SP273S from protein extract of different transformants. Figure 1 shows the result of a typical Western Blot Analyses. It demonstrates that the SP273 protein is produced in transformants, but not in wild type control strain.

Example 4: Preparation of Various Cassettes for Expression and Secretion of SP273S

[0180] Four signal peptides were selected to evaluate effective signal peptide sequences to facilitate the secretion of SP273S: LIP2 (GenBank Accession #; ADL57415; SEQ ID NOs: 5 and 6), XPR2 (GenBank Accession #: KAB8279805; SEQ ID NO: 7 & 8) of Y. lipolytica, MFa (GenBank Accession #: AAA88727; SEQ ID NOs: 7 and 10) and SLIC2 (GenBank Accession #: AAA35129; SEQ ID NOs: 11 and 12) of S. cerevisiae. The DNA fragments coding for these signal peptides were codon optimized, and then DNA was synthesized with SP273 S gene together as fusion proteins (SEQ ID NOs: 13, 14, IS and 16). The FBAINm promoter (SEQ P) NO: 3) and PEX20 terminator (SEQ ID NO: 4) were used to control the expression of these fusion proteins. The functional component of each plasmid is listed in Table 2. The only difference among these constructs is the DMA fragment coding for each signal peptide.

Table 2: Functional components in plasmids for SP273S secreted expression.

[0181] The nucleotide sequence of the codon optimized LIP2 signal peptide is SEQ ID

NO:5 and the amino acid sequence of the LIP2 signal peptide is SEQ ID NG:6. The nucleotide sequence of the codon optimized XPR2 signal peptide is SEQ ID NO: 7 and the amino acid sequence of the XPR2 signal peptide is SEQ ID NO: 8. The nucleotide sequence of the codon optimized MFa signal peptide is SEQ ID NO:9 and the amino acid sequence of the MFa signal peptide is SEQ ID NO: 10. The nucleotide sequence of the codon optimized SUC2 signal peptide is SEQ ID NO: 11 and the amino acid sequence of the SUC2 signa l peptide is SEQ ID NO:12. The ammo acid sequence of the LIP2 signal peptide fused with 5P273S is SEQ ID NO: 13. The amino acid sequence of the XPR2 signal peptide fused with SP273S is SEQ ID NO: 14. The amino acid sequence of the MFa signal peptide fused with SP273S is s SEQ ID NO: 15. The amino acid sequence of the SUC2 signal peptide fused with SP273S is SEQ ID NO:16.

Example 5: Secreted Expression of SP273S in Y. hvolvtica

[0182] Y. Hpoiyiica SP273S secretion transformants were generated by transforming Yarrowia strain Y2224 with the DMA fragment containing the SP273S secretion expression cassette and URA3 selection marker that was sandwiched with PQX25’ and 3’ arms of plasmid pL2SP2?3, pM†SP273, pS2SP273 & pX2SP273, individually (Table 2). it has been reported that Y. lipolytica strain ATCC#20362. the parent of strain Y2224, prefers non-homologons end joining mechanism to integrate the DNA fragment into its genome, therefore, several transformants were analyzed for each of these four different constructs.

[0183] SDS PAGE coupled with Western Blot analyses were performed to detect the secreted expression of SP273S from, concentrated supernatant of overnight cultures (1 OD of 1 rnL each) of these different transformants. Figures 2 aud 3 are the results of typical Western Blot analyses, showing SP273S protein secreted into supernatant by different transformants, hut not observed in wild type control (iintransfonned) strain. The degree of facilitated secretion of SP273S vanes for the four different signal peptides evaluated. The XPR2 signal peptide appears to be the most effective among the four signal peptides tested for secretion of SP273S protein. Note that a band of non-specific protein is detected by the primary antibody used in the Western blots.

