WO2021207420A1 - Vaccin à adn innovant contre les sars-cov, sars-cov-2 et mers-cov - Google Patents

Vaccin à adn innovant contre les sars-cov, sars-cov-2 et mers-cov Download PDF

Info

Publication number
WO2021207420A1
WO2021207420A1 PCT/US2021/026268 US2021026268W WO2021207420A1 WO 2021207420 A1 WO2021207420 A1 WO 2021207420A1 US 2021026268 W US2021026268 W US 2021026268W WO 2021207420 A1 WO2021207420 A1 WO 2021207420A1
Authority
WO
WIPO (PCT)
Prior art keywords
rbd
cov
sars
seq
albumin
Prior art date
Application number
PCT/US2021/026268
Other languages
English (en)
Inventor
T.C. Wu
Chien-Fu Hung
Ya Chea TSAI
Ssu-Hsueh TSENG
Richard B.S. Roden
Original Assignee
The Johns Hopkins University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Johns Hopkins University filed Critical The Johns Hopkins University
Publication of WO2021207420A1 publication Critical patent/WO2021207420A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • SARS-CoV-2 the novel coronavirus that causes COVID-19, has been rapidly spreading since the World Health Organization’ s first citation of the disease in December 2019. Since then the virus has spread throughout 196 countries (The World Health Organization, www.who.int/emergencies/diseases/novel-coronavirus-2019), and as of Monday, March 30, Johns Hopkins reported more than 700,000 worldwide-confirmed cases and cited the disease as causing over 35,000 deaths (Johns Hopkins University, coronavirus.jhu.edu). In the United States, all 50 states, Washington DC, and four territories have reported cases.
  • SARS-CoV-2 is not the first coronavirus found to infect humans, several distinct features make the virus particularly complicated to halt the outbreak.
  • COVID-19 is often compared to the 2003 Sudden Acute Respiratory Syndrome (SARS) and its respective virus, SARS-CoV. The two viruses cause similar respiratory tract infections and are from the same viral family; however, SARS and COVID-19 differ on biological and epidemiological levels.
  • SARS-CoV-2 and SARS-CoV share roughly 79% of genetic information, the structure and genetic coding for the binding spike protein are dissimilar (Wang, Y.,
  • Middle East Respiratory Syndrome is an illness also caused by a coronavirus called Middle East Respiratory Syndrome Coronavirus (MERS-CoV).
  • MERS Middle East Respiratory Syndrome Coronavirus
  • Most MERS patients developed severe respiratory illness with symptoms of fever, cough and shortness of breath. According to the CDC, about 3 or 4 out of every 10 patients reported with MERS have died. Health officials first reported the disease in Saudi Arabia in September 2012. Through retrospective (backward-looking) investigations, they later identified that the first known cases of MERS occurred in Jordan in April 2012. So far, all cases of MERS have been linked through travel to, or residence in, countries in and near the Arabian Peninsula. The largest known outbreak of MERS outside the Arabian Peninsula occurred in the Republic of Korea in 2015. The outbreak was associated with a traveler returning from the Arabian Peninsula.
  • CDC Centers for Disease Control and Prevention
  • the inventors now provide a vaccine to prevent new infections and to slow the spread of COVID-19.
  • the preventive vaccine is designed to produce neutralizing antibodies that would bind the SARS- CoV-2 spike protein receptor-binding domain (RBD).
  • the SARS-CoV-2 vims’ RBD binds ACE2 in order to infect human cells.
  • a vaccine that could rapidly produce neutralizing antibody to bind the SARS-CoV-2 RBD should render the pathogen unable to bind ACE2, resulting in the inability of SARS-CoV-2 to cause COVID-19. Therefore, the RBD of the spike protein serves as an important target for the development of a preventive vaccine against COVID-19.
  • the present invention provides a number of embodiments of a DNA vaccine which is directed to RBDs of SARS-CoV2 RBD, MERS-CoV RBD, and SARS-CoV RBD.
  • the present invention provides a DNA vaccine composition
  • a DNA vaccine composition comprising a synthetic polynucleotide encoding albumin protein, or a functional portion, or fragment or variant thereof, conjugated to SARS-CoV2 RBD, or a functional portion or fragment or variant thereof.
