HCoV VACCINE FOR IMPROVING IMMUNITY AGAINST SARS-COV-2 INFECTION

Abstract

Embodiments include a method of using inactivated human cold coronaviruses (HCoVs) particularly HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, alone or as a booster, for the immunization against SARS-CoV-2 infections. Vaccine embodiments further comprise HCoV virus envelope subunits which may be in the form of virus-like spheroids (VLS).

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34. An immunogenic composition comprising lipid virus-like spheroid particles wherein the virus like spheroid particles comprise immunogenic protein epitopes associated with glycoprotein spikes in the lipid membrane from HCoV-NL63 and HCoV-HKU1 and the lipid virus-like spheroid particles contain less immunogenic protein epitopes than associated with the full native virus selected, and the lipid virus-like spheroid particles are formed by: (a) selecting copper foam with a copper skeleton of pores around 50 μm; (b) immersing a portion of the copper foam into an aqueous solution of 0.03 M AgNO3 at room temperature; (c) treating the immersed silver foam with a mixed ethanol solution containing HS(CH.sub.2).sub.11CH.sub.3 and HS(CH.sub.2).sub.10COOH to form a treated copper foam; (d) drying the treated copper foam to form the final treated copper foam (FTCF); (e) running enveloped virus of HCoV-NL63 and HCoV-HKU1_ separately through untreated copper foam followed by the treated copper foam in a pH 7-7.4 solution; (f) releasing materials captured by the final treated copper foam by directing solution with a pH of 10.5-11 over said final treated foam; (g) separating out spheroids having a diameter between 90-150 nm.

35. The immunogenic composition of claim 34 further comprising an adjuvant from one at least of: alum, algammulin, monophosphoryl lipid A (MPL), resiquimod, muramyl dipeptide (MPD), N glycolyl dipeptide (GMDP), polyIC, CpG oligonucleotide, aluminum salts, water-in-oil emulsion and oil-in-water emulsion.

36. The immunogenic composition of claim 35 comprising an resiquimod adjuvant.

37. The immunogenic composition of claim 35 comprising an aluminum salt adjuvant.

38. The immunogenic composition of claim 35 comprising an alum adjuvant.

39. The immunogenic composition of claim 35 comprising a muramyl dipeptide adjuvant.

40. The immunogenic composition of claim 35 comprising an algammulin adjuvant.

41. The immunogenic composition of claim 35 a monophosphoryl lipid A adjuvant.

42. The immunogenic composition of claim 35 comprising N glycolyl dipeptide adjuvant.

43. The immunogenic composition of claim 35 comprising polyIC adjuvant.

44. The immunogenic composition of claim 35 comprising CpG oligonucleotide adjuvant.

45. The immunogenic composition of claim 35 comprising water-in-oil emulsion adjuvant.

46. The immunogenic composition of claim 35 comprising oil-in-water emulsion adjuvant.

47. The immunogenic composition of claim 35 comprising alum and MPD adjuvant.

Description

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0029] In one embodiment there is provided a vaccine for improving T-cell immunity, particularly. CD4+ T cell immunity, to SARS-CoV-2, said vaccine being prepared by (a) providing a plurality of population of cells in cell culture medium; (b) infecting each population of cells by inoculating the population of cells with the at least one of: HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 and incubating the inoculated population of cells to allow the virus in each cell culture medium to replicate and propagate; (c) collecting the virus from each cell culture medium; (d) purifying each of said virus from each cell culture; (e) inactivating each virus; and (f) preparing a pharmaceutical preparation for inoculation having different antigens from at least two or more of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-KHu1. In such embodiment, the antigens may comprise the whole virus, or part of such virus; from, for example, the virus envelope or a protein associated with the virus. The population of cells may be Vero cells. Inactivation may be at least one of beta propiolactone (BPL), hydrogen peroxide, formalin (formaldehyde) and copper. In one embodiment copper inactivation and hydrogen peroxide inactivation are sequential in order. The vaccine may further comprise an adjuvant one at least of which is selected from alum, Immodulon (IMM-101-containing a heat-killed whole cell Mobacterium obuense, a rapidly dividing harmless spaprophyte), algammulin, monphosphoryl lipid A (MPL), resiquimod, muramyl peptide (MPD), N glycolyl dipeptide (GMDP), polylC, CpG oligonucleotide, aluminum salts, water in oil emulsion and oil in water emulsion.