Example 6 Preparation of Various Cassettes for Expression and Surface Display of SP273S

[0184] To End an effective anchor to display SP273S protein on the cell surface, four candidates from Y. lipofytica were selected: the C-terrmnal 110 amino acids of ceil wall protein 1 (CWPi; GenBank Accession #: XP_504119; SEQ ID NO: 17); the C-temhmd 121 ammo acids of cell wall protein 3 (CWP3; GenBank Accession #: XP 503352; SEQ ID NO: 18), the C- terminal 173 amino acids of cell wall protein 6 (CWP6; GenBank. Accession #: XP_50557i; SEQ ID NO: 19), and anchor protein Piri (PIR1; GenBank Accession #: VBB85952; SEQ ID MO: 2d).

[0185] The DNA fragments coding for these cell wall-anchoring domains were codon optimized, and then synthesized with the XPR2::SP273S chimeric protein together to form 4 novel fusion proteins: XPR2::SP273S::CWP1 anchor (SEQ ID NO: 21); XPR2::SP273S::CWP3 anchor (SEQ ID NO: 22):, XPR2::SP273S::CWP6 anchor (SEQ ID NO: 23); and XPR2 : : SP273S : ; PIR1 anchor (SEQ ID NO: 24). The FBAINm promoter (SEQ ID NO: 3) and PEX20 terminator (SEQ ID NO: 4) sequences were used to control the expression of these fusion proteins.

[0186] Four constructs were generated to contain these 4 different expression cassettes. The functional component of each plasmid is listed in Table 3. The only difference among these constructs is the DNA fragment coding for display anchors.

Table 3: Functional components of 4 plasmids for SP273S displayed expression

Example 7: Surface Displayed Expression of SP273S in Y. lipolytyca

{0187] Y. kpolytica SP273S surface display transformants were generated by transforming strain Y2224 with the DMA fragments containing the SP273S displayed expression cassettes and URA3 selection marker that was sandwiched with POX25 and 3’ arms of plasmids pL2SP273SWl, pMfSP273SW3, pS2SP273SW6 & pX2SP273SPR, individually (described on Table 3). Since Y. lipolytica strain ATCC#20362, the parent of strain Y2224, prefers iion- homologous end joining mechanism to integrate the DNA fragment into its genome, several transformants were analyzed for each of these four different constructs.

[0188] SDS PAGE coupled with Western Blot, and cell sorting with imnmno-microseopic, and ELISA analyses were performed to detect the displayed expression of SP273S from different transformants. FIGS. 4 and 5 are the results of these analyses. These results demonstrate the displayed expression of SP273 on the surface of the transformed Yarrowia.

Example 8. Generation SP273S Intracellular Production Strain SP036

[0189] To increase the intracellular expression of SP273S, the SP273 expression cassette (Example 2) was coupled with Ura3 selection marker with 12 bp minimal promoter. Only those transformants with multiple integration of the DNA fragment containing the SP273 expression cassette and Ura3 selection marker with minimal promoter will glow with normal rate and form similar size of colonies as the single integration of the DNA fragment containing the SP273 expression cassette and Ura3 selection marker with frill length promoter. As described in Example 2, the FBAINm promoter, the synthetic SPC273S coding region and the PEX20 terminator were operably linked to generate an over expression cassette: FBAINmPro::SP273S;:Pex20ter. Expression vector pZP2-SP273AU (Table 4) contains the SP273S expression cassette, Ura3 selection marker (GenBank accession #: CAC32856) with 12 bp minimal promoter, and two arms of 810 bp 5’ region and 655 bp 3’ region of the POX2 locus (GenBank Accession #: KAB8282254).

Table 4: Functional components in plasmid pZP2-SP273AU for intracellular expression at high level

[0190] SDS PAGE coupled with Western Blot analyses were performed to detect the intracellular expression of SP273S from protein extract of different transformants. FIG. 6 shows the result of a typical Western Blot Analyses, in this case demonstrating transformant number 1 (designated as strain SP036) produced the SP273 protein at the highest level among different transformants.

