WO2022037248A1 - Live attenuated sars-cov-2 virus immunogenic compositions, methods of making and using - Google Patents
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- WO2022037248A1 WO2022037248A1 PCT/CN2021/101931 CN2021101931W WO2022037248A1 WO 2022037248 A1 WO2022037248 A1 WO 2022037248A1 CN 2021101931 W CN2021101931 W CN 2021101931W WO 2022037248 A1 WO2022037248 A1 WO 2022037248A1
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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Definitions
- the present invention is generally in the field of live attenuated viruses containing one or more antigens from Sars-CoV-2, immunogenic compositions including attenuated Sars-CoV-2 antigen, and methods of using such compositions.
- SARS-CoV and MERS-CoV show limit human to human transmission ability while cause severe disease and mortality.
- SARS-CoV-2 which uses human ACE2 as binding receptor as SARS-CoV6, is highly transmissible and causes variable severity of diseases from asymptomatic to severe and fatal outcome.
- SARS-CoV-2 vaccines are being developed for use in humans 30- 33 . However, there are concerns if current vaccine strategies will be able to provide sufficient and long last immunity to prevent infection and alleviate diseases. Studies have also showing that anti-SARS-CoV-2 antibodies decline rapidly in naturally infected individuals 34, 35 .
- compositions that can be used to elicit antibody responses against a SARS-CoV, preferably to levels that provide complete immunity.
- compositions for eliciting an immune response to SARS-CoV are provided.
- compositions continuing live attenuated Sars-CoV-2, Compositions immunogenic against Sars-CoV-2, methods of making and using, are provided.
- the compositions are based SARS-CoV-2 variant, Del-Mut-1 which contains a 30bp deletion at the S/S2 junction (with the PRRA polybasic cleavage motif removed) , passaged in Vero-E6 cells to obtain a variant with additional mutations in the multiple genes in the background of Del-Mut-1.
- This Del-Mut-1 variant is referred to herein as Ca-DelMut live attenuated SARS-CoV-2 virus (LASV) , hereinafter, Ca-DelMut-LASV (GenBank accession no: MT862537) .
- Ca-DelMut-LASV includes a mutation, which results in a deletion of ten amino acid sequence NSPRRARSVA (SEQ ID NO: 1) , which represents a deletion of amino acids 679-688 from the wild type SARS-CoV-2 spike protein from SARS-CoV-2 strain (NCBI Reference Sequence: YP_009724390.1).
- Human-1 SARS-CoV-2 or “Hu-1” DelMut-LASV additionally includes mutations which result in expressed proteins with the following mutations relative to "Hu-1 SARS-CoV-2”; an A578V mutation in the NSP3 protein; P25L mutation in the spike protein; a V367F in the spike protein; an F20S in the E protein and a V62L mutation in ORF8.
- Ca-DelMut has the genome encoded by SEQ ID NO: 3, with a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
- Ca-DelMut comprise a genome encoded by a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 3, with a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
- the method includes passaging the Del-Mut-1 virus under conditions that result in the introduction of the desired mutations in Del-Mut-1 i.e., the following mutations relative to - "Hu-1 SARS-CoV-2"; an A578V mutation in the NSP3 protein; P25L mutation in the spike protein; a V367F in the spike protein; an F20S in the E protein and a V62L mutation in ORF8.
- Del-Mut-1 is passaged at a first temperature, preferably not exceeding 33 °C and for at least 5 passages and subsequently passaged at a temperature, preferably not exceeding 30 °C for at least 3 passages. Each passage is preferably between 2-3 days.
- compositions of Ca-DelMut-LASV or a variant thereof are administered to a subject in need thereof by subcutaneous (s.c. ) , intradermal (i.d. ) , intramuscular (i.m. ) , intravenous (i.v. ) , oral, or intranasal administration; or by injection or by inhalation.
- the strain is administered intranasally.
- the compositions containing Ca-DelMut-LASV are administrated to a mammal in need of protective immunity against a Sars-CoV-2 infection.
- FIG. 1A Schematic diagram showing the deletion and mutations of Ca-DelMut in different regions of SARS-CoV-2 genome.
- Del-Mut-1 virus 15 was serially passaged in Vero E6 cells at 33°C (10 passages) and 30 °C (8 passages) .
- Virus from the 18 th passage was designated Ca-DelMut live attenuated SARS-CoV-2 virus (LASV) , and amplified to prepare virus stock.
- Ca-DelMut was sequenced by Sanger method; mutations are shown in the diagram and Table 1) .
- FIG. 1B is a sequence alignment of the S1/S2 junction regions of wild type SARS-CoV-2 (Hu-1) , and 30-bp deleted mutant, Del-Mut-1 14 .
- FIG. 2A shows replication efficiency of Ca-DelMut LASV is in vitro and in vivo.
- FIG. 2A Vero E6 cells or Calu-3 cells were infected with Ca-DelMut and other viruses at 0.01 moi at 37 °C. At the indicated time points, supernatants were collected, and virus titer determined by plaque assay in Vero E6 cells.
- FIG. 2B Ca-DelMut infection in hamsters. Hamsters were infected intranasally with either Ca-DelMut or wild type (WT) virus at different doses as indicated. Body weight change was monitored for 5 days.
- FIG. 2C Replication of Ca-DelMut in lung and nasal turbinate tissues.
- FIG. 2D Ca-DelMut LASV exhibits cold adaptation phenotype in vitro. Vero-E6 cells were infected with Ca-DelMut and other viruses at 0.01 moi and incubated at 30 °C. At indicated time points, supernatants were collected and virus titer was determined by plaque assay.
- FIGs. 2E-2F show histological data for Hamsters infected with 1x10 3 pfu of either Ca-DelMut, HK-13 or mock infected. At day 2 and 4 post infection, lungs and small intestine were collected and fixed in 10%formalin, and then processed in paraffin blocks and H&E staining.
- FIG. 2E. The top panel shows normal lung structures in the lungs of mock-infected control hamsters at 4x (left) and 20x (right) magnification.
- FIG. 2F shows representative histological images of hamster small intestines. The left-most image shows mock-infected control hamster small intestinal villi with normal structure.
- Ca-DelMut no apparent histopathological changes were detected in the small intestine (middle) .
- Figures 3A-3F show proinflammatory cytokine response in Ca-DelMut and wild type virus infected hamsters.
- Hamsters were infected intranasally with 1x10 3 pfu of either Ca-DelMut or WT HK-13 virus.
- RNA was extracted from lung tissues of infected hamsters and cDNA was synthesized using oligo dT primer.
- proinflammatory cytokines INF- ⁇ (FIG. 3A) , CCR4 (FIG. 3B) , IL-10 (FIG. 3C) , IL-21 (FIG. 3D) , TNF- ⁇ (FIG. 3E) and IL-6 (FIG.
- FIGs. 3G-3L show the effect of Ca-DelMut on proinflammatory cytokine response: TGF ⁇ 1 (FIG. 3G) , IL-12p40 (FIG. 3H) , CCL17 (FIG. 3I) , CCL22 (FIG. 3J) , IL-4 (FIG.
- Figures 4A and 4B are bar graphs showing the level of antibodies induced by Ca-DelMut immunization of hamsters (FIG. 4A) and neutralization activity against HK-13 virus strain (FIG. 4B) .
- Hamsters were immunized intranasally with 1.25x10 5 pfu of either Ca-DelMut LASV, Wild type virus (SZ-002) virus or mock immunized.
- SZ-002 Wild type virus
- Figures 5A and 5B are line and bar graphs showing the ability of Ca-DelMut immunization to protect against WT virus (HK-13 or HK 95) challenge in hamsters.
- Hamsters were inoculated with 1.25x105 pfu Ca-DelMut or mock immunized. At day 28 after immunization, hamsters were challenged with 1x10 3 pfu of either HK-13 or HK-95 virus.
- FIG. 5A Body weight and disease symptoms were monitored for 5 days.
- 5C and 5D are line and bar graphs showing the ability of lower dose of Ca-DelMut immunization to provide complete protection against WT HK-13 virus. measured as changes in body weight (FIG. 5C) and virus titer in lungs and nasal turbinate tissues (FIG. 5D) .
- FIGS. 6A-6B show histopathological analysis of lung pathology in Ca-DelMut LASV and mock immunized hamsters after WT virus challenge.
- hamsters were challenged with 1x10 3 pfu of either HK-13 or HK-95 virus.
- lung tissues were collected for histopathological study.
- FIG. 6A Challenge with HK-13.
- Day 2 Mock-vaccinated hamster lung showed bronchiolar epithelial cell death and the bronchiolar lumen filled with exudate and cell debris (arrow) . Diffuse alveolar infiltration and focal hemorrhage were also seen (arrowheads) .
- Ca-DelMut434 vaccinated hamster lungs showed regional alveolar septal infiltration and blood vessel congestion (arrowhead) , but no obvious bronchiolar epithelial cell death (arrow) .
- Day 5 Lungs of mock-vaccinated hamsters showed severe alveolar infiltration and exudation (arrowheads) , as well as bronchiolar luminal exudation (arrow) .
- Vaccinated hamster lungs showed focal alveolar septal infiltration (arrowheads) , while the bronchiolar epithelium appeared normal with no luminal secretion or cell debris (arrows) .
- FIG. 6B Challenge with HK-95.
- Vaccinated hamster lungs showed bronchiolar epithelial cell death with luminal cell debris (arrow) and diffuse alveolar infiltration with focalhemorrhage and exudation (arrowheads) , with a medium sized blood vessel showing severe endotheliitis (open arrow) .
- Vaccinated hamster lungs showed regional alveolar septal infiltration and blood vessel congestion (arrowhead) ; a blood vessel appeared to be normal (open arrow) , and no obvious bronchiolar epithelial cell death was observed (arrows) .
- FIG. 6C shows the effect of low inoculum of Ca-DelMut against reinfection of wild type SARS-CoV-2. Hamsters were infected intranasally with 1x103 pfu of either Ca-DelMut, HK-13 strain or mock immunized.
- hamsters were challenged with 1x103 pfu of HK-13 virus.
- lungs were collected for histopathological study. Lungs were fixed in 10%formalin, and then processed in paraffin blocks and H&E staining.
- Mock vaccinated hamster lung showed bronchiolar epithelial cells death and luminal secretion mixed with cell debris (arrows) ; diffuse alveolar infiltration and exudation (arrowheads) ; Ca-DelMut immunized lung showed no apparent bronchiolar epithelium cell death (arrows) , alveoli showed regional septal infiltrationIn HK-13 strain immunized hamster lung showed no apparent histopathology other than alveolar wall thickening (arrowheads) after re-challenge.
- each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
- any subset or combination of these is also specifically contemplated and disclosed.
- the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
- contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.
- These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions.
- steps in methods of making and using the disclosed compositions are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
- adjuvant refers to a compound or mixture that enhances an immune response.
- Attenuated refers to refers to procedures that weaken an agent of disease (a pathogen) .
- An attenuated virus is a weakened, less vigorous virus.
- a vaccine against a viral disease can be made from an attenuated, less virulent strain of the virus, a virus capable of stimulating an immune response and creating immunity but not causing illness or less severe illness.
- Attenuation can be achieved by chemical treatment of the pathogen, through radiation, or by genetic modification, using methods known to those skilled in the art. Attenuation may result in decreased proliferation, attachment to host cells, or decreased production or strength of toxins.
- the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease state being treated or to otherwise provide a desired pharmacologic effect.
- the precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc. ) , the disease, and the age of the subject.
- immunological composition or “composition” means that the composition can induce an immune response and is therefore antigenic.
- immune response means any reaction by the immune system. These reactions include the alteration in the activity of an organism's immune system in response to an antigen and can involve, for example, antibody production, induction of cell-mediated immunity, complement activation, or development of immunological tolerance.
- nasal administration refers to any form of administration whereby an active ingredient is propelled or otherwise introduced into the nasal passages of a subject so that it contacts the respiratory epithelium of the nasal cavity, from which it is absorbed into the systemic circulation.
- Nasal administration can also involve contacting the olfactory epithelium, which is located at the top of the nasal cavity between the central nasal septum and the lateral wall of each main nasal passage. The region of the nasal cavity immediately surrounding the olfactory epithelium is free of airflow. Thus, specialized methods must typically be employed to achieve significant absorption across the olfactory epithelium.
- oral refers to administration of a compound or composition to an individual by a route or mode along the alimentary canal.
- oral routes of administration of a composition include, without limitation, swallowing liquid or solid forms of a vaccine composition from the mouth, administration of a vaccine composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a vaccine composition, and rectal administration, e.g., using suppositories that release a live bacterial vaccine strain described herein.
- mammal as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.
- Topical administration refers to the application of a pharmaceutical agent to the external surface of the skin or the mucous membranes (including the surface membranes of the nose, lungs and mouth) , such that the agent crosses the external surface of the skin or mucous membrane and enters the underlying tissues. Topical administration can result in a limited distribution of the agent to the skin and surrounding tissues or, when the agent is removed from the treatment area by the bloodstream, systemic distribution of the agent.
- the agent is delivered by transdermal delivery, e.g., using a transdermal patch.
- Transdermal delivery refers to the diffusion of an agent across the skin (stratum corneum and epidermis) , which acts as a barrier few agents are able to penetrate.
- the dermis is permeable to absorption of many solutes and drugs, and topical administration therefor occurs more readily through skin which is abraded or otherwise stripped of the epidermis to expose the dermis.
- Absorption through intact skin can be enhanced by combining the active agent with an oily vehicle (e.g., creams, emollients, penetration enhancers, and the like, as described, e.g., in Remington's Pharmaceutical Sciences, current edition, Gennaro et al., eds. ) prior to application to the skin (a process known as inunction) .
- an oily vehicle e.g., creams, emollients, penetration enhancers, and the like, as described, e.g., in Remington's Pharmaceutical Sciences, current edition, Gennaro et al., eds.
- peptide refers to a class of compounds composed of amino acids chemically bound together.
- the amino acids are chemically bound together via amide linkages (CONH) ; however, the amino acids may be bound together by other chemical bonds known in the art.
- the amino acids may be bound by amine linkages.
- Peptide as used herein includes oligomers of amino acids and small and large peptides, including polypeptides.
- a “variant, ” “mutant, ” or “mutated” polynucleotide or polypeptide contains at least one polynucleotide or polypeptide sequence alteration as compared to the polynucleotide or polypeptide sequence of the corresponding wild-type or parent polynucleotide or polypeptide. Mutations may be natural, deliberate, or accidental. Mutations include substitutions, deletions, and insertions.