  • the present invention provides a DNA vaccine composition comprising a synthetic polynucleotide encoding human albumin protein, or a functional portion, or fragment or variant thereof, conjugated to SARS-CoV2 RBD, MERS- CoV RBD, and SARS-CoV RBD, or a functional portion or fragment or variant thereof.
  • the present invention provides a composition comprising a synthetic polypeptide consisting of albumin protein, or a functional portion, or fragment or variant thereof, conjugated to SARS-CoV2 RBD, or a functional portion or fragment or variant thereof.
  • the present invention provides a composition comprising a synthetic polypeptide consisting of SARS-CoV2 RBD, MERS-CoV RBD, and SARS-CoV RBD human albumin protein, or a functional portion, or fragment or variant thereof, conjugated to, or a functional portion or fragment or variant thereof.
  • the present invention provides a recombinant vector encoding the DNA vaccine compositions described herein.
  • the present invention provides a pharmaceutical composition comprising the DNA vaccine compositions described herein.
  • the present invention provides a cell or population of cells expressing the synthetic polypeptide compositions described herein.
  • the present invention provides a method for treating a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions described herein.
  • the present invention provides a method for treating a SARS-CoV-2 infection, and/or a MERS-CoV infection, and/or a SARS-CoV infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions described herein.
  • the present invention provides a method for treating COVID-19 in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions described herein.
  • the present invention provides a method for treating COVID-19 and/or a MERS-CoV infection, and/or a SARS-CoV infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions described herein.
  • the present invention provides a method for post-exposure prophylaxis or treatment of a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions described herein.
  • the present invention provides a method for post-exposure prophylaxis or treatment of a SARS-CoV-2 infection and/or a MERS-CoV infection, and/or a SARS-CoV infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions described herein.
  • Figure 1 Amino acid sequence of SARS-CoV-2 spike protein receptor binding domain linked to human albumin. Linked RBD and human albumin amino acid sequence. EF is the EcoRI cloning site between albumin and RBD within the pcDNA3 vector.
  • Figure 2 Genetic sequence of SARS-CoV-2 spike protein receptor binding domain linked to human albumin. Genetic Sequence of albumin-RBD protein inserted into pcDNA3 sequence. GAATTC is the EcoRI cloning site between albumin and RBD.
  • FIGS. 3A-3B pcDNA3-Hal-RBD expresses higher levels of RBD-albumin in a western blot.
  • 3 A Schematic of plasmids used to cell transfection and mice vaccination.
  • RBD SARS-CoV-2 Spike receptor binding domain.
  • Hal Human albumin.
  • CMV cytomegalovirus (CMV) immediate early promoter.
  • HEK 293 cells were transfected with lOug of each plasmid. After 48 hours transfection, cell lysates were collected for Western blot analysis. GAPDH as loading control.
  • FIGS 4A-4B pcDNA3 -Hal-RBD promotes higher levels of RBD antibody in vivo. Characterization of Hal-RBD DNA vaccination by ELISA.
  • FIGS 5A-5B pcDNA3 -Hal-RBD vaccination generate the highest levels of RBD-specific antibody in vaccinated mice among all of the various DNA vaccine methods tested. Characterization of Hal-RBD DNA vaccination by ELISA.
  • 5A is a graphic depiction of the dosing regimen for various vaccine compositions.
  • 5B Antibodies specific for the human SARS-CoV-2 Spike protein were analyzed by ELISA. Briefly, 1 ⁇ g/ml of human SARS-CoV-2 Spike protein in PBS were coated on BRANDplates® microplates for overnight.
  • Flow cytometry analysis to characterize the binding of the spike protein of SARS-CoV-2 by the antibody generated by vaccination with pcDNA3- Hal-RBD DNA.
  • HEK 293 cells were transfected with DNA encoding the spike protein of SARS- CoV-2.
  • binding to the spike protein at the cell surface was analyzed by incubation with sera from mice vaccinated pcDNA3-RBD DNA (light trace) (Panel A), pcDNA3-Hal-RBD DNA (light trace) (Panel B) or PBS (dark distribution curves in both Panels A and B) control followed by staining with PE-conjugated anti-mouse IgG. Representative figures of flow cytometry analysis are shown.