Example 9: Generation of SP273 Displayed Production Strains SP045 with CWP1 Anchor and

SP046 with PIR1 Anchor

[0191] As described in Example 8, to increase the displayed expression of SP273S, the SP273 displayed expression cassettes (Example 6, Table 3) XPR2::SP273S::CWP1 anchor and XPR2::SP273S:;PIR1 anchor was individually coupled with Ura3 selection marker with 12 bp minimal promoter (Table 5). Only those transformants with multiple integration of the DNA fragment containing the SP273 displayed expression cassette and Ura3 selection marker with minimal promoter will grow with normal rate and form similar size of colonies as the single integration of the DNA fragment containing the SP273 displayed expression cassette and Ura3 selection marker with frill length promoter.

Table 5: Functional components of 2 plasmids for SP273S displayed expression at high level

[0192] As described in Example 6, the chimeric genes encoding for the novel fusion proteins: XPR2::SP273S;:CWP1 anchor and XPR2::SP273S::PIR1 anchor were controlled by the same FBAINm promoter and PEX20 terminator (table 5). Expression vectors pZP2- X2CW1U and pZP2-X2PRU contains the two different SP273S display expression cassettes, and Ura3 selection marker (GenBank accession #: CAC32856) with 12 bp minimal promoter, and two arms of 810 bp 5’ region and 655 bp 3' region of the POX2 locus (GenBank Accession #: KAB8282254).

FACS analyses were employed to analyze the surface display of transformants with DNA fragment containing XPR2: :SP273S::CWP1 anchor or XPR2::SP273S::PåR1 expression cassette with Ura3 selection marker with 12 bp minimal promoter. Shown in the FIG. 7 is a histogram of Yarrowia WT, SP045 and SP046 strains, demonstrating that strains SP045 and SP046 displayed SP273 on the eel! surface of engineered Yarrowia at a high level.

SEQUENCES

Nucleotide sequence of the SP273S gene:

Amino acid sequence of tiie SP273S polypeptide:

Nucleotide sequence of the FBAINm Promoter:

Nucleotide sequence of the Pex20 terminator:

Nucleotide sequence of the codon optimized LIP2 signal peptide;

Amino acid sequence of the LEP2 signal peptide:

Nucleotide sequence of the codon optimized XPR2 signal peptide:

Amino acid sequence of theXPR2 signa l peptide:

Nucleotide sequence of the codon optimized MFa signal peptide :

Amino acid sequence of the MFa signal peptide: N

Nucleotide sequence of the codon optimized SUC2 signal peptide:

Amino acid sequence of the SUC2 signal peptide:

Ammo acid sequence of the LIP2 signal peptide fused with SP273S:

Amino acid sequence of the XPR2 signal peptide fused with SP273S:

Amine acid sequence of the MFa signal peptide fused with SP273S:

Amino acid sequence of the SUC2 signal peptide fused with SP273S:

Amine acid sequence of the CWPI anchor:

Ammo acid sequence of the CWP3:

Amino acid sequence of the CWP6 anchor:

Ammo acid sequence of the PM1 anchor:

Amino acid sequence of the XPR2::SP273S::CWP1 anchor fusion protein:

Amino acid sequence of the XPR2::SP273S::CWP3 anchor fusion protein:

Amino acid sequence of the XPR2::SP273S::CWP6 anchor fusion protein:

Amino acid sequence of the XPR2::SP273S::PIR1 anchor fusion protein:

Nucleotide sequence of the Spike protein:

Amino acid sequence of the Spike protein

Amino acid sequence of Severe acute respiratory syndrome coronavirus nucleocapsid phosphoprotein (2YP_009724397.2) :

Amino acid sequence of Severe acute respiratory syndrome coronavirus nucleocapsid phosphoprotein (2YP_009724397.2) 41-419:

Amino acid sequence of Severe acute respiratory syndrome corouavinis mieleoeapsid phosphoprotein (48-419):

Claims

We claim:

1 A recombinant Yarrowia cell comprising a nucleic acid encoding a coronavirus spike protein or a fragment thereof.

2. The recombinant cell of claim 1 , wherein the Yarrowia ceil is selected from the group consisting of Yarrowia bubula, Yarrowia deformans, Yarrowia hpolytica, Yarrowia porcina, Yarrowia yakushimensis, and Yarrowia parophonii.