- compositions including live attenuated SARS-CoV-2, herein, Ca-DelMut-LASV are provided.
- Ca-DelMut-LASV is provided in a pharmaceutically acceptable, alone or in combination with an adjuvant
- SARS-CoV-2 has four main structural proteins including spike (S) glycoprotein, small envelope (E) glycoprotein, membrane (M) glycoprotein, and nucleocapsid (N) protein, several accessory proteins and several non-structural proteins (NSP) .
- the spike or S glycoprotein is a transmembrane protein with a molecular weight of about 150 kDa found in the outer portion of the virus.
- S protein forms homotrimers protruding in the viral surface and facilitates binding of envelope viruses to host cells by attraction with angiotensin-converting enzyme 2 (ACE2) expressed in lower respiratory tract cells.
- ACE2 angiotensin-converting enzyme 2
- N protein The nucleocapsid known as N protein is the structural component of CoV localizing in the endoplasmic reticulum-Golgi region that structurally is bound to the nucleic acid material of the virus.
- M protein Another important part of this virus is the membrane or M protein, which is the most structurally structured protein and plays a role in determining the shape of the virus envelope.
- the last component is the envelope or E protein which is the smallest protein in the SARS-CoV structure that plays a role in the production and maturation of this virus.
- the virus includes several
- Ca-DelMut-LASV includes a deletion which results in expression of a spike protein without the amino acid sequence NSPRRARSVA (SEQ ID NO: 1) , which represents a deletion of amino acids 665-705 of the wild type SARS- CoV-2 spike protein of the Hu-1 isolate (Wu, et al., Nature, 579, 265-269 (2020) .
- Mutations in DelMut-LASV additionally includes the following amino acid changes relative to Hu-1 isolate; an A578V mutation in the NSP3 protein; P25L mutation in the spike protein; a V367F in the spike protein; an F20S in the E protein and a V62L mutation in ORF8.
- the complete sequence for the WT SARS-CoV-2 surface glycoprotein has been assigned the NCBI Reference: YP_009724390.1.
- the disclosed mutations are relative Hu-1 SARS-CoV-2 strain (Wu e Wu, et al., Nature, 579: 265-269 (2020) ) . Mutations in Ca-DelMut relative to Hu-1 SARS-CoV-2 strain are shown in Table 1.
- Ca-DelMut has the genome encoded by SEQ ID NO: 3, with a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
- Ca-DelMut comprise a genome encoded by a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 3, with a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
- DelMut-LASV or variants thereof can be included in a formulation for administration, in a carrier, and in some embodiments, in combination with an adjuvant.
- the adjuvant can serve as the carrier.
- immunogenic compositions containing the disclosed DelMut-LASV do not include an adjuvant.
- the disclosed Ca-DelMut-LASV or variants thereof can be administered in conjunction with other immunoregulatory agents, including adjuvants.
- adjuvants include, one or more set forth below:
- Mineral Containing Adjuvant Compositions include mineral salts, such as aluminum salts and calcium salts.
- Exemplary mineral salts include hydroxides (e.g., oxyhydroxides) , phosphates (e.g., hydroxyphosphates, orthophosphates) , sulfates, and the like or mixtures of different mineral compounds (e.g., a mixture of a phosphate and a hydroxide adjuvant, optionally with an excess of the phosphate) , with the compounds taking any suitable form (e.g., gel, crystalline, amorphous, and the like) , and with adsorption to the salt (s) being preferred.
- the mineral containing compositions can also be formulated as a particle of metal salt (WO/0023105) .
- Aluminum salts can be included in compositions of the invention such that the dose of Al 3+ is between 0.2 and 1.0 mg per dose.
- Oil-Emulsion Adjuvants suitable for use as adjuvants in the invention can include squalene-water emulsions, such as MF59 (5%Squalene, 0.5%Tween 80, and 0.5%Span 85, formulated into submicron particles using a microfluidizer) . See, e.g., WO90/14837, Podda, Vaccine 19: 2673-2680, 2001.
- the AS01 is a liposome-based adjuvant that is consist of monophosphoryl lipid A (MPL) and a saponin molecule (QS-21)
- AS03 is an ⁇ -tocopherol and squalene-based adjuvant that has been used in GSK's A/H1N1 pandemic flu vaccine
- MPL monophosphoryl lipid A
- QS-21 saponin molecule
- AS03 adjuvant system 03
- the MPL is extracted from Salmonella minnesota and QS-21 is purified from the bark of the South American tree Quillaja saponaria Molina.
- the MPL signals through Toll-like receptor-4 (TLR4) , which results in the activation of APCs and the production of cytokines and interferons (IFNs) .
- TLR4 Toll-like receptor-4
- submicron oil-in-water emulsions for use herein include squalene/water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 4-5%w/v squalene, 0.25-1.0%w/v Tween 80 (polyoxyelthylenesorbitan monooleate) , and/or 0.25-1.0%Span 85 (sorbitan trioleate) , and, optionally, N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2- (1'-2'-dipalmitoyl-s--n-glycero-3-huydroxyphosphophoryloxy) -ethylamine (MTP-PE) , for example, the submicron oil-in-water emulsion known as "MF59" (International Publication No.
- MF59 can contain 4-5%w/v Squalene (e.g., 4.3%) , 0.25-0.5%w/v Tween 80, and 0.5%w/v Span 85 and optionally contains various amounts of MTP-PE, formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass. ) .
- MTP-PE can be present in an amount of about 0-500 ⁇ g/dose, or 0-250 ⁇ g/dose, or 0-100 ⁇ g/dose.
- Submicron oil-in-water emulsions methods of making the same and immunostimulating agents, such as muramyl peptides, for use in the compositions, are described in detail in International Publication No. WO90/14837 and U.S. Pat. Nos. 6,299,884 and 6,451,325.
- CFA Complete Freund's adjuvant
- IFA incomplete Freund's adjuvant
- Saponin Adjuvant Formulations can also be used as adjuvants in the invention.
- Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla) , Gypsophilla paniculata (brides veil) , and Saponaria officianalis (soap root) .
- Saponin adjuvant formulations can include purified formulations, such as QS21, as well as lipid formulations, such as Immunostimulating Complexes (ISCOMs; see below) .
- Saponin compositions have been purified using High Performance Thin Layer Chromatography (HPLC) and Reversed Phase High Performance Liquid Chromatography (RP-HPLC) . Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C.
- a method of production of QS21 is disclosed in U.S. Pat. No. 5,057,540.
- Saponin formulations can also comprise a sterol, such as cholesterol (see WO96/33739) .
- ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine.
- Any known saponin can be used in ISCOMs.
- an ISCOM can include one or more of Quil A, QHA and QHC.
- ISCOMs are described in EP0109942, WO96/11711, and WO96/33739.
- the ISCOMS can be devoid of additional detergent. See WO00/07621.
- a description of the development of saponin based adjuvants can be found at Barr, et al., Advanced Drug Delivery Reviews 32: 247-27, 1998. See also Sjolander, et al., Advanced Drug Delivery Reviews 32: 321-338, 1998.
- Virosomes and Virus-Like Particles can also be used as adjuvants.
- These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome.
- the viral proteins can be recombinantly produced or isolated from whole viruses.
- viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA) , Hepatitis B virus (such as core or capsid proteins) , Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, QB-phage (such as coat proteins) , GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pl) .
- influenza virus such as HA or NA
- Hepatitis B virus such as core or capsid proteins
- Hepatitis E virus measles virus
- Sindbis virus Rotavirus
- Foot-and-Mouth Disease virus Retrovirus
- Norwalk virus Norwalk virus
- human Papilloma virus HIV
- RNA-phages such as coat proteins
- GA-phage f-phage
- Bacterial or Microbial Derivatives useful as adjuvants include: (i) Non-Toxic Derivatives of Enterobacterial Lipopolysaccharide (LPS) ; (ii) lipid derivatives, (iii) immunostimulatory oligonucleotides and ADP-Ribosylating Toxins and Detoxified Derivatives Thereof, (iv) ADP-Ribosylating Toxins and Detoxified Derivatives Thereof. Examples of Non-Toxic Derivatives of LPS Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3 dMPL) .
- MPL Monophosphoryl lipid A
- 3 dMPL 3-O-deacylated MPL
- 3 dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.
- An example of a "small particle” form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such "small particles" of 3 dMPL are small enough to be sterile filtered through a 0.22 micron membrane (see EP 0 689 454) .
- Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g., RC-529 (Johnson et al., Bioorg Med Chem Lett, 9: 2273-2278, 1999) .
- Examples of lipid A derivatives can include derivatives of lipid A from Escherichia coli such as OM-174.
- OM-174 is described for example in Meraldi et al., Vaccine 21: 2485-2491, 2003; and Pajak, et al., Vaccine 21: 836-842, 2003.
- immunostimulatory oligonucleotides nucleotide sequences containing a CpG motif (a sequence containing an unmethylated cytosine followed by guanosine and linked by a phosphate bond) , for example, CpG ODN (oligodeoxynucleotide) .
- RNA or oligonucleotides containing palindromic or poly (dG) sequences have also been shown to be immunostimulatory.
- the CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded.
- the guanosine can be replaced with an analog such as 2'-deoxy-7-deazaguanosine. See Kandimalla, et al., Nucleic Acids Research 31: 2393-2400, 2003; WO02/26757 and WO99/62923 for examples of analog substitutions.
- the adjuvant effect of CpG oligonucleotides is further discussed in Krieg, Nature Medicine (2003) 9 (7) : 831-835; McCluskie, et al., FEMS Immunology and Medical Microbiology (2002) 32: 179-185; WO98/40100; U.S. Pat. No. 6,207,646; U.S. Pat. No. 6,239,116 and U.S. Pat. No. 6,429,199.
- the CpG sequence can be directed to Toll-like receptor (TLR9) , such as the motif GTCGTT or TTCGTT. See Kandimalla, et al., Biochemical Society Transactions (2003) 31 (part 3) : 654- 658.
- TLR9 Toll-like receptor
- the CpG sequence can be specific for inducing a Th1 immune response, such as a CpG-A ODN, or it can be more specific for inducing a B cell response, such a CpG-B ODN.
- CpG-A and CpG-B ODNs are discussed in Blackwell, et al., J. Immunol. 170: 4061-4068, 2003; Krieg, TRENDS in Immunology 23: 64-65, 2002, and WO01/95935.
- the CpG oligonucleotide can be constructed so that the 5' end is accessible for receptor recognition.
- two CpG oligonucleotide sequences can be attached at their 3' ends to form "immunomers" .
- Bacterial ADP-ribosylating toxins and detoxified derivatives thereof can be used as adjuvants in the invention.
- the toxin can be derived from E. coli (i.e., E. coli heat labile enterotoxin (LT) ) , cholera (CT) , or pertussis (PTX) .
- the adjuvant can be a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G.
- ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in the following references, each of which is specifically incorporated by reference herein in their entirety: Beignon, et al., Infection and Immunity 70: 3012-3019, 2002; Pizza, et al., Vaccine 19: 2534-2541, 2001; Pizza, et al., Int. J. Med.
- Bioadhesives and mucoadhesives can also be used as adjuvants in the invention.
- Suitable bioadhesives can include esterified hyaluronic acid microspheres (Singh et al., J. Cont. Rel. 70: 267-276, 2001) or mucoadhesives such as cross-linked derivatives of poly (acrylic acid) , polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof can also be used as adjuvants in the invention disclosed for example in WO99/27960.
- Microparticles can also be used as adjuvants.
- Microparticles i.e., a particle of about 100 nm to about 150 ⁇ m in diameter, or 200 nm to about 30 ⁇ m in diameter, or about 500 nm to about 10 ⁇ m in diameter
- materials that are biodegradable and/or non-toxic e.g., a poly (alpha-hydroxy acid) , a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, and the like
- a negatively-charged surface e.g., with SDS
- a positively-charged surface e.g., with a cationic detergent, such as CTAB
- liposome formulations suitable for use as adjuvants are described in U.S. Pat. No. 6,090,406, U.S. Pat. No. 5,916,588, and EP 0 626 169.
- PCPP formulations for use as adjuvants are described, for example, in Andrianov et al., Biomaterials 19: 109-115, 1998.1998.
- muramyl peptides suitable for use as adjuvants in the invention can include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) , N-acetyl-normuramyl-1- alanyl-d-isoglutamine (nor-MDP) , and N-acetylmuramyl-1-alanyl-d-isoglutaminyl-1-alanine-2- (1'-2'-dipalmitoyl-s--n-glycero-3-hydroxyphosphoryloxy) -ethylamine MTP-PE) .
- thr-MDP N-acetyl-muramyl-L-threonyl-D-isoglutamine
- imidazoquinolone compounds suitable for use as adjuvants in the invention can include Imiquimod and its homologues, described further in Stanley, “Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential” Clin Exp Dermatol 27: 571-577, 2002 and Jones, “Resiquimod 3M” , Curr Opin Investig Drugs 4: 214-218, 2003.
- Human immunomodulators suitable for use as adjuvants in the invention can include cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, and the like) , interferons (e.g., interferon-gamma) , macrophage colony stimulating factor, and tumor necrosis factor.
- cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, and the like)
- interferons e.g., interferon-gamma
- macrophage colony stimulating factor e.g., macrophage colony stimulating factor
- tumor necrosis factor e.g., tumor necrosis factor.
- adjuvant compositions can include: a saponin and an oil-in-water emulsion (WO99/11241) ; a saponin (e.g., QS21) +a non-toxic LPS derivative (e.g., 3 dMPL) (see WO94/00153) ; a saponin (e.g., QS21) +a non-toxic LPS derivative (e.g., 3 dMPL) +a cholesterol; a saponin (e.g., QS21) +3 dMPL+IL-12 (optionally+a sterol) (WO98/57659) ; combinations of 3 dMPL with, for example, QS21 and/or oil-in-water emulsions (See European patent applications 0835318, 0735898 and 0761231) ; SAF, containing 10%Squalane, 0.4%Tween 80, 5%pluronic-block
- Ribi adjuvant system Ribi Immunochem
- Ribi Immunochem containing 2%Squalene, 0.2%Tween 80
- one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL) , trehalose dimycolate (TDM) , and cell wall skeleton (CWS) , preferably MPL+CWS (Detox)
- MPL+CWS trehalose dimycolate
- CWS cell wall skeleton
- mineral salts such as an aluminum salt
- a non-toxic derivative of LPS such as 3 dPML
- Aluminum salts and MF59 are examples of adjuvants for use with injectable viral vaccines.