  • Figures 7A-7B Genetic sequence of alternative embodiment of SARS-CoV-2 spike protein receptor binding domain linked to human albumin showing nucleotide (7 A) and amino acid (7B) sequences. Genetic Sequence of albumin-RBD protein inserted into pNGVL4a vector sequence. GAATTC is the EcoRI cloning site between albumin and RBD.
  • Figures 8A-8B Genetic sequence of another embodiment of SARS-CoV-2 spike protein receptor binding domain, the MERS-CoV RBD, and the SARS-CoV RBD linked to human albumin showing nucleotide (8A) and amino acid (8B) sequences. Genetic Sequence of albumin-RBD protein inserted into pNGVL4a vector sequence. GAATTC is the EcoRI cloning site between albumin and RBD.
  • Figures 9A-9B Genetic sequence of another embodiment of SARS-CoV-2 spike protein receptor binding domain, the MERS-CoV RBD, and the SARS-CoV RBD linked to human albumin showing nucleotide (9 A) and amino acid (9B) sequences. Genetic Sequence of albumin-RBD protein inserted into pNGVL4a vector sequence. GAATTC is the EcoRI cloning site between albumin and RBD.
  • Figure 9A discloses SEQ ID NO: 21.
  • FIG. 10 pcDNA3-Hal-RBD and pNGVL4a-Hal RBD expresses higher levels of secreted protein in a western blot.
  • HEK 293 cells were transfected with 10 ⁇ g of each plasmid. After 48 hours transfection, cell lysates and supernatant were collected for Western blot analysis.
  • Lane 1 cell pellet from pcDNA3 -Hal-RBD transfected cells.
  • Lane 2 cell pellet from pNGVL4a-Hal- RBD transfected cells.
  • Lane 3 supernatant from pcDNA3-Hal-RBD transfected cells.
  • Lane 4 supernatant from pNGVL4a -Hal-RBD transfected cells.
  • Hal-RBD protein generates higher levels of RBD antibody in vivo.
  • ELISA assays using sera derived from mice vaccinated with RBD, RBD-FC or Hal-RBD protein.
  • C57BL/6 mice (5 per group) were vaccinated subcutaneously with 10 ⁇ g of RBD, 20 ⁇ g RBD-FC or 40 ⁇ g of Hal-RBD protein vaccine with 5 ⁇ g MPL/mouse three times at 1 week interval.
  • Sera from each group of mice were collected through tail vein 1 week after the last vaccination.
  • Serial dilution of the sera was used for ELISA using 96 well plate coated with RBD protein.
  • naked DNA vaccines have emerged as a potentially promising strategy due to their simplicity, stability, and safety profiles.
  • naked DNA vaccine does not amplify in the transfected cells and spread throughout the body, there is limited potency.
  • DNA vaccine strategies require techniques to improve potency.
  • a common approach to increasing potency is to administer the vaccine via electroporation.
  • electroporation is uncomfortable and difficult, making it unlikely to become a widely adopted vaccine.
  • the present inventors modified the DNA vaccine itself to increase potency.
  • the inventors provide herein an innovative strategy to improve naked DNA vaccine potency by conjugating albumin protein with the target antigen: RBD of the spike protein of SARS-CoV-2.
  • the inventors now show that the linkage of albumin with the target antigen leads to the enrichment of targeted antigen protein, specifically within the draining lymph nodes. These elevated target antigen protein levels within the lymph nodes leads to high titers of antibody against the linked antigen in vaccinated mice.
  • the linking of albumin therefore increases potency of a DNA vaccine strategy while avoiding cumbersome electroporation, making the vaccine a more feasible approach.
  • the inventors now also provide a broader spectrum coronavirus vaccine that can potentially protect subject from all three know disease-causing coronaviruses, by linking albumin with the SARS-CoV-2 RBD antigen, MERS-CoV RBD antigen, and SARS-CoV RBD antigen.