[0030] Deactivation is preferably by exposing the HCoV at one time to copper ions (particularly cupric) and at a distinct time hydrogen peroxide (sequential deactivation). Copper has a free electron in its outer orbital shell of electrons that allows it to easily take part in oxidation-reduction reactions. The copper pokes holes in the Coronavirus lipid coating which allows lower concentrations of other inactivating agents such as beta-propiolactone, formalin or H.sub.20.sub.2 to be used to inactivate the virus. The present inventors have recognized concomitant use is sub-optimal. Whole killed virus, or protein/glycoprotein submits of the viruses may be used in the making of the vaccine.

[0031] In another embodiment there is a combination vaccine for immunization against SARS-CoV-2 infection comprising at least one unique epitope from each of a plurality in the group of HCoV-NL63, HCoV-OC42, HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, or the subgroup HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, of such unique epitope being unique to all other HCoVs in such group, or genetic instructions to make such one unique epitope from each of viruses in the group, wherein the unique epitopes have epitope homology with SARS-CoV-2 of greater than or equal to 60%, more preferably greater than 70%, yet more preferably greater than 80%, and yet more preferably greater than 90%, or even more preferably greater than 95%. In a preferred embodiment at least two unique epitopes from each virus of one, two, three, or four in the group is combined, and in a more preferred embodiment at least three unique epitopes from each virus of one, two, three, or four in the group is combined. The unique epitope in each case may be from the RNA virus envelope. The unique epitopes may be limited to cross-reactivity with epitopes of SARS-CoV-2 in the SARS-COV-2 spike, N, nsp8, nsp12 and nsp13. The unique epitopes are preferably limited to epitopes found associated with the native HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 lipid bilayer. The unique epitopes may be naturally-derived, synthetically manufactured, or recombinantly produced. Such unique HCoV epitope vaccine may be used with any of the vaccines noted in the WHO World Health Organization, Draft Landscape of COVID-19 Candidate Vaccines, 31 Jul. 2020, and similar technology based vaccines. In a preferred embodiment, such unique HCoV epitope vaccine is used as a booster such COVID-19 Candidate Vaccines set forth by the WHO 31 Jul. 2020 to provide prolonged cell mediated immunity, particularly through CD4.sup.+ T-cells.

[0032] Also provided in a vaccine for immunization against SARS-CoV-2 infection comprising at least one unique protein epitope, or genetic instructions to make such one unique protein epitope, from a plurality of the group of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, such unique protein epitope being unique to all other HCoVs in such group, wherein the unique epitopes have epitope homology with SARS-CoV-2 of greater than or equal to 60%. The vaccine may comprises at least two unique protein epitopes, or genetic instruction make such two unique protein epitopes, are selected from a plurality of the group of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1. The plurality of the group of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 may be three HCoVs. The unique protein epitopes have a homology with SARS-CoV-2 epitope of at least greater than or equal to 50%, 60%, 67%, 70%, 80% 90%, or 95%. The vaccine may be used as a booster to a SARS-CoV-2 specific vaccine. Preferably the unique protein epitopes have homology of at least 60% with at least one of SARS-CoV-2 spike, N, nsp8, nsp12 or nsp13, and yet more preferably the SARS-CoV-spike protein.