- Bacterial toxins and bioadhesives are examples of adjuvants for use with mucosally-delivered vaccines, such as nasal vaccines. All adjuvants noted above and others as generally known in the art to one of ordinary skill can be formulated for intranasal administration using techniques well known in the art.
- compositions of the invention can be formulated in pharmaceutical compositions.
- These compositions can comprise, in addition to the Ca-DelMut-LASV, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. Such materials should typically be non-toxic and should not typically interfere with the efficacy of the active ingredient.
- the precise nature of the carrier or other material can depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes.
- compositions for oral administration can be in tablet, capsule, powder or liquid form.
- a tablet can include a solid carrier such as gelatin or an adjuvant.
- Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, dextrose, or other saccharide solution or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol can be included.
- carrier refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition (e.g., immunogenic or vaccine formulation) is administered.
- An excipient is a pharmacologically inert substance by itself, but when used in combination with an active ingredient it can provide several benefits.
- Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
- Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. The formulation should be selected according to the mode of administration.
- the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity, and stability.
- a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity, and stability.
- isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection.
- Preservatives, stabilizers, buffers, antioxidants, and/or other additives can be included, as required.
- Examples include, but are not limited to 2-Phenoxyethanol albumin (bovine, bovine serim, cal serum, human) , ovalbumin, ascorbic acid, monosodium glutamate, sucrose, D-mannose, D-fructose, dextrose, potassium phosphate, plasdone C, anhydrous lactose, microcrystalline cellulose, polacrilin potassium, magnesium stearate, cellulose acetate phthalate, alcohol, acetone, castor oil, sugar, gelatin, formaldehyde etc.
- Administration is preferably in a “therapeutically effective amount” or “prophylactically effective amount” (as the case can be, although prophylaxis can be considered therapy) , this being sufficient to show benefit to the individual.
- the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of disease being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. ( “Remington's” ) .
- Ca-DelMut-LASV is obtained by passaging the Del-Mut-1 virus under conditions that result in the introduction of the desired mutations in Del-Mut-1, discussed above.
- DelMut-1 can be obtained following methods known in the art. 14 .
- TheDelmut-1 virus includes a 10 aa deletion in the spike protein when compared to the spike protein (SEQ ID NO: NO: 2) of Hu-1 Sars-CoV-2 (Fig. 1B) .
- the Delmut-1 virus is serially passaged in a suitable cell line used for viral passaging, and for a sufficient amount of time and culture conditions, to obtain passage adapted mutations in Delmut-1.
- a preferred cell type is Vero E6 cells, where the cells are initially passaged at a temperature not exceeding 36 °C, more preferably, not exceeding 35 °C, more preferably, not exceeding 34 °C, and even more preferably, not exceeding 33 °C.
- the virus is passaged under these conditions for at least 5-15 times, preferably, at least 10 times with each passage being preferably between 2-3 days.
- the culture conditions are changed to a temperature greater than 27 °C and not exceeding 33°C, preferably, exceeding 32 °C, not exceeding 31 °C, and more preferably, not exceeding 30°C.
- the virus is passaged at this second temperature for at least 3-13 times, preferably, at least 8 times.
- the virus After passage at the disclosed temperatures, the virus is amplified and titered.
- the virus is preferably passaged until virus titer is stabilized, with virus titer maintained without meaningful change for three consecutive passages.
- without meaningful change refers to changes including no change or no statistically significant change
- the virus is serially passaged in Vero E6 cells 10 times at 33 °C and then passaged at 30 °C for 8 times. For each passage, incubation time is 2-3 days depending on the occurrence of cytopathic effects. After the 18 th passage, the virus is amplified and titered by plaque assay.
- cells used for the cultivation of viruses using a cultivation medium can be cells that can grow in vitro in synthetic media and can be used for the propagation of viruses.
- These can be for example BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, MDBK cells, , MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NS0, PerC6 (human retina cells) , chicken embryo cells or derivatives, embryonated egg cells, embryonated chicken eggs or derivatives thereof.
- the cells can be used to substitute the Vero cells exemplified herein.
- the cells are used to passaged the Del-Mut-1 virus in for an effective amount of time to obtain a stable viral titer.
- the rescued virus is passaged in a virus-producing cell cells for a period of time until viral titre remains unchanged for 3 consecutive passaged.
- the cultivation medium used for the production of viruses can be any medium known from prior art that is applicable for virus cultivation.
- the medium is a synthetic medium.
- This can be for example basal media as Modified Eagle's media MEM, minimum essential media MEM, Dulbecco's modified Eagle's media D-MEM, D-MEM-F12 media, William's E media, RPMI media and analogues and derivative thereof.
- basal media as Modified Eagle's media MEM, minimum essential media MEM, Dulbecco's modified Eagle's media D-MEM, D-MEM-F12 media, William's E media, RPMI media and analogues and derivative thereof.
- These can also be specialty cell cultivation and virus growth media as VP-SFM, OptiPro TM SFM, AIM media, HyQ SFM4 MegaVir TM , EX-CELL TM Vero SFM, EPISERF, ProVero, any 293 or CHO media and analogues and derivatives thereof.
- media can be supplemented by any additive known from prior art that is applicable for cell and virus cultivation as for example animal sera and fractions or analogues thereof, amino acids, growth factors, hormones, buffers, trace elements, trypsin, sodium pyruvate, vitamins, L-glutamine and biological buffers.
- Preferable medium is OptiPRO TM SFM supplemented with L-glutamine and trypsin.
- the disclosed Ca-DelMut-LASV can be used to effectively increase viral titer or elicit an immune response in a subject in need thereof.
- subjects can include the elderly (e.g., >65 years old) , young children (e.g., ⁇ 5 years old) .
- Methods for improving immune response in children using adjuvanted formulations are disclosed for example in U.S. Publication 2017/0202955.
- the Ca-DelMut-LASV can generally be administered directly to a mammal in need thereof to increase viral titer in the mammal and elicit an immune response.
- the subject is a young child, less than 5 years of age. In other embodiments, the subject is a young child, less than two years of age.
- the composition is administered intranasally. In other embodiments the subject is elderly, and the subject can be between the ages of 5 and 65.
- Direct delivery can be accomplished by parenteral injection (e.g., subcutaneously, intraperitoneally, intradermal, intravenously, intramuscularly, or to the interstitial space of a tissue) , or mucosally, such as by rectal, oral (e.g., tablet, spray) , vaginal, topical, transdermal (See e.g., WO99/27961) or transcutaneous (See e.g., WO02/074244 and WO02/064162) , inhalation, intranasal (See e.g., WO03/028760) , ocular, aural, pulmonary or other mucosal administration.
- parenteral injection e.g., subcutaneously, intraperitoneally, intradermal, intravenously, intramuscularly, or to the interstitial space of a tissue
- mucosally such as by rectal, oral (e.g., tablet, spray) , vaginal, topical, trans
- compositions can also be administered topically by direct transfer to the surface of the skin. Topical administration can be accomplished without utilizing any devices, or by contacting naked skin with the composition utilizing a bandage or a bandage-like device (see, e.g., U.S. Pat. No. 6,348,450) .
- the mode of administration is parenteral, mucosal, or a combination of mucosal and parenteral immunizations.
- the mode of administration is parenteral, mucosal, or a combination of mucosal and parenteral immunizations in a total of 1-2 vaccinations 1-3 weeks apart.
- the route of administration includes but is not limited to intranasal delivery.
- composition is administered in an effective amount to induce an immune response against a one or more Sars-CoV-2 antigens encoded by the Ca-DelMut-LASV.
- an effective amount of virus generally results in production of antibody and/or activated T cells that kill or limit proliferation of or infection by the Sars-CoV-2.
- the composition can typically be used to elicit systemic and/or mucosal immunity, for example to elicit an enhanced systemic and/or mucosal immunity.
- the immune response can be characterized by the induction of a serum IgG and/or intestinal IgA immune response.
- the level of protection against Sars-CoV-2 infection can be more than 50%, e.g., 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In one aspect, the level of protection can be 100%.
- the immune response induced by the invention can be one or both of a TH1 immune response and a TH2 response.
- the immune response can be an improved or an enhanced or an altered immune response.
- the immune response can be one or both of a systemic and a mucosal immune response.
- the immune response can be an enhanced systemic and/or mucosal response.
- An enhanced systemic and/or mucosal immunity is reflected in an enhanced TH1 and/or TH2 immune response.
- the enhanced immune response can include an increase in the production of IgG1 and/or IgG2a and/or IgA.
- the mucosal immune response can be a TH2 immune response.
- the mucosal immune response can include an increase in the production of IgA.
- activated TH2 cells enhance antibody production and are therefore of value in responding to extracellular infections.
- Activated TH2 cells can typically secrete one or more of IL-4, IL-5, IL-6, and IL-10.
- a TH2 immune response can also result in the production of IgG1, IgE, IgA, and/or memory B cells for future protection.
- a TH2 immune response can include one or more of an increase in one or more of the cytokines associated with a TH2 immune response (such as IL-4, IL-5, IL-6 and IL-10) , or an increase in the production of IgG1, IgE, IgA and memory B cells.
- an enhanced TH2 immune response can include an increase in IgG1 production.
- a TH1 immune response can include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with a TH1 immune response (such as IL-2, IFN-gamma, and TNF-alpha) , an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a.
- the enhanced TH1 immune response can include an increase in IgG2a production.
- the Ca-DelMut-LASV can be used either alone or in combination with other agents optionally with an immunoregulatory agent capable of eliciting a Th1 and/or Th2 response.
- the precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc. ) , and age of the subject being treated. Appropriate dosages can be determined by a person skilled in the art, considering the therapeutic context, age, and general health of the recipient. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired.
- the physician may evaluate circulating plasma levels of virus, and/or the production of existing antibodies against the antigen (s) . Active virus can also be measured in terms of plaque-forming units (PFU) .
- PFU plaque-forming units
- a plaque-forming unit can be defined as areas of cell lysis (CPE) in monolayer cell culture, under overlay conditions, initiated by infection with a single virus particle.
- CPE cell lysis
- dosage levels of virus between 10 2 and 10 12 pfu are administered to humans.
- the dosage range is from 10 4 to 10 10 pfu, 10 5 to 10 9 pfu, 10 6 to 10 8 pfu, or any dose within these stated ranges.
- the amount of each vaccine agent can be within their described ranges.
- Virus is typically administered in a liquid suspension, in a volume ranging between 10 ⁇ l and 100 ⁇ l depending on the route of administration. Vaccine volumes commonly practiced range from 0.1 mL to 0.5 mL. Generally, dosage and volume will be lower for local injection as compared to systemic administration or infusion.
- the vaccine composition can be administered in a single dose or a multi-dose format.
- Vaccines can be prepared with adjuvant hours or days prior to administrations, subject to identification of stabilizing buffer (s) and suitable adjuvant composition.
- the dose will be 100 ⁇ l administered locally in multiple doses, while systemic or regional administration via subcutaneous, intramuscular, intra-organ, intravenous or intranasal administration can be from for example, 10 to 100 ⁇ l.
- Kits including the disclosed Ca-DelMut-LASV are also provided.
- the kit can include a separate container containing a suitable carrier, diluent or excipient. Additionally, the kit can include instructions for mixing or combining ingredients and/or administration.
- compositions can be in liquid form or can be lyophilized.
- suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes.
- Containers can be formed from a variety of materials, including glass or plastic.
- a container can have a sterile access port (for example, the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) .
- the kit can further include a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device (s) .
- a pharmaceutically-acceptable buffer such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device (s) .
- the kit can further include a third component comprising an adjuvant.
- the kit can also include a package insert containing written instructions for methods of inducing immunity, preventing infections, or for treating infections.
- the package insert can be an unapproved draft package insert or can be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body.
- FDA Food and Drug Administration
- the invention also provides a delivery device pre-filled with the compositions of the invention.
- compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
- GMP Good Manufacturing Practice
- compositions, and methods can be further understood through the following numbered paragraphs and examples.
- a live attenuated SAR-CoV-2 virus comprising genome encoded by SEQ ID NO: 3, comprising a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
- the live attenuated virus of paragraph 1 or 2 comprising proteins with the following mutations relative to Hu-1 SARS-CoV-2; an A578V mutation in the NSP3 protein; P25L mutation in the spike protein; a V367F in the spike protein; an F20S in the E protein and a V62L mutation in ORF8.
- composition comprising the live attenuated virus of any one of paragraphs 1-3 or a variant thereof.
- composition of paragraph 6, wherein the variant comprises a genome encoded by a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 3, with a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
- a pharmaceutical composition comprising an effective amount of the attenuated virus of anyone of paragraph s 1-4.
- composition of anyone of paragraphs 5-7 further comprising an adjuvant.
- composition of any one of paragraphs 5-8 in a form suitable for nasal administration.
- composition of paragraph 9 wherein the adjuvant is selected from the group consisting of Montanide ISA-51 and CpG ODN (oligodeoxynucleotide) , AS01 (adjuvant system 01) , aluminum hydroxide or a TLR (Toll-like receptor) agonist.
- the adjuvant is selected from the group consisting of Montanide ISA-51 and CpG ODN (oligodeoxynucleotide) , AS01 (adjuvant system 01) , aluminum hydroxide or a TLR (Toll-like receptor) agonist.
- a method for increasing an immune response to Sars-CoV-2 in a subject in need thereof comprising administering the composition of any one of paragraphs 1-11, to the subject.
- composition is administered subcutaneously, intradermal, intravenously, or intramuscularly.
- DelMut virus were prepared as previously described 14 .
- the Delmut-1 virus was serially passaged in Vero E6 cells for 10 times at 33 °C and then passaged at 30 °C for 8 times. For each passage, incubation time is 2-3 days depending on the occurrence of cytopathic effects.
- the virus was amplified in a T75 Flask and titered by plaque assay. Also, the virus was sequenced by Sanger method.
- Vero E6 Confluent Vero E6, Calu-3 cells were infected at 0.01 moi of the indicated viruses for 3 days. Virus supernatant was collected at the indicated time points. Virus titer was determined by plaque assay using Vero E6 cells.
- Confluent Vero E6 wells were incubated with 10 fold serially diluted virus for 1 h. After adsorption, virus was discarded. Cells were washed and overlaid with 1%agarose DMEM and incubated for 3 days at 37 °C. Cells were fixed with 10%formaldehyde for 1 day. Agarose gels were removed and plaques were stained with 1%crystal violet and counted.