  • the present invention provides a DNA vaccine composition
  • a DNA vaccine composition comprising a synthetic polynucleotide encoding albumin protein, or a functional portion, or fragment or variant thereof, conjugated to RBD, or a functional portion or fragment or variant thereof.
  • DNA vaccine compositions of the present invention can be used for other SARS coronavirus infections having the same or similar RBD.
  • the DNA vaccine composition comprises a synthetic polynucleotide encoding human albumin protein, or a functional portion, or fragment or variant thereof, conjugated to RBD, or a functional portion or fragment or variant thereof.
  • albumin means the most abundant circulating protein in the plasma of most mammals. Human albumin (Hal) is present in the plasma of healthy individuals at a concentration of (3.5-5 g/dL) and it represents approximately 50% of the total protein content.
  • HA is a small globular protein (molecular weight: 66.5 kDa), consisting of a single chain of 585 amino acids organized in three repeated homologue domains (sites I, II, and III), each of which comprises two separate sub-domains (A and B).
  • albumin protein means the full-length expressed polypeptide of a nucleic acid encoding the albumin gene, or a functional portion or fragment, or variant thereof.
  • albumin isoforms include, but are not limited to, human albumin isoforms (NM_000477, AAH41789.1, and AAH35969), for example and mouse albumin ligand isoform (AAH49971), for example.
  • the DNA sequence of Hal used in the present invention is detailed in Fig. 2 and has the following sequence:
  • TCAAGCTGCCTTAGGCTTA SEQ ID NO: 1.
  • the term “functional portion or fragment thereof,” with respect to the albumin protein, means that the portion or fragment of the albumin polypeptide retains its ability to bind to the neonatal Fc receptor and traffic through the lymphatic system.
  • the albumin protein is mammalian. In certain embodiments, the albumin protein can be murine, porcine, ovine, bovine, human, or combinations thereof.
  • the amino acid sequence of Hal used in the present invention is detailed in Fig. 1 and has the following sequence:
  • SARS coronavirus infection means a subject who is infected with one or more coronaviruses (CoVs).
  • CoVs are enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy.
  • Coronaviruses cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs and upper respiratory disease chickens to potentially lethal human respiratory infections.
  • the initial attachment of the CoV virion to the host cell is initiated by interactions between the S protein and its receptor.
  • the sites of receptor binding domains (RBD) within the SI region of a coronavirus S protein vary depending on the virus, with some having the RBD at the N-terminus of SI (MHV) while others (SARS-CoV) have the RBD at the C- terminus of SI.
  • the S -protein/receptor interaction is the primary determinant for a coronavirus to infect a host species and governs the tissue tropism of the virus.
  • Many coronaviruses utilize peptidases as their cellular receptor. It is unclear why peptidases are used, as entry occurs even in the absence of the enzymatic domain of these proteins.
  • a- coronaviruses utilize aminopeptidase N (APN) as their receptor, SARS-CoV, SARS-CoV-2, and HCoV-NL63 use angiotensin-converting enzyme 2 (ACE2) as their receptor, MHV enters through CEACAM1, and the recently identified MERS-CoV binds to dipeptidyl- peptidase 4 (DPP4) to gain entry into human cells.
  • APN aminopeptidase N
  • SARS-CoV-2 SARS-CoV-2
  • HCoV-NL63 use angiotensin-converting enzyme 2 (ACE2) as their receptor
  • MHV enters through CEACAM1
  • DPP4 dipeptidyl- peptidase 4
  • the subject is infected with SARS-CoV-2, which causes the COVID-19 disease.
  • SARS-CoV-2 is an enveloped virus carrying a positive-sense, single- stranded RNA genome and a nucleocapsid with helical symmetry.
  • the pathogenesis of COVID-19 is complex but begins with the binding of the spike (S) protein to the Angiotensin Converting Enzyme 2 (ACE2) receptor on Type I and Type II pneumocytes in the lower respiratory tract. This binding event facilitates viral entry into respiratory epithelial cells, whereupon the viral genome replicates inside the cell and newly assembled virions bud from the plasma membrane to infect other cells.