[0033] Also provided is a vaccine for immunization against SARS-CoV-2 infection comprising at three or more unique protein epitopes associated with the lipid membrane from at least two of the group of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKUL such protein epitopes being unique to all other HCoVs in such group, wherein the unique epitopes have epitope homology with SARS-CoV-2 of greater than or equal to 65%. The unique protein epitopes may be associated with the lipid membrane. The vaccine of said unique protein epitopes for each virus from the group may be in the form of lipid virus like spheroid particles. The lipid virus like spheroid particles are formed by: (a) selecting copper foam with a copper skeleton of pores around 50 um; (b) immersing the copper foam an aqueous solution of 0.03 M AgNO3 at room temperature; (c) treating the immersed silver foam with a mixed ethanol solution containing HS(CH.sub.2).sub.11CH.sub.3 and HS(CH.sub.2).sub.10COOH to form a treated copper foam; (d) drying the treated copper foam to form the final treated copper foam (FTCF); (e) running enveloped virus from the group through untreated copper foam followed by the treated copper foam in a 7-7.4 solution; (f) releasing materials captured by the final treated copper foam by directing solution with a pH of 10.5-11 over said final treated foam; (g) separating out spheroids having a diameter between 90 nm-150 nm.

[0034] Coronaviruses are enveloped with a lipid bilayer, and are believed to induce fusion of the viral envelope with the cell membrane to target cells. Viral fusion glycoproteins are the key epitopes to induce the membrane fusion reaction that allows viral entry. U.S. Pat. No. 6,455,050 to Aventis Pasteur Limited teaches techniques for obtaining viral envelope glycoproteins by means of an appropriate detergent (e.g. Triton X-100 or octylglucoside). Nucleopcapsids are taught to be removable by centrifugation, with viral surface glycoproteins being purified from a glycoprotein enriched fraction by affinity chromatography, such as lentil-lectin and concanavlin A covalently coupled to cross-linked Sepharose or cellulosic microporous membranes. Viral surface glycoproteins are taught to be eluted from the column in the presence of an appropriate competing sugar, such as methyl-D-mannopyranoside, in the presence or absence of salt. Highly purified. glycoprotein preparations is said to be obtained in accord with such process (as judged by Coomassie blue or silver stained SDS polyacrylamide gels).

[0035] Taught herein is another technique for isolating natural glycoproteins of the lipid bilayer of coronaviruses from the nucleocapsid. Such technique provides lipid bound glycoproteins that may be reannealed into virus like spheroids (VLS). Such as system makes use of highly lipophilic surfaces that can by pH made to switch to highly hydrophilic surfaces. Such technique may be used in conjunction with the with a HCoV vaccine or SARS-CoV-2 specific vaccine to provide more immunogenicity as seen with VLP (virus like particles). The present inventors propose that ADR is more likely when the antibody pool is more limited to just a few proteins, such as those found on the spike protein of SARS-CoV-2, and that irrespective of contrary thought, a more robust pool. of antibodies may actually reduce ADRs. Such technique makes use of a switchable copper foam having silver deposition followed by surface modification with a mixed solution of thiol containing carboxylic groups and methyl groups (HS(CH.sub.2).sub.11CH.sub.3 and HS(CH.sub.2).sub.10COOH) to provide for pH reversibility between a superhydrophobicity surface and a hydrophilicity surface prposed for removing oil from water. See, Liu et al., A Smart Switchable Bioinspired Copper Foam Responding to Different pH Droplets for Reversible Oil-Water Separation, J. Mater. Chem. 2017, (5) 2603-2612, as explained in Example 2 below. The VLS can be formed form at least one of the group of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, more preferably at least two of the group, yet more preferably three of the group, or even more preferably form all of the viruses in the group. The VLS spheroids may also be formed from SARS-CoV-2 itself. The VLS spheroids can be separated by chromatography, with cryo-electron microscopy being used to detect the same which generally should have diameters of 90-150 nm with glycoprotein protrusions/spikes. The VLS spheroids are then manufactured conventionally into vaccines, which, for example, are used to provide immunity to SARS-CoV-2 infections.