- hamsters were immunized either 10 3 pfu of Ca-DelMut or WT HK-13 virus. All experiments involving SARS-CoV-2 were conducted in a biosafety level 3 laboratory. All animal studies were approved by the Committee on the Use of Live Animals in Teaching and Research, Hong Kong University.
- Hamster Anti-spike RBD IgG detection kit (Wantai-Bio, China) was used to detect RBD specific antibodies. Procedures were done according to the manual. Briefly, heat inactivated sera were 10-fold serially diluted and added to the plate and incubated at 37 °C for 30 mins. Sera from and WT challenged hamsters were done as controls. The plate was washed for 5 times and then incubated with secondary antibody reagent at 37 °C for 30 mins. After washing, color development solution was added and incubated at 37 °C for 15 mins. Stop solution was added and absorbance at 450 nm was measured.
- RNAzol RT reagent MRC
- cDNA was synthesized using High capacity cDNA reverse transcription kit (Invitrogen) and oligo dT primers following the protocol.
- qPCR was done using SYBR Premix Ex Taq (Takara) reagent and gene specific primers in LC480 PCR machine (Roche) .
- PCR condition was as follows: Initial denaturation: 95 °C for 5 min, 45 cycles of amplification: 95 °C for 10s, 60 °C for 10s, 72 °C for 10s, and melting curve analysis, 65 °C to 97°C at 0.1°C/s.
- Expression of the target genes was normalized to the internal reference gene (hamster ⁇ -actin) and comparative Ct (2- ⁇ Ct) method was utilized to calculate the cytokine expression profile.
- Ca-DelMut variant of SARS-CoV-2 groups of hamsters were infected with Ca-DelMut, Del-Mut-1 or wild type HK13 strain. While inoculation with 10 3 pfu HK13 caused significant body weight loss post infection, no apparent body weight loss was observed in Ca-DelMut (1.25 x 10 5 and 10 3 pfu) infected hamsters ( Figure 2B) .
- Ca-DelMut As a live attenuated virus vaccine to prevent SARS-CoV-2 virus infection and disease.
- hamsters were re-challenged with wild type SARS-CoV-2 viruses.
- HK-95 contains a D614G substitution in the spike protein, which has been suggested to render SARS-CoV-2 with higher infectivity in humans 23 .
- SARS-CoV-2 contains a PRRA polybasic motif not seen in most the closely related bat and pangolin coronavirus which are currently known 3, 5, 8 .
- the presence of such polybasic cleavage site at the S1/S2 junction of the spike of SARS-CoV-2 virus is considered as a critical property for enhanced coronavirus virus infectivity in humans and zoonotic potential 14, 26 .
- a peptide assay showed that the polybasic cleavage site harbored in SARS-CoV-2 would make the cleavage site more accessible to proteases which are known to activate coronavirus spike protein 10 .
- SARS-CoV and SARS-CoV-2 utilize ACE2 to mediate infection
- the distinctive infectivity and pathogenicity displayed by SARS-CoV-2 are likely to associate with acquisition of polybasic furin cleavage site in the protein which, together with enhanced binding affinity for ACE2 by the RBD of SARS-CoV-2, would significantly broaden the tissue tropism of infection.
- Deregulated innate immunity during the early stage of infection in the upper respiratory may determine the subsequent outcome of dissemination to the lower respiratory tract and the disease severity 17, 28 .
- Ca-DelMut was found to replicate at levels comparable to wild type virus in the nasal turbinate but less effectively than wild type virus, in the lung (Figure 2C) .
- RNAaemia RNAaemia
- IL-6 interleukin 6
- Luo, M., et al. IL-6 and CD8+ T cell counts combined are an early predictor of in-hospital mortality of patients with COVID-19. JCI Insight 5 (2020) .
Abstract
Provided are a live attenuated SAR-CoV-2 virus (Ca-DelMut-LASV), compositions comprising the live attenuated virus and methods for increasing an immune response to Sars-CoV-2. Ca-DelMut-LASV is characterized by a deletion of the amino acid sequence NSPRRARSVA (SEQ ID NO: 1), which represents a deletion of amino acids 665-705 of the wild type SARS-CoV-2 spike. Ca-DelMut-LASV additionally includes the following mutations relative to wild type SARS-CoV-2: an A578V mutation in the NSP3 protein; a P25L mutation in the spike protein; a V367F in the spike protein; an F20S in the E protein and a V62L mutation in ORF8.
Description
The present invention is generally in the field of live attenuated viruses containing one or more antigens from Sars-CoV-2, immunogenic compositions including attenuated Sars-CoV-2 antigen, and methods of using such compositions.
Emergence of SARS-CoV-2, a novel zoonotic origin β-coronavirus, has led to the first documented pandemic caused by a coronavirus
1, 2. The virus continues to circulate in humans with rapidly increase of infections and casualties globally each day (https: //coronavirus. jhu. edu/map. html) . While coronaviruses from bat and pangolin have been found to be closely related to SARS-CoV-2
3-6, the real ancestral virus which caused cross species transmission or the intermediate host which mediate infection in humans has not been defined. In the past two decades, three coronavirus jump species barrier and infect humans
7. SARS-CoV and MERS-CoV show limit human to human transmission ability while cause severe disease and mortality. In contrast, SARS-CoV-2, which uses human ACE2 as binding receptor as SARS-CoV6, is highly transmissible and causes variable severity of diseases from asymptomatic to severe and fatal outcome.
Several SARS-CoV-2 vaccines are being developed for use in humans
30-
33. However, there are concerns if current vaccine strategies will be able to provide sufficient and long last immunity to prevent infection and alleviate diseases. Studies have also showing that anti-SARS-CoV-2 antibodies decline rapidly in naturally infected individuals
34, 35.
There remains a need for compositions that can be used to elicit antibody responses against a SARS-CoV, preferably to levels that provide complete immunity.
It is an object of the present invention to provide compositions for eliciting an immune response to SARS-CoV.
It is also an object of the present invention to provide a methods of eliciting an immune response against SARS-CoV in a subject in need thereof.
SUMMARY OF THE INVENTION
Compositions continuing live attenuated Sars-CoV-2, Compositions immunogenic against Sars-CoV-2, methods of making and using, are provided. The compositions are based SARS-CoV-2 variant, Del-Mut-1 which contains a 30bp deletion at the S/S2 junction (with the PRRA polybasic cleavage motif removed) , passaged in Vero-E6 cells to obtain a variant with additional mutations in the multiple genes in the background of Del-Mut-1. This Del-Mut-1 variant is referred to herein as Ca-DelMut live attenuated SARS-CoV-2 virus (LASV) , hereinafter, Ca-DelMut-LASV (GenBank accession no: MT862537) . Ca-DelMut-LASV includes a mutation, which results in a deletion of ten amino acid sequence NSPRRARSVA (SEQ ID NO: 1) , which represents a deletion of amino acids 679-688 from the wild type SARS-CoV-2 spike protein from SARS-CoV-2 strain (NCBI Reference Sequence: YP_009724390.1). Hereafter denote as "Hu-1 SARS-CoV-2" or "Hu-1" DelMut-LASV additionally includes mutations which result in expressed proteins with the following mutations relative to "Hu-1 SARS-CoV-2"; an A578V mutation in the NSP3 protein; P25L mutation in the spike protein; a V367F in the spike protein; an F20S in the E protein and a V62L mutation in ORF8. Ca-DelMut has the genome encoded by SEQ ID NO: 3, with a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144. Variants of Ca-DelMut as also provided. Ca-DelMut comprise a genome encoded by a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 3, with a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
Also disclosed is a method of making Ca-DelMut-LASV. The method includes passaging the Del-Mut-1 virus under conditions that result in the introduction of the desired mutations in Del-Mut-1 i.e., the following mutations relative to - "Hu-1 SARS-CoV-2"; an A578V mutation in the NSP3 protein; P25L mutation in the spike protein; a V367F in the spike protein; an F20S in the E protein and a V62L mutation in ORF8. Del-Mut-1 is passaged at a first temperature, preferably not exceeding 33 ℃ and for at least 5 passages and subsequently passaged at a temperature, preferably not exceeding 30 ℃ for at least 3 passages. Each passage is preferably between 2-3 days.
Pharmaceutical compositions which are immunogenic are also provided. The pharmaceutical compositions include the disclosed Ca-DelMut-LASV or a variant thereof, preferably produced according to the disclosed methods. The pharmaceutical compositions typically include an effective amount of Ca-DelMut-LASV to induce an immune response in subject in need thereof when administered to the subject. The pharmaceutical compositions can include additional agents, for example adjuvants to enhance the immune response. In some embodiments, the pharmaceutical compositions do not include an adjuvant. In one embodiment, the composition includes an effective mount of the Ca-DelMut-LASV.
Methods of treating a subject in need thereof by administering the pharmaceutical composition to the subject are also provided. The methods can be a vaccine protocol. Thus, in some embodiments, the subject is administered the composition to provide prophylactic or therapeutic protection against Sars-CoV-2. Pharmaceutical compositions of Ca-DelMut-LASV or a variant thereof, are administered to a subject in need thereof by subcutaneous (s.c. ) , intradermal (i.d. ) , intramuscular (i.m. ) , intravenous (i.v. ) , oral, or intranasal administration; or by injection or by inhalation. In other aspects, the strain is administered intranasally. The compositions containing Ca-DelMut-LASV are administrated to a mammal in need of protective immunity against a Sars-CoV-2 infection.
Figure 1A. Schematic diagram showing the deletion and mutations of Ca-DelMut in different regions of SARS-CoV-2 genome. Del-Mut-1 virus 15 was serially passaged in Vero E6 cells at 33℃ (10 passages) and 30 ℃ (8 passages) . Virus from the 18 th passage was designated Ca-DelMut live attenuated SARS-CoV-2 virus (LASV) , and amplified to prepare virus stock. Ca-DelMut was sequenced by Sanger method; mutations are shown in the diagram and Table 1) . FIG. 1B is a sequence alignment of the S1/S2 junction regions of wild type SARS-CoV-2 (Hu-1) , and 30-bp deleted mutant, Del-Mut-1 14.
Figures 2A-2C show replication efficiency of Ca-DelMut LASV is in vitro and in vivo. FIG. 2A. Vero E6 cells or Calu-3 cells were infected with Ca-DelMut and other viruses at 0.01 moi at 37 ℃. At the indicated time points, supernatants were collected, and virus titer determined by plaque assay in Vero E6 cells. FIG. 2B. Ca-DelMut infection in hamsters. Hamsters were infected intranasally with either Ca-DelMut or wild type (WT) virus at different doses as indicated. Body weight change was monitored for 5 days. FIG. 2C. Replication of Ca-DelMut in lung and nasal turbinate tissues. After virus challenge (1 x 10
3 pfu) , lung and nasal turbinate tissues of hamsters were collected at day 2 and 4, then homogenized and virus titer determined. Error bars represent mean ± s.d. (n=3) . Statistical comparisons between means were performed by Student’s t-test: ***p<0.001, **p<0.01, *p<0.05, NS: not significant. FIG. 2D. Ca-DelMut LASV exhibits cold adaptation phenotype in vitro. Vero-E6 cells were infected with Ca-DelMut and other viruses at 0.01 moi and incubated at 30 ℃. At indicated time points, supernatants were collected and virus titer was determined by plaque assay. Error bars represent mean ± s.d. (n=3) . h.p.i.: hours post infection. FIGs. 2E-2F show histological data for Hamsters infected with 1x10
3 pfu of either Ca-DelMut, HK-13 or mock infected. At day 2 and 4 post infection, lungs and small intestine were collected and fixed in 10%formalin, and then processed in paraffin blocks and H&E staining. FIG. 2E. The top panel shows normal lung structures in the lungs of mock-infected control hamsters at 4x (left) and 20x (right) magnification. At day 2 after infection with Ca-DelMut, lung tissues showed only mild regional alveolar septal infiltration and blood vessel congestion. No obvious bronchiolar epithelium desquamation or luminal debris (arrows) , and no alveolar space infiltration or exudation were observed. At day 4, no deleterious progression of histopathology was observed. For WT virus at day 2 post-infection, the low magnification image (left) showed regional lung consolidation and focal pulmonary hemorrhage (arrows) . The higher magnification image (right) showed massive alveolar space infiltration (arrowheads) and hemorrhage, with a little bronchiolar luminal cell debris (arrows) visible. At day 4 post WT-infection, the low magnification image (left) showed intensive alveolar exudation, infiltration and hemorrhage resulting in pulmonary consolidation (arrows) , while the higher magnification image (right) showed intensive protein rich exudates filling the alveolar space (arrows) , in addition to massive infiltration and alveolar hemorrhage; a blood vessel shows moderate infiltration (arrowheads) .. FIG. 2F shows representative histological images of hamster small intestines. The left-most image shows mock-infected control hamster small intestinal villi with normal structure. At day 4 post infection with Ca-DelMut, no apparent histopathological changes were detected in the small intestine (middle) . For WT virus infection, small intestinal lamina propria blood vessel congestion, infiltration and edema resulting in swelling of the villi (solid arrows) and enterocyte desquamation (open arrows) were observed.
Figures 3A-3F show proinflammatory cytokine response in Ca-DelMut and wild type virus infected hamsters. Hamsters were infected intranasally with 1x10
3 pfu of either Ca-DelMut or WT HK-13 virus. At day 2 and 4, RNA was extracted from lung tissues of infected hamsters and cDNA was synthesized using oligo dT primer. Expression of different proinflammatory cytokines (INF-γ (FIG. 3A) , CCR4 (FIG. 3B) , IL-10 (FIG. 3C) , IL-21 (FIG. 3D) , TNF-α (FIG. 3E) and IL-6 (FIG. 3F) ) was examined by qPCR, was normalized to the internal reference gene (hamster γ-actin) , and comparative Ct (2-ΔΔCt) method was utilized to calculate the cytokine expression profile. Statistical comparisons between means were performed by Student’s t-test: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns: not significant. FIGs. 3G-3L show the effect of Ca-DelMut on proinflammatory cytokine response: TGFβ1 (FIG. 3G) , IL-12p40 (FIG. 3H) , CCL17 (FIG. 3I) , CCL22 (FIG. 3J) , IL-4 (FIG. 3K) and IL-13 (FIG. 3L) in hamsters measured from RNA extracted from lung and nasal turbinate tissues using RNAsol RT procedures. Statistical comparisons between means were performed by Student’s t-test: ***p<0.001, **p<0.01, *p<0.05, ns:not significant.