  • S spike
  • ACE2 Angiotensin Converting Enzyme 2
  • SARS-CoV-2 RBD means the receptor-binding domain of the spike protein of a SARS corona vims, and in some embodiments, it is the RBD of the SARS-CoV-2 virus, or a functional portion or fragment thereof. In one embodiment, the RBD has the following nucleotide sequence:
  • amino acid sequence of RBD used in the present invention is detailed in Fig. 1 and has the following sequence:
  • MERS-CoV RBD means the receptor-binding domain of the spike protein of a MERS-CoV corona virus, and in some embodiments, it is the RBD of the MERS-CoV virus, or a functional portion or fragment thereof. In one embodiment, the
  • RBD is shown in Fig. 8A, and has the following nucleotide sequence:
  • the amino acid sequence of MERS-CoV RBD used in the present invention is detailed in Fig. 8B and has the following sequence: FEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDF TCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATV PHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYR KQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIAS QLGNCVEYR (SEQ ID NO: 16) or a functional portion or fragment thereof.
  • SARS-CoV RBD means the receptor-binding domain of the spike protein of a SARS-CoV corona vims, and in some embodiments, it is the RBD of the SARS-CoV virus, or a functional portion or fragment thereof. In one embodiment, the
  • RBD is shown in Fig. 8A, and has the following nucleotide sequence:
  • the amino acid sequence of SARS-CoV RBD used in the present invention is detailed in Fig. 8B and has the following sequence: VVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTF KCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGC VLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYW PLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFN (SEQ ID NO: 18) or a functional portion or fragment thereof.
  • the DNA vaccine composition comprises a first polynucleotide sequence encoding a synthetic polypeptide encoding albumin, or a functional portion, or fragment or variant thereof, linked via one or more linking polynucleotides, to polynucleotide encoding a synthetic polypeptide encoding RBD, or a functional portion, or fragment or variant thereof.
  • the conjugate composition has the albumin amino acid sequence domain at the N- terminal side of the polypeptide followed by a linker, and then the RBD domain at the C- terminal end of the polypeptide.
  • the linker comprises a polynucleotide which encodes one or more amino acids.
  • the linker can be a polynucleotide encoding 1 to 50 amino acids, including, for example, 2, 3, 4, 5, 10, 15, 20, 30, 40, up to 50 amino acids.
  • the linker is a dipeptide.
  • the linker is the dipeptide Glu-Phe.
  • the DNA vaccine composition comprises at least the polynucleotide sequence of SEQ ID NO: 5.
  • the synthetic polynucleotide encoding the DNA vaccine composition has the nucleic acid sequence of SEQ ID NO: 5, or a polynucleotide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 5.
  • the DNA vaccine composition comprises at least the polynucleotide sequence of SEQ ID NO: 11 (Albumin-RBD (lowercase single underlined sequence is albumin, italicized sequence is linker, lowercase double underlined sequence RBD, and remainder of the sequence is vector)).
  • the synthetic polynucleotide encoding the DNA vaccine composition has the nucleic acid sequence of SEQ ID NO: 11, or a polynucleotide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 11:
  • the DNA vaccine composition comprises at least the polynucleotide sequence of SEQ ID NO: 13 (Albumin- COVID19 RBD- Mers RBD- Sars
  • RBD single underlined polynucleotide sequence is albumin, italicized polynucleotide sequence is COVID19 RBD, bold polynucleotide sequence is Mers RBD, double underlined polynucleotide sequence is Sars RBD, and the remainder of the polynucleotide sequence is the vector pNGVL4a).
  • DNA vaccine composition has the nucleic acid sequence of SEQ ID NO: 13, or a polynucleotide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 13:
  • the DNA vaccine composition comprises at least the polynucleotide sequence of SEQ ID NO: 19 (Albumin- COVID19 RBD- Mers RBD- Sars RBD (single underline is albumin, uppercase bold and italicized is EcoRI cloning site, lowercase italicized is COVID19 RBD, uppercase bold is Mers RBD, double underline is Sars RBD, and remainder is vector pNGVL4a)).
  • the synthetic polynucleotide encoding the DNA vaccine composition has the nucleic acid sequence of SEQ ID NO: 19, or a polynucleotide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 19:
  • the DNA vaccine composition can be comprised within an expression cassette.