3. The recombinant cell of cla im i or claim 2, wherein the spike protein or fra gment thereof is expressed mtracellularly, is secreted extraeellularly, or is displayed on the surface of the Yarrowia cell.

4. The recombinant cell of any one of claim 1-3, wherein the coronavirus spike protein or fragment thereof is derived from a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and SARS-CoV-2 virus.

5. The recombinant cell of claim 4, wherein the coronavirus is a SARS-CoV-2 vires.

6. The recombinant cell of any one of claims 1-5, wherein the nucleic acid encoding the coronavirus spike protein comprises SEQ ID NO: 1.

7. The recombinant cell of any one of claims 1-5, wherein the corona vires spike protein comprises the amino acid sequence of SEQ ID NO:2.

8. The recombinant cell of claim 7, wherein the coronavirus spike protein further comprises a signal peptide selected from the group consisting of signal peptides encoded by SEQ åD NO: 6, SEQ ID NO:8, SEQ ID NO: 10, and SEQ ID NO: 12.

9. The recombinant cell of any one of claims 1-8 further comprising a nucleic acid encoding one or more additional coronavirus proteins or fragment thereof.

10. The recombinant cell of claim 9, wherein the additional coronavirus protein is a coronavirus nucleocapsid phosphoprotein or fragment thereof.

11. The recombinant cell of claim 10, wherein the coronavirus nucleocapsid phosphoprotein comprises the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO:29.

12. The recombinant cell of any one of cl aims 7-11, wherein the coronavirus spike protein and/or additional coronavirus proteins farther comprises a cell surface anchoring peptide selected from the group consisting of cell surface anchoring peptides encoded by SEQ ID NO: 17, SEQ ID NO: IS, SEQ ID NO: 19, and SEQ ID NO:20.

13. A vector comprising the nucleic acid encoding a coronavirus spike protein or fragment thereof for expression in the recombinant cell of any one of claims 1-12.

14. The vector of claim 13, farther comprising a promoter encoded by SEQ ID NO:3 and/or a terminator encoded by SEQ ID NO: 4

15. A composition comprising the recombinant cell of any one of claim 1-12 and a pharmaceutically acceptable earner or excipient.

16. The composition of claim 15, wherein the composition is lyophi!ized or freeze dried.

17. The composition of claim 16, wherein the composition is encapsulated or coated.

18. The composition of any one of claims 15-17, wherein the composition is a food product, food ingredient, dietary supplement or medicament.

19. The composition of any one of cla ims 15 -18, wherein at least about 100 mg composition to at least about 10 g composition of recombinant YarrowM cells are present in the composition,

20. The composition of any one of claims 15-19, wherein the composition is a probiotic,

21. The composition of any one of cla ims 15-20, wherein the composition has been pasteurized or heat heated.

22, A tablet prolonged-release capsule, prolonged-release grannie, powder, sachet, or gummy comprising the composition of any one of claims 15-21.

23, The composition of any one of claims 15-21, wherein the composition is formulated for intranasal administration.

24, A mechanical inhaler comprising the composition of claim 23.

25, A kit comprising one or more of (a)(1) the composition of any one of claims 15-21; C ii) the tablet, prolonged-release capsule, prolonged- release granule, powder, sachet, or gummy of claim 22; or (iii) the mechanical inhaler of claim 24; and b) mitten instructions for administration to a subject.