Figures 4A and 4B are bar graphs showing the level of antibodies induced by Ca-DelMut immunization of hamsters (FIG. 4A) and neutralization activity against HK-13 virus strain (FIG. 4B) . Hamsters were immunized intranasally with 1.25x10
5 pfu of either Ca-DelMut LASV, Wild type virus (SZ-002) virus or mock immunized. At day 21, blood was collected from hamsters and tested for anti-S1 RBD specific IgG titer and neutralization activity against HK-13 virus strain. FIGs. 4C and 4D Lower dose (103 pfu) Ca-DelMut immunization is able to induce a strong humoral response in hamsters. Hamsters were infected intranasally with 1x103 pfu of either Ca-DelMut or HK-13 virus, or mock immunized. At day 21, blood was collected from hamsters and tested for anti-S1 RBD IgG titers (FIG. 4C) and neutralization activity against HK-13 virus (FIG. 4D) . Error bars represent mean ± s.d. (n=3) . LOD: level of detection.
Figures 5A and 5B are line and bar graphs showing the ability of Ca-DelMut immunization to protect against WT virus (HK-13 or HK 95) challenge in hamsters. Hamsters were inoculated with 1.25x105 pfu Ca-DelMut or mock immunized. At day 28 after immunization, hamsters were challenged with 1x10
3 pfu of either HK-13 or HK-95 virus. FIG. 5A. Body weight and disease symptoms were monitored for 5 days. FIG. 5B. At days 2 and 5 post infection, lungs and nasal turbinate tissues were collected for virus titration and histopathological study. Error bars represent mean ± s.d. (n=3) . FIGs. 5C and 5D are line and bar graphs showing the ability of lower dose of Ca-DelMut immunization to provide complete protection against WT HK-13 virus. measured as changes in body weight (FIG. 5C) and virus titer in lungs and nasal turbinate tissues (FIG. 5D) . Hamsters were infected intranasally with 1x103 pfu of either Ca-DelMut or HK-13 virus, or mock immunized. At day 28 after immunization, hamsters were challenged with 1x103 pfu of HK-13 virus. Body weight change and disease symptoms were monitored for 5 days. At day 5 post-infection, lungs and nasal turbinate tissues were collected for virus titration. Error bars represent mean ± s.d. (n=3) .
Figures 6A-6B show histopathological analysis of lung pathology in Ca-DelMut LASV and mock immunized hamsters after WT virus challenge. At day 28 after immunization, hamsters were challenged with 1x10
3 pfu of either HK-13 or HK-95 virus. At day 2 and 5 post infection, lung tissues were collected for histopathological study. FIG. 6A. Challenge with HK-13. Day 2: Mock-vaccinated hamster lung showed bronchiolar epithelial cell death and the bronchiolar lumen filled with exudate and cell debris (arrow) . Diffuse alveolar infiltration and focal hemorrhage were also seen (arrowheads) . Ca-DelMut434 vaccinated hamster lungs showed regional alveolar septal infiltration and blood vessel congestion (arrowhead) , but no obvious bronchiolar epithelial cell death (arrow) . Day 5: Lungs of mock-vaccinated hamsters showed severe alveolar infiltration and exudation (arrowheads) , as well as bronchiolar luminal exudation (arrow) . Vaccinated hamster lungs showed focal alveolar septal infiltration (arrowheads) , while the bronchiolar epithelium appeared normal with no luminal secretion or cell debris (arrows) . FIG. 6B. Challenge with HK-95. Day 2: Mock-vaccinated hamster lungs showed bronchiolar epithelial cell death with luminal cell debris (arrow) and diffuse alveolar infiltration with focalhemorrhage and exudation (arrowheads) , with a medium sized blood vessel showing severe endotheliitis (open arrow) . Vaccinated hamster lungs showed regional alveolar septal infiltration and blood vessel congestion (arrowhead) ; a blood vessel appeared to be normal (open arrow) , and no obvious bronchiolar epithelial cell death was observed (arrows) . Day 5: Lungs of mock-vaccination control hamsters showed severe alveolar infiltration and exudation (arrowheads) and bronchiolar luminal cell debris (arrow) . Vaccinated hamster lungs showed focal alveolar septal infiltration (arrowheads) , while the bronchiolar epithelium appeared normal without luminal secretion (arrows) . Scale bar: 100μm. FIG. 6C shows the effect of low inoculum of Ca-DelMut against reinfection of wild type SARS-CoV-2. Hamsters were infected intranasally with 1x103 pfu of either Ca-DelMut, HK-13 strain or mock immunized. At day 28 after immunization, hamsters were challenged with 1x103 pfu of HK-13 virus. At day 5 post infection, lungs were collected for histopathological study. Lungs were fixed in 10%formalin, and then processed in paraffin blocks and H&E staining. Mock vaccinated hamster lung showed bronchiolar epithelial cells death and luminal secretion mixed with cell debris (arrows) ; diffuse alveolar infiltration and exudation (arrowheads) ; Ca-DelMut immunized lung showed no apparent bronchiolar epithelium cell death (arrows) , alveoli showed regional septal infiltrationIn HK-13 strain immunized hamster lung showed no apparent histopathology other than alveolar wall thickening (arrowheads) after re-challenge.
Several SARS-CoV-2 vaccines are being developed for use in humans
30-
33. However, there are concerns if current vaccine strategies will be able to provide sufficient and long last immunity to prevent infection and alleviate diseases. It was found that anti-SARS-CoV-2 antibodies decline rapidly in naturally infected individuals
34, 35. This study showed that Ca-DelMut induces a different innate response compared to that observed with wild type virus in animal model and provide sterilizing protective immunity to challenge of wild type virus. Because attenuated Ca-DelMut does not provoke proinflammatory cytokines which could interfere with induction of adaptive immunity, it is possible that Ca-DelMut variant may be able to more balanced immunity. Given the high replication efficiency and low pathogenic properties of Ca-DelMut, it can serve as an ideal strain for production to make an inactivated vaccine. Methods for making and using Ca-DelMut and variants thereof are disclosed herein.
I. DEFINITIONS
Materials
Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials. These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
As used herein, the term "adjuvant" refers to a compound or mixture that enhances an immune response.
As used herein, “attenuated” refers to refers to procedures that weaken an agent of disease (a pathogen) . An attenuated virus is a weakened, less vigorous virus. A vaccine against a viral disease can be made from an attenuated, less virulent strain of the virus, a virus capable of stimulating an immune response and creating immunity but not causing illness or less severe illness. Attenuation can be achieved by chemical treatment of the pathogen, through radiation, or by genetic modification, using methods known to those skilled in the art. Attenuation may result in decreased proliferation, attachment to host cells, or decreased production or strength of toxins.
As used herein, the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease state being treated or to otherwise provide a desired pharmacologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc. ) , the disease, and the age of the subject.
The term “immunogenic composition” or “composition” means that the composition can induce an immune response and is therefore antigenic. By “immune response” means any reaction by the immune system. These reactions include the alteration in the activity of an organism's immune system in response to an antigen and can involve, for example, antibody production, induction of cell-mediated immunity, complement activation, or development of immunological tolerance.
The term “nasal administration” refers to any form of administration whereby an active ingredient is propelled or otherwise introduced into the nasal passages of a subject so that it contacts the respiratory epithelium of the nasal cavity, from which it is absorbed into the systemic circulation. Nasal administration can also involve contacting the olfactory epithelium, which is located at the top of the nasal cavity between the central nasal septum and the lateral wall of each main nasal passage. The region of the nasal cavity immediately surrounding the olfactory epithelium is free of airflow. Thus, specialized methods must typically be employed to achieve significant absorption across the olfactory epithelium.
The terms “oral” , “enteral” , “enterally” , “orally” , “non-parenteral” , “non-parenterally” , and the like, refer to administration of a compound or composition to an individual by a route or mode along the alimentary canal. Examples of “oral” routes of administration of a composition include, without limitation, swallowing liquid or solid forms of a vaccine composition from the mouth, administration of a vaccine composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a vaccine composition, and rectal administration, e.g., using suppositories that release a live bacterial vaccine strain described herein.
The term “mammal” as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.
The term “topical administration” refers to the application of a pharmaceutical agent to the external surface of the skin or the mucous membranes (including the surface membranes of the nose, lungs and mouth) , such that the agent crosses the external surface of the skin or mucous membrane and enters the underlying tissues. Topical administration can result in a limited distribution of the agent to the skin and surrounding tissues or, when the agent is removed from the treatment area by the bloodstream, systemic distribution of the agent. In a preferred form, the agent is delivered by transdermal delivery, e.g., using a transdermal patch. Transdermal delivery refers to the diffusion of an agent across the skin (stratum corneum and epidermis) , which acts as a barrier few agents are able to penetrate. In contrast, the dermis is permeable to absorption of many solutes and drugs, and topical administration therefor occurs more readily through skin which is abraded or otherwise stripped of the epidermis to expose the dermis. Absorption through intact skin can be enhanced by combining the active agent with an oily vehicle (e.g., creams, emollients, penetration enhancers, and the like, as described, e.g., in Remington's Pharmaceutical Sciences, current edition, Gennaro et al., eds. ) prior to application to the skin (a process known as inunction) .
As used herein, the term “peptide” refers to a class of compounds composed of amino acids chemically bound together. In general, the amino acids are chemically bound together via amide linkages (CONH) ; however, the amino acids may be bound together by other chemical bonds known in the art. For example, the amino acids may be bound by amine linkages. Peptide as used herein includes oligomers of amino acids and small and large peptides, including polypeptides.
As used herein, a “variant, ” “mutant, ” or “mutated” polynucleotide or polypeptide contains at least one polynucleotide or polypeptide sequence alteration as compared to the polynucleotide or polypeptide sequence of the corresponding wild-type or parent polynucleotide or polypeptide. Mutations may be natural, deliberate, or accidental. Mutations include substitutions, deletions, and insertions.
II. COMPOSITIONS
Immunogenic compositions including live attenuated SARS-CoV-2, herein, Ca-DelMut-LASV are provided. Ca-DelMut-LASV is provided in a pharmaceutically acceptable, alone or in combination with an adjuvant
A. Attenuated SARS-CoV-2
SARS-CoV-2 has four main structural proteins including spike (S) glycoprotein, small envelope (E) glycoprotein, membrane (M) glycoprotein, and nucleocapsid (N) protein, several accessory proteins and several non-structural proteins (NSP) . The spike or S glycoprotein is a transmembrane protein with a molecular weight of about 150 kDa found in the outer portion of the virus. S protein forms homotrimers protruding in the viral surface and facilitates binding of envelope viruses to host cells by attraction with angiotensin-converting enzyme 2 (ACE2) expressed in lower respiratory tract cells. The nucleocapsid known as N protein is the structural component of CoV localizing in the endoplasmic reticulum-Golgi region that structurally is bound to the nucleic acid material of the virus. Another important part of this virus is the membrane or M protein, which is the most structurally structured protein and plays a role in determining the shape of the virus envelope. The last component is the envelope or E protein which is the smallest protein in the SARS-CoV structure that plays a role in the production and maturation of this virus. The virus includes several
Ca-DelMut-LASV includes a deletion which results in expression of a spike protein without the amino acid sequence NSPRRARSVA (SEQ ID NO: 1) , which represents a deletion of amino acids 665-705 of the wild type SARS- CoV-2 spike protein of the Hu-1 isolate (Wu, et al., Nature, 579, 265-269 (2020) . Mutations in DelMut-LASV additionally includes the following amino acid changes relative to Hu-1 isolate; an A578V mutation in the NSP3 protein; P25L mutation in the spike protein; a V367F in the spike protein; an F20S in the E protein and a V62L mutation in ORF8. The complete sequence for the WT SARS-CoV-2 surface glycoprotein has been assigned the NCBI Reference: YP_009724390.1.
1 mfvflvllpl vssqcvnltt rtqlPpaytn sftrgvyypd kvfrssvlhs tqdlflpffs
61 nvtwfhaihv sgtngtkrfd npvlpfndgv yfasteksni irgwifgttl dsktqslliv
121 nnatnvvikv cefqfcndpf lgvyyhknnk swmesefrvy ssannctfey vsqpflmdle
181 gkqgnfknlr efvfknidgy fkiyskhtpi nlvrdlpqgf saleplvdlp iginitrfqt
241 llalhrsylt pgdsssgwta gaaayyvgyl qprtfllkyn engtitdavd caldplsetk
301 ctlksftvek giyqtsnfrv qptesivrfp nitnlcpfge vfnatrfasv yawnrkrisn
361 cvadysVlyn sasfstfkcy gvsptklndl cftnvyadsf virgdevrqi apgqtgkiad
421 ynyklpddft gcviawnsnn ldskvggnyn ylyrlfrksn lkpferdist eiyqagstpc
481 ngvegfncyf plqsygfqpt ngvgyqpyrv vvlsfellha patvcgpkks tnlvknkcvn
541 fnfngltgtg vltesnkkfl pfqqfgrdia dttdavrdpq tleilditpc sfggvsvitp
601 gtntsnqvav lyqdvnctev pvaihadqlt ptwrvystgs nvfqtragcl igaehvnnsy
661 ecdipigagicasyqtqtNSPRRARSVAsqssiiaytmslgaensvaysnn siaiptnfti
721 svtteilpvs mtktsvdctm yicgdstecs nlllqygsfc tqlnraltgi aveqdkntqe
781 vfaqvkqiyk tppikdfggf nfsqilpdps kpskrsfied llfnkvtlad agfikqygdc
841 lgdiaardli caqkfngltv lpplltdemi aqytsallag titsgwtfga gaalqipfam
901 qmayrfngig vtqnvlyenq klianqfnsa igkiqdslss tasalgklqd vvnqnaqaln
961 tlvkqlssnf gaissvlndi lsrldkveae vqidrlitgr lqslqtyvtq qliraaeira
1021 sanlaatkms ecvlgqskrv dfcgkgyhlm sfpqsaphgv vflhvtyvpa qeknfttapa
1081 ichdgkahfp regvfvsngt hwfvtqrnfy epqiittdnt fvsgncdvvi givnntvydp
1141 lqpeldsfke eldkyfknht spdvdlgdis ginasvvniq keidrlneva knlneslidl
1201 qelgkyeqyi kwpwyiwlgf iagliaivmv timlccmtsc csclkgccsc gscckfdedd
1261 sepvlkgvkl hyt (SEQ ID NO: 2) , with the residues deleted in DelMut-LASV, shown in bold font and capital letters.