  • expression cassette or "expression vector” as used herein refers to a nucleotide sequence which is capable of affecting expression of a protein coding sequence in a host compatible with such sequences.
  • Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be included, e.g., enhancers.
  • “Operably linked' refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence.
  • expression cassettes include plasmids, recombinant viruses, any form of a recombinant "naked DNA" vector, and the like.
  • immunogen or “immunogenic composition” is synonymous with “antigen or antigenic” and refers to a compound or composition comprising a peptide, polypeptide or protein which is "immunogenic,” i.e., capable of eliciting, augmenting or boosting a cellular and/or humoral immune response, either alone or in combination or linked or fused to another substance.
  • An immunogenic composition can be a peptide of at least about 5 amino acids, a peptide of 10 amino acids in length, a fragment 15 amino acids in length, a fragment 20 amino acids in length or greater; smaller immunogens may require presence of a "carrier" polypeptide e.g., as a fusion protein, aggregate, conjugate or mixture, preferably linked (chemically or otherwise) to the immunogen.
  • the immunogen can be recombinantly expressed from a vaccine vector, which can be naked DNA comprising the immunogen's coding sequence operably linked to a promoter, e.g., an expression cassette.
  • the immunogen includes one or more antigenic determinants or epitopes, which may vary in size from about 3 to about 15 amino acids.
  • the immunogen or antigen is a synthetic polypeptide encoding RBD or portion or fragment thereof.
  • the RBD antigen is from one or more different viruses or can contain more than one copy of the RBD antigen. In some embodiments, the RBD antigen is from three different coronaviruses, such as SARS-Cov2, MERS-CoV, and SARS-CoV. [0073] In accordance with an embodiment, the present invention provides a recombinant vector encoding the DNA vaccine compositions described herein.
  • nucleic acid includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
  • the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
  • the nucleic acids of the invention are recombinant.
  • the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above.
  • the replication can be in vitro replication or in vivo replication.
  • the nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra, and Ausubel et al., supra.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine- substituted nucleotides).
  • modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine
  • the substituted nucleic acid sequence may be optimized. Without being bound to a particular theory, it is believed that optimization of the nucleic acid sequence increases the translation efficiency of the mRNA transcripts. Optimization of the nucleic acid sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. Optimization of the nucleic acid sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.
  • the invention also provides an isolated or purified nucleic acid comprising a nucleotide sequence which is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.
  • the nucleic acids of the invention can be incorporated into a recombinant expression vector. In this regard, the invention provides recombinant expression vectors comprising any of the nucleic acids of the invention.
  • the term "recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell.
  • the vectors of the invention are not naturally occurring as a whole. However, parts of the vectors can be naturally occurring.
  • the inventive recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single- stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vectors can comprise naturally occurring, non-naturally occurring internucleotide linkages, or both types of linkages.
  • the non-naturally occurring or altered nucleotides or internucleotide linkages does not hinder the transcription or replication of the vector.
  • the DNA vaccine composition or expression cassette will be inserted into a DNA vector or plasmid.
  • the recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
  • the vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).
  • Bacteriophage vectors such as ⁇ GT10, ⁇ GT11, ⁇ Zap I I (Stratagene), /.EMBL4, and lNM1149, also can be used.
  • plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).
  • the plasmid can be one of the pcDNA3 family of plasmids known in the art.
  • the plasmid can be the pNGVL4a expression vector.
  • the pcDNA3 contains the ampicillin resistant gene, rendering it inappropriate for clinical usage.
  • the inventors have previously used a mammalian cell expression vector, pNGVL4a DNA construct for our clinical studies.
  • the pNGVL4a DNA construct does not contain ampicillin resistant gene.
  • Applicants’ have cloned various constructs including the albumin-SARS-CoV2 RBD fusion construct, and two albumin- SARS-CoV2 RBD, MERS-CoV RBD and SARS-CoV fusion constructs into the pNGVL4a vector and are analyzing the expression and immunogenicity of these DNA constructs.
  • the recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra.
  • Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell.
  • Replication systems can be derived, e.g., from ColEl, 2 m plasmid, l, SV40, bovine papilloma virus, and the like.