EXAMPLE 1: HCoV Vaccine for Prolonging T-Cell Immunity to SARS-CoV-2

[0036] HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 are isolated from lavage samples, each from multiple persons suffering therefrom to cover the phylogenic tree. Different strains of each HCoV of each is plaque purified and passaged once in Vero cells to generate a P1 stock. The P1 stock is adaptively cultured, passed and expanded on Vero cells. Additional passages are performed to generate, for example, P2 to PS5stocks, Growth kinetics are measured to assure efficient replication and to reach a peak titre of about 6 to 7 log.sub.10 median tissue culture infections dose by 3 or 4 days post infection at a multiplicity of infection of, for example, 0.001 to 0.01 and temperatures between 33° and 37° C. Additional passages are performed to obtain the Puma stock (such as P10). Multiple P stocks are sequenced to assure genetic integrity that might affect NAb epitopes. Whole genome of each strain and the P.sub.final undergo deep sequencing analysis are undertaken to assure sequence homology of more than about 99.95%. P.sub.final stock is propagated in a culture of Vero cells (e.g. 50 liters) using the Cell Factory system. Inactivation is brought about by inactivation with at least one of beta propiolactone (BPL), hydrogen peroxide, formalin (formaldehyde) and copper. Purification is by depth filtration and multiple (e.g. two) steps of chromatography to yield highly pure HCoV stock. Ultrafiltration, size exclusion chromatography and sucrose gradient centrifugation may be used in the purification process. B-propionolactone, for example, may be thoroughly mixed with harvested viral solution at a ratio of 1:4,00 at 2° C.-8° C. Western blot analysis is used to show vaccine stock contains viral structural proteins. Two or more, preferably three or more, of the purified and inactivated HCoV-299E, HCoV-OC43, HCoV-Nt63 and HCoV-HKU1 are mixed with an adjuvant one at least of which is selected from alum, Immodulon (IMM-101-containing a heat-killed whole cell Mobacterium obuense, a rapidly dividing harmless saprophyte), algammulin, monphosphoryl lipid A (MPL), resiquimod, muramyl peptide (MPD), N glycolyl dipeptide (GMDP), polylC, CpG oligonucleotide, aluminum salts, water in oil emulsion and oil in water emulsion, and pharmaceutical excipients to form an administrable vaccine. Inactivation should be measured checking for immunogenic response before and after immunization in animals. Dose may be selected by looking at different doses and determining Nab levels at, for example, 7, 14 and 21 days in each of multiple dosing groups. Whole killed virus, or protein/glycoprotein submits of the viruses may be used in the making of the vaccine.

EXAMPLE 2: Cornavirus Envelope with attached Glycoprotein Separation from RNA and Nucleoproteins

[0037] Copper foam with a copper skeleton of pores around 50 um is selected. The foam are immersed in an aqueous solution of 0.03 M AgNO3 at room temperature. The silver treated foam is then treated with a mixed ethanol solution containing HS(CH.sub.2).sub.11CH.sub.3 and HS(CH.sub.2).sub.10COOH to form the Final Treated Copper Foam (FTCF). The enveloped virus is exposed to first to untreated copper foam followed by the treated copper foam in a pH 7 solution. Untreated copper ions blast hole into the viral coating, while destroying RNA inside of the virus. Passage through the FTCF at pH 7 makes the FTCF highly hydrophobic while at the same time highly lipophilic. The coronavirus flows through the pores of the copper foam. The coronavirus envelope is deposited along the FTCF at pH about 7—about 7.4 with the RNA and nucleoproteins being washed away in the stream. Release of the attracted lipid from the envelope upon change of pH to more basic pHs may form virus like spheres of natural glycoprotein covered lipid membrane (by changing the pH about the FTCF to about 10.5 to no more than about pH 11 which makes the surface much less lipophilic and more hydrophilic). The pH is carefully controlled to avoid irreversible denaturation of the glycoproteins.