The disclosed mutations are relative Hu-1 SARS-CoV-2 strain (Wu e Wu, et al., Nature, 579: 265-269 (2020) ) . Mutations in Ca-DelMut relative to Hu-1 SARS-CoV-2 strain are shown in Table 1.
Table 1. Deletion and mutations of Ca-DelMut mutant in virus genome compared to Hu-1
*Details of deletion and mutations in the Ca-DelMut variant with reference to the Hu-1 SARS-CoV-2 strain (Wu et al., Nature 579, 265-269. 2020)
Thus, Ca-DelMut has the genome encoded by SEQ ID NO: 3, with a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
NCBI Reference Sequence: NC_045512.2, Severe acute respiratory syndrome coronavirus 2 isolate Hu-1, complete genome, provides the nucleic acid sequence:
Variants of Ca-DelMut as also provided. Ca-DelMut comprise a genome encoded by a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 3, with a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
DelMut-LASV or variants thereof can be included in a formulation for administration, in a carrier, and in some embodiments, in combination with an adjuvant. The adjuvant can serve as the carrier. In some embodiments, immunogenic compositions containing the disclosed DelMut-LASV do not include an adjuvant.
B. Adjuvant
The disclosed Ca-DelMut-LASV or variants thereof can be administered in conjunction with other immunoregulatory agents, including adjuvants. Useful adjuvants but are not limited to, one or more set forth below:
Mineral Containing Adjuvant Compositions include mineral salts, such as aluminum salts and calcium salts. Exemplary mineral salts include hydroxides (e.g., oxyhydroxides) , phosphates (e.g., hydroxyphosphates, orthophosphates) , sulfates, and the like or mixtures of different mineral compounds (e.g., a mixture of a phosphate and a hydroxide adjuvant, optionally with an excess of the phosphate) , with the compounds taking any suitable form (e.g., gel, crystalline, amorphous, and the like) , and with adsorption to the salt (s) being preferred. The mineral containing compositions can also be formulated as a particle of metal salt (WO/0023105) . Aluminum salts can be included in compositions of the invention such that the dose of Al
3+ is between 0.2 and 1.0 mg per dose.
Oil-Emulsion Adjuvants suitable for use as adjuvants in the invention can include squalene-water emulsions, such as MF59 (5%Squalene, 0.5%Tween 80, and 0.5%Span 85, formulated into submicron particles using a microfluidizer) . See, e.g., WO90/14837, Podda, Vaccine 19: 2673-2680, 2001. The AS01 (adjuvant system 01) is a liposome-based adjuvant that is consist of monophosphoryl lipid A (MPL) and a saponin molecule (QS-21) , whereas AS03 (adjuvant system 03) is an α-tocopherol and squalene-based adjuvant that has been used in GSK's A/H1N1 pandemic flu vaccine
The MPL is extracted from Salmonella minnesota and QS-21 is purified from the bark of the South American tree Quillaja saponaria Molina. The MPL signals through Toll-like receptor-4 (TLR4) , which results in the activation of APCs and the production of cytokines and interferons (IFNs) . Q-21 is reported to induce antigen-specific antibody response as well as cell-mediated immunity. Gupta, et al., Int Immunopharmacol. 2020 86: 106717. Additional adjuvants for use in the compositions are submicron oil-in-water emulsions. Examples of submicron oil-in-water emulsions for use herein include squalene/water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 4-5%w/v squalene, 0.25-1.0%w/v Tween 80 (polyoxyelthylenesorbitan monooleate) , and/or 0.25-1.0%Span 85 (sorbitan trioleate) , and, optionally, N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2- (1'-2'-dipalmitoyl-s--n-glycero-3-huydroxyphosphophoryloxy) -ethylamine (MTP-PE) , for example, the submicron oil-in-water emulsion known as "MF59" (International Publication No. WO90/14837; U.S. Pat. Nos. 6,299,884 and 6,451,325, incorporated herein by reference in their entirety. MF59 can contain 4-5%w/v Squalene (e.g., 4.3%) , 0.25-0.5%w/v Tween 80, and 0.5%w/v Span 85 and optionally contains various amounts of MTP-PE, formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass. ) . For example, MTP-PE can be present in an amount of about 0-500 μg/dose, or 0-250 μg/dose, or 0-100 μg/dose. Submicron oil-in-water emulsions, methods of making the same and immunostimulating agents, such as muramyl peptides, for use in the compositions, are described in detail in International Publication No. WO90/14837 and U.S. Pat. Nos. 6,299,884 and 6,451,325.
Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) can also be used as adjuvants in the invention.
Saponin Adjuvant Formulations can also be used as adjuvants in the invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla) , Gypsophilla paniculata (brides veil) , and Saponaria officianalis (soap root) . Saponin adjuvant formulations can include purified formulations, such as QS21, as well as lipid formulations, such as Immunostimulating Complexes (ISCOMs; see below) . Saponin compositions have been purified using High Performance Thin Layer Chromatography (HPLC) and Reversed Phase High Performance Liquid Chromatography (RP-HPLC) . Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. A method of production of QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponin formulations can also comprise a sterol, such as cholesterol (see WO96/33739) . Combinations of saponins and cholesterols can be used to form unique particles called ISCOMs. ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. For example, an ISCOM can include one or more of Quil A, QHA and QHC. ISCOMs are described in EP0109942, WO96/11711, and WO96/33739. Optionally, the ISCOMS can be devoid of additional detergent. See WO00/07621. A description of the development of saponin based adjuvants can be found at Barr, et al., Advanced Drug Delivery Reviews 32: 247-27, 1998. See also Sjolander, et al., Advanced Drug Delivery Reviews 32: 321-338, 1998.
Virosomes and Virus-Like Particles (VLPs) can also be used as adjuvants. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins can be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA) , Hepatitis B virus (such as core or capsid proteins) , Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, QB-phage (such as coat proteins) , GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pl) .
Bacterial or Microbial Derivatives useful as adjuvants include: (i) Non-Toxic Derivatives of Enterobacterial Lipopolysaccharide (LPS) ; (ii) lipid derivatives, (iii) immunostimulatory oligonucleotides and ADP-Ribosylating Toxins and Detoxified Derivatives Thereof, (iv) ADP-Ribosylating Toxins and Detoxified Derivatives Thereof. Examples of Non-Toxic Derivatives of LPS Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3 dMPL) . 3 dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. An example of a "small particle" form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such "small particles" of 3 dMPL are small enough to be sterile filtered through a 0.22 micron membrane (see EP 0 689 454) . Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g., RC-529 (Johnson et al., Bioorg Med Chem Lett, 9: 2273-2278, 1999) . Examples of lipid A derivatives can include derivatives of lipid A from Escherichia coli such as OM-174. OM-174 is described for example in Meraldi et al., Vaccine 21: 2485-2491, 2003; and Pajak, et al., Vaccine 21: 836-842, 2003. Examples of immunostimulatory oligonucleotides nucleotide sequences containing a CpG motif (a sequence containing an unmethylated cytosine followed by guanosine and linked by a phosphate bond) , for example, CpG ODN (oligodeoxynucleotide) . Bacterial double stranded RNA or oligonucleotides containing palindromic or poly (dG) sequences have also been shown to be immunostimulatory. The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. Optionally, the guanosine can be replaced with an analog such as 2'-deoxy-7-deazaguanosine. See Kandimalla, et al., Nucleic Acids Research 31: 2393-2400, 2003; WO02/26757 and WO99/62923 for examples of analog substitutions. The adjuvant effect of CpG oligonucleotides is further discussed in Krieg, Nature Medicine (2003) 9 (7) : 831-835; McCluskie, et al., FEMS Immunology and Medical Microbiology (2002) 32: 179-185; WO98/40100; U.S. Pat. No. 6,207,646; U.S. Pat. No. 6,239,116 and U.S. Pat. No. 6,429,199. The CpG sequence can be directed to Toll-like receptor (TLR9) , such as the motif GTCGTT or TTCGTT. See Kandimalla, et al., Biochemical Society Transactions (2003) 31 (part 3) : 654- 658. The CpG sequence can be specific for inducing a Th1 immune response, such as a CpG-A ODN, or it can be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in Blackwell, et al., J. Immunol. 170: 4061-4068, 2003; Krieg, TRENDS in Immunology 23: 64-65, 2002, and WO01/95935. In some aspects, the CpG oligonucleotide can be constructed so that the 5' end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences can be attached at their 3' ends to form "immunomers" . See, for example, Kandimalla, et al., BBRC 306: 948-95, 2003; Kandimalla, et al., Biochemical Society Transactions 31: 664-658, 2003; Bhagat et al., "BBRC 300: 853-861, 2003, and WO03/035836. Bacterial ADP-ribosylating toxins and detoxified derivatives thereof can be used as adjuvants in the invention. For example, the toxin can be derived from E. coli (i.e., E. coli heat labile enterotoxin (LT) ) , cholera (CT) , or pertussis (PTX) . The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO95/17211 and as parenteral adjuvants in WO98/42375. In some aspects, the adjuvant can be a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G. The use of ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in the following references, each of which is specifically incorporated by reference herein in their entirety: Beignon, et al., Infection and Immunity 70: 3012-3019, 2002; Pizza, et al., Vaccine 19: 2534-2541, 2001; Pizza, et al., Int. J. Med. Microbiol 290: 455-461, 2003; Scharton-Kersten et al., Infection and Immunity 68: 5306-5313, 2000; Ryan et al., Infection and Immunity 67: 6270-6280, 2003; Partidos et al., Immunol. Lett. 67: 09-216, 1999; Peppoloni et al., Vaccines 2: 285-293, 2003; and Pine et al., J. Control Release 85: 263-270, 2002.
Bioadhesives and mucoadhesives can also be used as adjuvants in the invention. Suitable bioadhesives can include esterified hyaluronic acid microspheres (Singh et al., J. Cont. Rel. 70: 267-276, 2001) or mucoadhesives such as cross-linked derivatives of poly (acrylic acid) , polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof can also be used as adjuvants in the invention disclosed for example in WO99/27960.
Adjuvant Microparticles: Microparticles can also be used as adjuvants. Microparticles (i.e., a particle of about 100 nm to about 150 μm in diameter, or 200 nm to about 30 μm in diameter, or about 500 nm to about 10 μm in diameter) formed from materials that are biodegradable and/or non-toxic (e.g., a poly (alpha-hydroxy acid) , a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, and the like) , with poly (lactide-co-glycolide) are envisioned, optionally treated to have a negatively-charged surface (e.g., with SDS) or a positively-charged surface (e.g., with a cationic detergent, such as CTAB) .
Examples of liposome formulations suitable for use as adjuvants are described in U.S. Pat. No. 6,090,406, U.S. Pat. No. 5,916,588, and EP 0 626 169.
Additional adjuvants include polyoxyethylene ethers and polyoxyethylene esters. WO99/52549. Such formulations can further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WO 01/21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 01/21152) . In some aspects, polyoxyethylene ethers can include: polyoxyethylene-9-lauryl ether (laureth 9) , polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, or polyoxyethylene-23-lauryl ether.
PCPP formulations for use as adjuvants are described, for example, in Andrianov et al., Biomaterials 19: 109-115, 1998.1998. Examples of muramyl peptides suitable for use as adjuvants in the invention can include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) , N-acetyl-normuramyl-1- alanyl-d-isoglutamine (nor-MDP) , and N-acetylmuramyl-1-alanyl-d-isoglutaminyl-1-alanine-2- (1'-2'-dipalmitoyl-s--n-glycero-3-hydroxyphosphoryloxy) -ethylamine MTP-PE) . Examples of imidazoquinolone compounds suitable for use as adjuvants in the invention can include Imiquimod and its homologues, described further in Stanley, "Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential" Clin Exp Dermatol 27: 571-577, 2002 and Jones, "Resiquimod 3M" , Curr Opin Investig Drugs 4: 214-218, 2003. Human immunomodulators suitable for use as adjuvants in the invention can include cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, and the like) , interferons (e.g., interferon-gamma) , macrophage colony stimulating factor, and tumor necrosis factor.
Adjuvant Combinations: The adjuvants are used in come preferred embodiments as combinations. For example, adjuvant compositions can include: a saponin and an oil-in-water emulsion (WO99/11241) ; a saponin (e.g., QS21) +a non-toxic LPS derivative (e.g., 3 dMPL) (see WO94/00153) ; a saponin (e.g., QS21) +a non-toxic LPS derivative (e.g., 3 dMPL) +a cholesterol; a saponin (e.g., QS21) +3 dMPL+IL-12 (optionally+a sterol) (WO98/57659) ; combinations of 3 dMPL with, for example, QS21 and/or oil-in-water emulsions (See European patent applications 0835318, 0735898 and 0761231) ; SAF, containing 10%Squalane, 0.4 %Tween 80, 5%pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion; and Montanide is a water-in-oil (w/o) emulsion that contains mineral oil and surfactant from mannide monooleate family.. Ribi adjuvant system (RAS) , (Ribi Immunochem) containing 2%Squalene, 0.2%Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL) , trehalose dimycolate (TDM) , and cell wall skeleton (CWS) , preferably MPL+CWS (Detox) ; and one or more mineral salts (such as an aluminum salt) +a non-toxic derivative of LPS (such as 3 dPML) .
Aluminum salts and MF59 are examples of adjuvants for use with injectable viral vaccines. Bacterial toxins and bioadhesives are examples of adjuvants for use with mucosally-delivered vaccines, such as nasal vaccines. All adjuvants noted above and others as generally known in the art to one of ordinary skill can be formulated for intranasal administration using techniques well known in the art.
C. Formulations and carriers
The composition of the invention can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to the Ca-DelMut-LASV, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. Such materials should typically be non-toxic and should not typically interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes.
Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, dextrose, or other saccharide solution or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol can be included. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition (e.g., immunogenic or vaccine formulation) is administered. An excipient, by definition, is a pharmacologically inert substance by itself, but when used in combination with an active ingredient it can provide several benefits. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. The formulation should be selected according to the mode of administration.