26, A method of heating or preventing illness and/or symptoms associated with a coronavirus infection in a subject in need thereof, said method comprising administering a. therapeutically effective dose of the recombinant cell of any one of claim 1 12 or the composition of any one of claims 15-21 to the subject.

27, The method of claim 26, wherein said illness is a respiratory illness.

28, The method of claim 27, wherein the respiratory illness is acute respiratory distress syndrome (ARDS).

29, The method of claim 27, wherein the respiratory illness is pneumonia.

30, The method of any one of claims 26-29, wherein said symptoms are one or more of shortness of breath or difficulty breathing (dyspnea), fatigue, muscle or body aches, nausea or vomiting, diarrhea, cough, fever, loss or change of sense of smell (anosmia) and loss or change of sense of taste (ageusia).

31, The method according to any one of the claims 26-30, wherein said preventing or heating is achieved by stimulation of the immune system in the subject when in contact with recombinant cell of any one of claims 1-12.

32, The method of claim 31 , wherein said stimulation of the immune system is a T-celT mediated immune response and/or a B-cell-mediated immune response.

33, The method according to any one of claims 26-32, wherein the coronavirus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and SARS-CoV-2 viras.

34, The method according to claim 33, wherein the coronavirus is a SARS-CoV-2 viras.

35, The method according to any one of claims 26-34, wherein the subject has one or more pre-existing conditions selected from the gr oup consisting of obesity, type II diabetes, chronic lung disease or moderate to severe asthma, heart conditions, immunocompromised, chronic kidney disease and liver disease.

36, The method according to any one of claims 26-35, wherein the subject is 65 years of age or older and/or is a resident in a nursing home or long-term care facility or jail or prison or university.

37, The method according to any one of claims 26-36, wherein the subject is a human.

38, The method according to any one of claims 26-36, wherein the subject is a non-human animal.

39, A recombinant cell of any one of claims 1-12 for use hi preventing or treating illness and/or symptoms associated with coronaviruses in a subject in need thereof.

40, The recombinant cell for use of claim 39, wherein said illness is a respiratory illness,

41, The recombinant cell for use of claim 39 or claim 40, wherein the respiratory illness is acute respiratory distress syndrome (ARDS).

42, The recombinant cell for use of claim 40, wherein the respiratory illness is pneumonia.

43, The recombinant cell for use according to any one of claims 39-42, wherein said symptoms are one or more of cough, fever, shortness of breath or difficulty breathing (dyspnea), fatigue, muscle or body aches, nausea or vomiting, diarrhea, loss or change of sense of smell (anosmia), and loss or change of sense of taste (ageusia).

44, The recombinant cell for use according to any one of claims 39-42, wherein said preventing or treating is achieved by stimulation of the immune system in the subject when in contact with the recombinant cell of any one of claims 1-12,

45. The method of claim 44, wherein said stimulation of the immune system is a T-eell- mediated immune response and/or a B-cell-mediated immune response.

46, The recombinant cell for use according to any one of c laims 39-45, wherein the coronavirus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and SARS-CoV-2 vims.

47. The recombinant cell for use according to claim 46, wherein the coronavirus is a SARS- CoV-2 virus.

48. The recombinant cell for use according to any one of claims 39-47. wherein the subject has one or more pre-existing conditions selected from the group consisting of obesity , type II diabetes, chronic lung disease or moderate to severe asthma, heart conditions, immunocompromised, chronic kidney disease, liver disease.

49. The recombinant cell for use according to any one of claims 39-48, wherein the subject is 65 years of age or older and/or is a resident in a nursing home or long-term care facility or jail or prison or a university.

50. The recombinant cell for use according to any one of claims 39-49. wherein the subject is human.

51. A composition of any one of claims 15-18 for use in preventing or treating illness and/or symptoms associated with coronaviruses in a subject in need thereof.