For intravenous, cutaneous, or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity, and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants, and/or other additives can be included, as required. Examples include, but are not limited to 2-Phenoxyethanol albumin (bovine, bovine serim, cal serum, human) , ovalbumin, ascorbic acid, monosodium glutamate, sucrose, D-mannose, D-fructose, dextrose, potassium phosphate, plasdone C, anhydrous lactose, microcrystalline cellulose, polacrilin potassium, magnesium stearate, cellulose acetate phthalate, alcohol, acetone, castor oil, sugar, gelatin, formaldehyde etc.
Administration is preferably in a “therapeutically effective amount” or “prophylactically effective amount” (as the case can be, although prophylaxis can be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of disease being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. ( “Remington's” ) .
III. METHODS OF MAKING
Ca-DelMut-LASV is obtained by passaging the Del-Mut-1 virus under conditions that result in the introduction of the desired mutations in Del-Mut-1, discussed above. DelMut-1 can be obtained following methods known in the art. 14. TheDelmut-1 virus includes a 10 aa deletion in the spike protein when compared to the spike protein (SEQ ID NO: NO: 2) of Hu-1 Sars-CoV-2 (Fig. 1B) .
Passage of Del-Mut-1 Virus
The Delmut-1 virus is serially passaged in a suitable cell line used for viral passaging, and for a sufficient amount of time and culture conditions, to obtain passage adapted mutations in Delmut-1. A preferred cell type is Vero E6 cells, where the cells are initially passaged at a temperature not exceeding 36 ℃, more preferably, not exceeding 35 ℃, more preferably, not exceeding 34 ℃, and even more preferably, not exceeding 33 ℃. The virus is passaged under these conditions for at least 5-15 times, preferably, at least 10 times with each passage being preferably between 2-3 days. Following this initial passaging, the culture conditions are changed to a temperature greater than 27 ℃ and not exceeding 33℃, preferably, exceeding 32 ℃, not exceeding 31 ℃, and more preferably, not exceeding 30℃. The virus is passaged at this second temperature for at least 3-13 times, preferably, at least 8 times.
After passage at the disclosed temperatures, the virus is amplified and titered. The virus is preferably passaged until virus titer is stabilized, with virus titer maintained without meaningful change for three consecutive passages. As used herein, without meaningful change refers to changes including no change or no statistically significant change
In a particular preferred embodiment, the virus is serially passaged in Vero E6 cells 10 times at 33 ℃ and then passaged at 30 ℃ for 8 times. For each passage, incubation time is 2-3 days depending on the occurrence of cytopathic effects. After the 18
th passage, the virus is amplified and titered by plaque assay.
Although viral passaging is exemplified using Vero cells (which are disclosed as a preferred embodiment) , cells used for the cultivation of viruses using a cultivation medium can be cells that can grow in vitro in synthetic media and can be used for the propagation of viruses. These can be for example BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, MDBK cells, , MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NS0, PerC6 (human retina cells) , chicken embryo cells or derivatives, embryonated egg cells, embryonated chicken eggs or derivatives thereof. These cells can be used to substitute the Vero cells exemplified herein. The cells are used to passaged the Del-Mut-1 virus in for an effective amount of time to obtain a stable viral titer. In preferred embodiment, the rescued virus is passaged in a virus-producing cell cells for a period of time until viral titre remains unchanged for 3 consecutive passaged.
The cultivation medium used for the production of viruses can be any medium known from prior art that is applicable for virus cultivation. Preferably the medium is a synthetic medium. This can be for example basal media as Modified Eagle's media MEM, minimum essential media MEM, Dulbecco's modified Eagle's media D-MEM, D-MEM-F12 media, William's E media, RPMI media and analogues and derivative thereof. These can also be specialty cell cultivation and virus growth media as VP-SFM, OptiPro
TM SFM, AIM
media, HyQ SFM4 MegaVir
TM, EX-CELL
TM Vero SFM, EPISERF, ProVero, any 293 or CHO media and analogues and derivatives thereof. These media can be supplemented by any additive known from prior art that is applicable for cell and virus cultivation as for example animal sera and fractions or analogues thereof, amino acids, growth factors, hormones, buffers, trace elements, trypsin, sodium pyruvate, vitamins, L-glutamine and biological buffers. Preferable medium is OptiPRO
TM SFM supplemented with L-glutamine and trypsin.
IV. METHODS OF USE
The disclosed Ca-DelMut-LASV can be used to effectively increase viral titer or elicit an immune response in a subject in need thereof. In some aspects, subjects can include the elderly (e.g., >65 years old) , young children (e.g., <5 years old) . Methods for improving immune response in children using adjuvanted formulations are disclosed for example in U.S. Publication 2017/0202955.
The Ca-DelMut-LASV can generally be administered directly to a mammal in need thereof to increase viral titer in the mammal and elicit an immune response. In some embodiments the subject is a young child, less than 5 years of age. In other embodiments, the subject is a young child, less than two years of age. In the embodiments, the composition is administered intranasally. In other embodiments the subject is elderly, and the subject can be between the ages of 5 and 65.
Viruses are typically administered to a patient in need thereof in a pharmaceutical composition. Pharmaceutical compositions containing virus may be for systemic or local administration. Dosage forms for administration by parenteral (intramuscular (IM) , intraperitoneal (IP) , intravenous (IV) or subcutaneous injection (SC) ) , or transmucosal (nasal, vaginal, pulmonary, or rectal) routes of administration can be formulated. In the most preferred embodiments, the immunizing virus is delivered peripherally by intranasally or by intramuscular injection, and the therapeutic virus is delivered by local injection.
Direct delivery can be accomplished by parenteral injection (e.g., subcutaneously, intraperitoneally, intradermal, intravenously, intramuscularly, or to the interstitial space of a tissue) , or mucosally, such as by rectal, oral (e.g., tablet, spray) , vaginal, topical, transdermal (See e.g., WO99/27961) or transcutaneous (See e.g., WO02/074244 and WO02/064162) , inhalation, intranasal (See e.g., WO03/028760) , ocular, aural, pulmonary or other mucosal administration. Compositions can also be administered topically by direct transfer to the surface of the skin. Topical administration can be accomplished without utilizing any devices, or by contacting naked skin with the composition utilizing a bandage or a bandage-like device (see, e.g., U.S. Pat. No. 6,348,450) . In some aspects, the mode of administration is parenteral, mucosal, or a combination of mucosal and parenteral immunizations. In other aspects, the mode of administration is parenteral, mucosal, or a combination of mucosal and parenteral immunizations in a total of 1-2 vaccinations 1-3 weeks apart. In related aspects, the route of administration includes but is not limited to intranasal delivery.
A. Effective amounts
Typically the composition is administered in an effective amount to induce an immune response against a one or more Sars-CoV-2 antigens encoded by the Ca-DelMut-LASV. For example, an effective amount of virus generally results in production of antibody and/or activated T cells that kill or limit proliferation of or infection by the Sars-CoV-2.
The composition can typically be used to elicit systemic and/or mucosal immunity, for example to elicit an enhanced systemic and/or mucosal immunity. For example, the immune response can be characterized by the induction of a serum IgG and/or intestinal IgA immune response. Typically, the level of protection against Sars-CoV-2 infection can be more than 50%, e.g., 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In one aspect, the level of protection can be 100%.
The immune response induced by the invention can be one or both of a TH1 immune response and a TH2 response. The immune response can be an improved or an enhanced or an altered immune response. The immune response can be one or both of a systemic and a mucosal immune response. For example, the immune response can be an enhanced systemic and/or mucosal response. An enhanced systemic and/or mucosal immunity is reflected in an enhanced TH1 and/or TH2 immune response. For example, the enhanced immune response can include an increase in the production of IgG1 and/or IgG2a and/or IgA. In another aspect the mucosal immune response can be a TH2 immune response. For example, the mucosal immune response can include an increase in the production of IgA.
Typically, activated TH2 cells enhance antibody production and are therefore of value in responding to extracellular infections. Activated TH2 cells can typically secrete one or more of IL-4, IL-5, IL-6, and IL-10. A TH2 immune response can also result in the production of IgG1, IgE, IgA, and/or memory B cells for future protection. In general, a TH2 immune response can include one or more of an increase in one or more of the cytokines associated with a TH2 immune response (such as IL-4, IL-5, IL-6 and IL-10) , or an increase in the production of IgG1, IgE, IgA and memory B cells. For example, an enhanced TH2 immune response can include an increase in IgG1 production. A TH1 immune response can include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with a TH1 immune response (such as IL-2, IFN-gamma, and TNF-alpha) , an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a. For example, the enhanced TH1 immune response can include an increase in IgG2a production.
The Ca-DelMut-LASV can be used either alone or in combination with other agents optionally with an immunoregulatory agent capable of eliciting a Th1 and/or Th2 response.
B. Dosages
The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc. ) , and age of the subject being treated. Appropriate dosages can be determined by a person skilled in the art, considering the therapeutic context, age, and general health of the recipient. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. In determining the effective amount of the virus to be administered for the prophylaxis, the physician may evaluate circulating plasma levels of virus, and/or the production of existing antibodies against the antigen (s) . Active virus can also be measured in terms of plaque-forming units (PFU) . A plaque-forming unit can be defined as areas of cell lysis (CPE) in monolayer cell culture, under overlay conditions, initiated by infection with a single virus particle. Generally, dosage levels of virus between 10
2 and 10
12 pfu are administered to humans. In different embodiments, the dosage range is from 10
4 to 10
10 pfu, 10
5 to 10
9 pfu, 10
6 to 10
8 pfu, or any dose within these stated ranges. When more than one vaccine is to be administered (i.e., in combination vaccines) , the amount of each vaccine agent can be within their described ranges.
Virus is typically administered in a liquid suspension, in a volume ranging between 10 μl and 100 μl depending on the route of administration. Vaccine volumes commonly practiced range from 0.1 mL to 0.5 mL. Generally, dosage and volume will be lower for local injection as compared to systemic administration or infusion.
The vaccine composition can be administered in a single dose or a multi-dose format. Vaccines can be prepared with adjuvant hours or days prior to administrations, subject to identification of stabilizing buffer (s) and suitable adjuvant composition. Typically, the dose will be 100 μl administered locally in multiple doses, while systemic or regional administration via subcutaneous, intramuscular, intra-organ, intravenous or intranasal administration can be from for example, 10 to 100 μl.
V. KITS
Kits including the disclosed Ca-DelMut-LASV are also provided. The kit can include a separate container containing a suitable carrier, diluent or excipient. Additionally, the kit can include instructions for mixing or combining ingredients and/or administration.
Compositions can be in liquid form or can be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container can have a sterile access port (for example, the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) .
The kit can further include a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device (s) . The kit can further include a third component comprising an adjuvant.
The kit can also include a package insert containing written instructions for methods of inducing immunity, preventing infections, or for treating infections. The package insert can be an unapproved draft package insert or can be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body.
The invention also provides a delivery device pre-filled with the compositions of the invention.
The compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
The disclosed compositions, and methods can be further understood through the following numbered paragraphs and examples.
1. A live attenuated SAR-CoV-2 virus comprising genome encoded by SEQ ID NO: 3, comprising a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
2 The attenuated virus of paragraph 1 comprising a spike protein of SEQ ID NO: 2, wherein the sequence NSPRRARSVA (SEQ ID NO: 1) , is deleted
3. The live attenuated virus of
4. The live attenuated virus of any one of paragraphs 1-3, comprising genome encoded by SEQ ID NO: 3, comprising a deletion of nucleotides 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
5. A composition comprising the live attenuated virus of any one of paragraphs 1-3 or a variant thereof.
6. The composition of paragraph 6, wherein the variant comprises a genome encoded by a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 3, with a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
7. A pharmaceutical composition comprising an effective amount of the attenuated virus of anyone of paragraph s 1-4.
8. The composition of anyone of paragraphs 5-7, further comprising an adjuvant.
9. The composition of any one of paragraphs 5-8, in a form suitable for nasal administration.
10. The composition of paragraph 9, wherein the adjuvant is selected from the group consisting of Montanide ISA-51 and CpG ODN (oligodeoxynucleotide) , AS01 (adjuvant system 01) , aluminum hydroxide or a TLR (Toll-like receptor) agonist.
11. The composition of any one of paragraphs 1-10, wherein the composition is lyophilized.
12. A method for increasing an immune response to Sars-CoV-2 in a subject in need thereof, comprising administering the composition of any one of paragraphs 1-11, to the subject.
13. The method of paragraph 12, wherein the composition is administered by parenteral injection
14. The method of paragraph 13, wherein the composition is administered subcutaneously, intradermal, intravenously, or intramuscularly.
15. The method of paragraph 14 wherein the composition is administered intranasally.
EXAMPLES
Materials and methods
Generation of cell adapted DelMut (Ca-DelMut) virus
DelMut virus were prepared as previously described
14. To generate Ca-DelMut virus, the Delmut-1 virus was serially passaged in Vero E6 cells for 10 times at 33 ℃ and then passaged at 30 ℃ for 8 times. For each passage, incubation time is 2-3 days depending on the occurrence of cytopathic effects. After the 18
th passage, the virus was amplified in a T75 Flask and titered by plaque assay. Also, the virus was sequenced by Sanger method.
Growth kinetics
Confluent Vero E6, Calu-3 cells were infected at 0.01 moi of the indicated viruses for 3 days. Virus supernatant was collected at the indicated time points. Virus titer was determined by plaque assay using Vero E6 cells.
Plaque assay
Confluent Vero E6 wells were incubated with 10 fold serially diluted virus for 1 h. After adsorption, virus was discarded. Cells were washed and overlaid with 1%agarose DMEM and incubated for 3 days at 37 ℃. Cells were fixed with 10%formaldehyde for 1 day. Agarose gels were removed and plaques were stained with 1%crystal violet and counted.
Immunization and challenge of Hamsters
7-8 weeks old golden Syrian hamsters were anesthetized intraperitoneally with ketamine and xylazine and then immunized intranasally with 1.25x10
5 pfu of Ca-Delmut virus or mock immunized with PBS. Body weight and diseases symptoms were monitored daily. At day 21, sera were collected from hamsters for anti-Spike RBD IgG and neutralizing antibody determination. At day 28, hamsters were challenged either WT viruses: HK-13 and HK-95 at a dose of 1x10
3 pfu. Body weight and diseases symptoms were monitored daily. At day 2 and day 5, lung and nasal turbinate were collected for histopathology and virus titration by plaque assay. For low dose immunization, hamsters were immunized either 10
3 pfu of Ca-DelMut or WT HK-13 virus. All experiments involving SARS-CoV-2 were conducted in a biosafety level 3 laboratory. All animal studies were approved by the Committee on the Use of Live Animals in Teaching and Research, Hong Kong University.
Pathogenicity of Ca-DelMut and WT HK-13 virus in hamster Hamsters were challenged intranasally with 1x10
3 pfu of either Ca-DelMut or WT virus. Body weight and diseases symptoms were monitored daily. At day 2 and day 4, lung and nasal turbinate were collected for histopathology study, or homogenized to determine proinflammatory cytokine expression and virus titration.
Neutralization assay
Heat inactivated sera were 2 fold serially diluted in DMEM medium and incubated with 100 pfu of the indicated virus at 37 ℃ for 1 h. The mix was added to confluent Vero E6 cells and incubated for 4 days at 37 ℃.
and WT challenged sera were used as controls. After 4 days, cytopathic effects (CPE) was detected under microscope, and neutralization endpoint is the highest dilution with 50%of inhibition of CPE.
ELISA
Hamster Anti-spike RBD IgG detection kit (Wantai-Bio, China) was used to detect RBD specific antibodies. Procedures were done according to the manual. Briefly, heat inactivated sera were 10-fold serially diluted and added to the plate and incubated at 37 ℃ for 30 mins. Sera from
and WT challenged hamsters were done as controls. The plate was washed for 5 times and then incubated with secondary antibody reagent at 37 ℃ for 30 mins. After washing, color development solution was added and incubated at 37 ℃ for 15 mins. Stop solution was added and absorbance at 450 nm was measured. Quantification of expression of proinflammatory cytokines and chemokines Quantification of expression of proinflammatory cytokines was studied by qRT-PCR similar to a previous study
36. Briefly, total RNA was extracted from hamster lungs by RNAzol RT reagent (MRC) according to the manual. cDNA was synthesized using High capacity cDNA reverse transcription kit (Invitrogen) and oligo dT primers following the protocol. qPCR was done using SYBR Premix Ex Taq (Takara) reagent and gene specific primers in LC480 PCR machine (Roche) . PCR condition was as follows: Initial denaturation: 95 ℃ for 5 min, 45 cycles of amplification: 95 ℃ for 10s, 60 ℃ for 10s, 72 ℃ for 10s, and melting curve analysis, 65 ℃ to 97℃ at 0.1℃/s. Expression of the target genes was normalized to the internal reference gene (hamster γ-actin) and comparative Ct (2-ΔΔCt) method was utilized to calculate the cytokine expression profile.
Histopathology
Organs were fixed in 10%PBS buffered formalin and processed to paraffin embedded blocks. Tissue sections were stained with haematoxylin and eosin (H&E) and examined by light microscopy according to a previous study
36.
Results
Growth and pathogenic properties of Ca-DelMut in vitro and in vivo A panel of SARS-CoV-2 variants containing 15-30bp deletion at the S1/S2 junction of spike protein have been previously identified. One of the variants, Del-Mut-1 which contains a 30bp deletion (in which the PRRA polybasic cleavage motif is removed) , was shown to be attenuated in hamster model
15. This mutant was further passaged in Vero-E6 cells, resulting in a variant with additional mutations in the multiple genes in the background of Del-Mut-1. This variant is designated herein as Ca-DelMut (Figure 1A and Table 1) .
Growth properties of Ca-DelMut, parental Del-Mut-1 and a wild type (HK13) SARS-Cov-2 were analyzed in Vero and Calu-3 cells. It is interesting to note that both Del-Mut-1 and Ca-DelMut grow to a significant higher titer at 24 and 48-hour time points than the wild type virus, with Ca-DelMut showing the strongest growth ability in both cells (Figure 2A) . Notably, Ca-DelMut exhibits a cold adaptation phenotype in vitro, as demonstrated by comparatively high level of replication at 30℃ , whereas wild type virus (HK13) replicates poorly at this temperature (Figure 2D) . To further characterize Ca-DelMut variant of SARS-CoV-2, groups of hamsters were infected with Ca-DelMut, Del-Mut-1 or wild type HK13 strain. While inoculation with 10
3pfu HK13 caused significant body weight loss post infection, no apparent body weight loss was observed in Ca-DelMut (1.25 x 10
5 and 10
3 pfu) infected hamsters (Figure 2B) . Histopathological analysis showed only mild regional alveolar septal infiltration and blood vessel congestion with no obvious bronchiolar epithelium desquamation or luminal debris, no alveolar space infiltration or exudation and no intestinal pathological changes were observed in Ca-DelMut infected hamsters, a marked difference to the pathology observed in wild type virus infected animals (FIG. 2E) . For intestinal damage, no apparent histopathological changes were observed in the small intestine following infection with the Ca-DelMut (Fig. 2F) . Small intestinal lamina propria blood vessel congestion, infiltration and edema (resulting in swelling of the villi and enterocyte desquamation (indicated by open arrows on right panel in Fig. 2F) could be seen following infection with wild type virus. Examination of virus replication in the lung and nasal turbinate tissues found Ca-DelMut replicates actively in the nasal turbinate tissues but replication efficiency is much lower compared to the HK13, in hamster lungs (Figure 2C) . These results indicate that Ca-DelMut has altered tissue tropism and is more likely to infect and replicate in the upper respiratory tract.
Immune response to infection of Ca-DelMut in hamsters
Impaired or dysfunctional immune response has been characterized as an important mechanism of pathogenesis of SARS-CoV-2 infection in humans
17-19. Research on SARS-CoV and MERS-CoV has revealed that interferon-mediated antiviral response is a double-edge sword which can produce both protective and pathogenic effect in humans. How Ca-DelMut infection induces host interferon response is of interest for understanding molecular basis of its attenuation. To test if infection with Ca-DelMut induces differential immune response compared to that of wild type SARS-CoV-2, interferon and cytokine expression in lung tissues from virus infected hamsters was examined. In contrast to infection with wild type HK13 virus, Ca-DelMut does not provoke elevated level of cytokines in infected hamsters (Figures 3A-3L) . Aberrant activation of IL6 has been recognized as an important biomarker of disease severity of SARS-CoV-2 infected patients
19-21. Remarkably, activation of IL-6 was only observed in HK-13 infected hamsters but not in those infected with Ca-DelMut. The adaptive immune response from hamsters infected with Ca-DelMut and recovered from wild type virus infection was then analyzed. Levels of Receptor binging domain (RBD) specific antibodies in sera collected three weeks after infection were determined. Sera from both hamsters infected with Ca-DelMut or that had recovered from wild type virus (HK-13)
22 infection showed robust induction of RBD specific antibodies (Figures 4A and 4C) . Cell base neutralization assay also demonstrated strong neutralizing activity against wild type virus strain HK-13 in sera collected from both Ca-DelMut variant and wild type SARS-CoV-2 virus (HK-13) infected hamsters (Figures 4B and 4D) . These results indicate that infection with Ca-DelMut leads to an altered immune response which does not induce elevated levels of proinflammatory cytokines seen with wild type SARS-CoV-2 virus infection, but nonetheless elicits a strong adaptive immune response of neutralizing antibodies.
Infection of Ca-DelMut confers full protection of SARS-CoV-2 infection with sterilizing immunity
Because infection with Ca-DelMut causes no apparent disease in hamsters while inducing a strong neutralizing antibody response, subsequent studies examined the potential of Ca-DelMut as a live attenuated virus vaccine to prevent SARS-CoV-2 virus infection and disease. Four weeks after infection with Ca-DelMut live attenuated virus, hamsters were re-challenged with wild type SARS-CoV-2 viruses. Two strains of SARS-CoV-2, HK-13 and HK-95, were used in the challenge experiment. HK-95 contains a D614G substitution in the spike protein, which has been suggested to render SARS-CoV-2 with higher infectivity in humans
23. Negligible body weight loss was observed in Ca-DelMut vaccinated hamsters infected with either strain of wild type virus, whereas control (no Ca-DelMut immunization) hamsters lost about 12-15 percent of body weight by day 5 post infection (Figure 5A and 5C-D) . Analyses of virus replication in the lung and nasal turbinate tissues from re-challenged hamsters showed that administration of Ca-DelMut provides sterilizing immunity against subsequent re-challenge with either HK13 or HK95 SARS-CoV-2 strain, with the quantity of virus (being either very low, or below the level of detection in both lung and nasal tissues on day 2 and 5 post-infection (Figure 5B and 5C-5D) . Histopathological analysis showed that mock vaccinated hamster lung collected at day 5 post infection with either wild type virus strain showed extensive alveolar exudation and infiltration; bronchiolar epithelial cell death with luminal exudation and cell debris were also observed. In contrast, in Ca-DelMut LASV inoculated hamsters, challenging with either strain of SARS-CoV-2 virus only caused mild regional alveolar septal infiltration and blood vessel congestion at day 2 and day 5 post infection. No other pulmonary histopathological changes were observed (Figure 6A and B) . In contrast, Ca-DelMut inoculated hamsters challenged with either strain of wild type SARS-CoV-2virus only experienced mild regional alveolar septal infiltration and blood vessel congestion at days 2 and5 post-infection. No other pulmonary histopathological changes were observed. Low inoculum of CA-DelMUT (1 x 103 pfu) is sufficient to provide full protection against wild type challenge (FIg. 6C) . These data indicated that prior infection with live attenuated Ca-DelMut variant provide completely protection to infection of wild type virus in hamsters.
Discussion
Coronaviruses are zoonotic pathogens with distinct ability to cause cross species transmission in humans 24. Besides 229E, OC43, HKU-1 and NL63 which have long been circulating in humans, SARS-CoV, MERS-CoV and SARS-CoV-2 have jumped species barrier to infect humans in recent years 7. SARS-CoV-2 virus utilizes same cellular receptor, ACE2, for mediating infection as SARS-CoV, but has exhibited a distinctive infectivity and transmissibility 6. There is strong interest in understanding the unique abilities that SARS-CoV-2 has acquired, that allow highly efficient infection and transmission in humans. SARS-CoV-2 contains a PRRA polybasic motif not seen in most the closely related bat and pangolin coronavirus which are currently known 3, 5, 8. The presence of such polybasic cleavage site at the S1/S2 junction of the spike of SARS-CoV-2 virus is considered as a critical property for enhanced coronavirus virus infectivity in humans and zoonotic potential 14, 26. A peptide assay showed that the polybasic cleavage site harbored in SARS-CoV-2 would make the cleavage site more accessible to proteases which are known to activate coronavirus spike protein 10. This study characterized one of the mutants of SARS-CoV-2 variants (with a deletion at the S1/S2 junction) , Ca-DelMut, and show it has low pathogenicity, it does not provoke inflammatory immune response in hamsters and it induces adaptive immunity to protect subsequent infection with more pathogenic strains. Ca-DelMut attenuated virus is a useful tool for studying SARS-CoV-2 replication, host tissue tropism and transmissibility of SARS-CovV-2.
Although both SARS-CoV and SARS-CoV-2 utilize ACE2 to mediate infection, the distinctive infectivity and pathogenicity displayed by SARS-CoV-2 are likely to associate with acquisition of polybasic furin cleavage site in the protein which, together with enhanced binding affinity for ACE2 by the RBD of SARS-CoV-2, would significantly broaden the tissue tropism of infection. Deregulated innate immunity during the early stage of infection in the upper respiratory may determine the subsequent outcome of dissemination to the lower respiratory tract and the disease severity
17, 28. Ca-DelMut was found to replicate at levels comparable to wild type virus in the nasal turbinate but less effectively than wild type virus, in the lung (Figure 2C) . Importantly, while replication of Ca-DelMut variant was observed in the lung, the elevated expression of proinflammatory cytokines elicited in wild type virus infected hamsters was not detected. These onservations clearly suggest the polybasic cleavage is a virulence element in the SARS-CoV-2 and that its removal would makes SARS-CoV-2 less pathogenic, and more similar to a common cold respiratory coronavirus virus. It is believed that SARS-CoV-2 will continue to undergo further adaptation through circulation in humans. A previous study revealed variants with deletion at the S1/S2 junction present at low level in clinical specimens
16. In 2003, the SARS-like coronavirus characterized from civet cats and the early-outbreaks human SARS-CoV isolates contained a 29-bp sequence in the ORF8 sequence which was deleted following subsequent circulation in humans
29. Deletions in ORF7b and ORF8 have also been observed in SARS-CoV-2, although the significance of these alterations remain unknown
30, 31. It remains to be seen if continued evolution of SARS-CoV-2 in humans will subsequently select less pathogenic variant like Ca-DelMut or other mutants.
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Claims (16)
- A live attenuated SAR-CoV-2 virus comprising genome encoded by SEQ ID NO: 3, comprising a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
- The attenuated virus of claim 1 comprising a spike protein of SEQ ID NO: 2, wherein the sequence NSPRRARSVA (SEQ ID NO: 1) is deleted
-
The live attenuated virus of claim 1 or 2, comprising proteins with the following mutations relative to Hu-1 SARS-CoV-2; an A578V mutation in the NSP3a protein; P25L mutation in the spike protein; a V367F in the spike protein; an F20S in the E protein and a V62L mutation in ORF8. - The live attenuated virus of any one of claims 1-3, comprising genome encoded by SEQ ID NO: 3, comprising a deletion of nucleotides 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
- A composition comprising the live attenuated virus of any one of claims 1-3 or a variant thereof.
- The composition of claim 6, wherein the variant comprises a genome encoded by a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 3, with a deletion of nucleotides selected from the group consisting of 1663, 4455, 8782, 21636, 22661, 23598-23627, 24034, 26303, 26729, 58077 and 28144.
- A pharmaceutical composition comprising an effective amount of the attenuated virus of anyone of claims 1-4.
- The composition of claim 7, further comprising a preservative.
- The composition of anyone of claims 5-7, further comprising an adjuvant.
- The composition of any one of claims 5-9, in a form suitable for nasal administration.
- The composition of claim 9, wherein the adjuvant is selected from the group consisting of Montanide ISA-51 and CpG ODN (oligodeoxynucleotide) , AS01 (adjuvant system 01) , aluminum hydroxide or a TLR (Toll-like receptor) agonist.
- The composition of any one of claims 1-11, wherein the composition is lyophilized.
- A method for increasing an immune response to Sars-CoV-2 in a subject in need thereof, comprising administering the composition of any one of claims 1-12, to the subject.
- The method of claim 13, wherein the composition is administered by parenteral injection
- The method of claim 14, wherein the composition is administered subcutaneously, intradermal, intravenously, or intramuscularly.
- The method of claim 14 wherein the composition is administered intranasally.
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