52. The composition for use of claim 51. wherein said illness is a respiratory illness.

53, The composition for use of claim 51 or claim 52. wherein ihe respiratory illness is acuterespiratory distress syndrome (ARDS).

54, The composition for use of claim 52, wherein the respiratory illness is pneumonia.

55, The composition for use according to any one of claims 51-54, wherein said symptoms are one or more of cough, fever, shortness of breath or difficulty breathing (dyspnea), fatigue, muscle or body aches, nausea or vomiting, diarrhea, loss or change of sense of smell (anosmia), and loss or change of sense of taste (ageusia).

56, The composition for use according to any one of claims 51-54, wherein said preventing or treating is a chieved by stimulation of the immune system in the subject when in contact with the composition of any one of claims 12-18.

57, The method of claim 56, wherein said stimulation of the immune system is a T-cell- mediated immune response and/or a B-cell-mediated immune response.

58, The composition for use according to any one of claims 51-57, wherein the coronavirus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus),

OC43 (beta coronavirus), HK.U1 (beta coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and SARS-CoV-2 virus.

59, The composition for use according to claim 58, wherein the coronavirus is a SARS-CoV- 2 virus.

60, The composition for use according to any one of claims 51-59, wherein the subject has one or more pre-existing conditions selected from the group consisting of obesity, type II diabetes, chronic lung disease or moderate to severe astlmia, heart conditions, immunocompromised, chronic kidney disease, liver disease.

61, The composition for use according to any one of claims 51-60, wherein the sub ject is human.

62, The composition for use according to any one of claims 47-61, wherein the subject is 65 years of age or older and/or is a resident in a nursing home or long-term care facility or jail or prison or a university.

63, Use of a recombinant cell of any one of claims 1-12 for preventing or treating illness and/or symptoms associated with coronaviruses in a subject in need thereof.

64, Use of a composition of any one of claims 15-21 for preventing or treating illness and/or symptoms associated with coronaviruses in a subject in need thereof.

65, The use of claim 60 or claim 64, wherein said illness is a respiratory illness.

66, The use of any one of claims 63-65 , wherein the respiratory illness is acute respiratory distress syndrome (ARDS).

67, The use of any one of claims 63-65 , wherein the respiratory illness is pneumonia.

68, The use of any one of claims 63-67 , wherein said symptoms are one or more of cough, fever, shortness of breath or difficulty breathing (dyspnea), fatigue, muscle or body aches, nausea or vomiting, diarrhea, loss or change of sense of smell ( anosmia), and loss or change of sense of taste (ageusia).

69, The use of any one of claims 63-68, wherein said preventing or treating is achieved by stimulation of the immune system in the subject when in contact with the recombinant of any one of claims 1-12.

70, The use of claim 69, wherein said stimulation of the immune system is a T-cell-mediaied immune response and/or a B-cell-mediated immune response.

71, The use of any one of claims 63 -70, wherein the coronavirus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (Middle East Respiratory Syndrome coronavirus), SARS-CoV, and SARS-CoV-2 virus.

72, The use of claim 71, wherein the coronavirus is SARS-CoV-2 virus.

73. The use of any one of claims 63-72, wherein the subject has one or more pre-existing conditions selected from the group consisting of obesity, type II diabetes, chronic lung disease or moderate to severe asthma, heart conditions, immunocompromised, chronic kidney disease, liver disease,

74. The u se of any one of claims 63-73, wherein the subject is 65 years of age or older and/or is a resident in a nursing home or long-term care facility or jail or prison or a university.

75. The use of any one of claims 73-74, wherein the subject is human.

76. The use of any one of claims 76-75, wherein the subject is a non-human animal.

77. A recombinant Yarrowia cell comprising one or more nucleic acids encoding one or more viral proteins or fragments thereof.

78. The recombinant cell of claim 77, wherein the Yarrowia cell is selected from the group consisting of Yarrowia bubula, Yarrowia deformans, Yarrowia Hpolytica, Yarrowia porcina, Yarrowia yakushimensis, and Yarrowia parophonii.

79. The recombinant cell of claim 77 or claim 78, wherein the viral protein(s) or fragment(s) thereof are expressed intracellularly, secreted extra cellularly, or displayed on the surface of the Yarrowia cell.

80. The recombinant cell of any one of claims 77-79, wherein the viral protein is derived from an influenza virus.

81. The recombinant cell of claim 80, wherein the influenza virus is influenza virus A, influenza virus B, influenza virus C, or influenza virus D.

82. The recombinant cell of claim 81, wherein the influenza virus is influenza virus A and the viral proiem(s) or fragments thereof are derived from strain HINT H2N2, H3N2, H5N1, H7N7, H1N2. H9N2, H7N2, H7N3, H10N7, H7N9, and/or H6N1.

83. The recombinant cell of any one of claims 77-82, wherein the viral protein(s) are hemagglutinin and/or neuraminidase.

84, The recombinant cell of any one of claims 77-83, wherein the viral protein(s) further comprise one or more cell surface anchoring peptide selected from the group consisting of cell surface anchoring peptides encoded by SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:20.

85, A vector comprising the nucleic acid encoding one or more viral proteins or fragment thereof for expression in the recombinant cell of any one of claims 77-83.

86, A composition comprising the recombinant cell of any one of claim 77-83 and a pharmaceutically acceptable carrier or excipient.

87. The composition of claim 86, wherein the composition is formulated for intranasal administration.

88. A tablet, prolonged-release capsule, prolonged-release granule, powder, sachet, or gummy comprising the composition of any one of claims 77-84.

89. A mechanical inhaler comprising the composition of claim 86.

90, A method of treating or preventing illness and/or symptoms associated with a respiratory viral infection in a subject in need thereof said method comprising administering a therapeutically effective dose of the recombinant cell of any one of claim 77-84 or the composition of any one of claims 85-86 to the subject.

91. A recombinant cell of any one of claims 77-84 for use hi preventing or treating illness and/or symptoms associated with respiratory viral infection in a subject in need thereof.

92. A method for producing a coronavirus spike protein or fragment thereof the method comprising culturing the recombinant cell of any one of claims 1-11 in media under suitable conditions for the production of the spike protein or fragment thereof wherein the spike protein or fragment thereof is secreted extracellularly.

93, The method of claim 93, further comprising recovering the spike protein or fragment thereof from the media. 34, A composition comprising the recombinant coronavirus spike protein or fragment thereof produced by the method of claim.92 or claim 93 and a pharmaceutically acceptable earner or excipient.

95, A tablet, prolonged-release capsule, prolonged-release granule, powder, sachet, or gummy comprising the composition of claim 94.

96, A mechanical inhaler comprising the composition of claim 94.

97, A method of treating or preventing illness and/or symptoms associated with a coronavirus infection in a subject m need thereof, said method comprising administering a therapeutically effective dose of composition of claim 94 to the subject .

98, A method for producing one or more viral proteins or fragments thereof, the method comprising culturing the recombinant cell of any one of claims 77-83 in media under suitable conditions for the production of oue or more viral proteins or fragments thereof, wherein the one or more viral proteins or fragments thereof are secreted extracellularly.

99, The method of claim 98, further comprising recovering the one or more viral proteins or fragments thereof from the media.

100, A composition comprising the one or more viral proteins or fragments thereof produced by the method of claim 98 or claim 99 and a pharmaceutically acceptable earner or excipient.

101, A tablet, prolonged-release capsule, prolonged-release granule, powder, sachet, or gummy comprising the composition of claim 100.

102, A mechanical inhaler comprising the composition of claim 100.

103, A method of treating or preventing illness and/or symptoms associated with a respiratory viral infection in a subject in need thereof, said method comprising administering a therapeutically effective dose of composition of claim 100 to the subject.