  • the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
  • regulatory sequences such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
  • the recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts.
  • Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.
  • Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
  • the recombinant expression vector can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the fusion proteins, polypeptide, or protein (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the fusion proteins, polypeptide, or protein.
  • promoters e.g., strong, weak, inducible, tissue- specific and developmental- specific, is within the ordinary skill of the artisan.
  • the combining of a nucleotide sequence with a promoter is also within the skill of the artisan.
  • the promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.
  • CMV cytomegalovirus
  • the present invention provides a composition comprising a synthetic polypeptide encoding albumin protein, or a functional portion, or fragment or variant thereof, conjugated to RBD, or a functional portion or fragment or variant thereof.
  • the synthetic polypeptide encoding albumin protein, or a functional portion, or fragment or variant thereof, conjugated to RBD, or a functional portion or fragment or variant thereof is a fusion polypeptide which acts as an immunogen to the immune system and is expressed in the cells of the subject that have taken up the DNA vaccine of the present invention.
  • the synthetic polypeptide encoding human albumin protein has the amino acid sequence of SEQ ID NO: 2, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 2.
  • the synthetic polypeptide encoding RBD has the amino acid sequence of SEQ ID NO: 4, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 4.
  • the synthetic polypeptide encoding RBD, or a functional portion, or fragment or variant thereof, conjugated to human albumin protein, or a functional portion or fragment or variant thereof comprises the amino acid sequence of at least SEQ ID NO: 6, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 6.
  • the synthetic polypeptide molecules described herein such as SEQ ID NO: 6, or a functional portion or fragment or variant thereof comprises the amino acid sequence of at least SEQ ID NO: 6, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 6, can also be used as a vaccine.
  • the synthetic polypeptide molecules described herein such as SEQ ID NO: 12, or a functional portion or fragment or variant thereof comprises the amino acid sequence of at least SEQ ID NO: 12, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 12, can also be used as a vaccine.
  • the synthetic polypeptide molecules described herein, such as SEQ ID NO: 14, or a functional portion or fragment or variant thereof comprises the amino acid sequence of at least SEQ ID NO: 14, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 14, can also be used as a vaccine.
  • the synthetic polypeptide molecules described herein such as SEQ ID NO: 20, or a functional portion or fragment or variant thereof comprises the amino acid sequence of at least SEQ ID NO: 20, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 20, can also be used as a vaccine.
  • the term "functional portion" when used in reference to the antigenic epitope or immunogen refers to any part or fragment, which part or fragment retains the biological activity of which it is a part (the parent molecule, antibody, or antigen).
  • Functional portions encompass, for example, those parts that retain the ability to specifically bind to the antigen (e.g., in an MHC-independent manner), or detect, treat, or prevent the disease, to a similar extent, the same extent, or to a higher extent, as the parent molecule.
  • the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent molecule.
  • the invention also provides a synthetic polypeptide molecule comprising at least one of the polypeptides described herein along with at least one other polypeptide.
  • the other polypeptide can exist as a separate polypeptide of the fusion protein, or can exist as a polypeptide, which is expressed in frame (in tandem) with one of the inventive polypeptides described herein.
  • the other polypeptide can encode any peptidic or proteinaceous molecule, or a portion thereof. Suitable methods of making fusion proteins are known in the art, and include, for example, recombinant methods. See, for instance, Choi et al., Mol. Biotechnol. 31: 193-202 (2005).
  • fusion proteins, polypeptides, and proteins described herein include functional variants of the inventive fusion proteins, and polypeptides, and proteins described herein.
  • the term "functional variant” as used herein refers to fusion proteins, polypeptides, or proteins having substantial or significant sequence identity or similarity to a parent fusion proteins, polypeptides, or proteins, which functional variant retains the biological activity of the fusion proteins, polypeptides, or proteins of which it is a variant.
  • the functional variant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the parent fusion proteins, polypeptide, or protein.
  • the functional variant can, for example, comprise the amino acid sequence of the parent fusion proteins, polypeptide, or protein with at least one conservative amino acid substitution.
  • Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same chemical or physical properties.
  • the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, lie, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc.
  • an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain e.g., Ala, Gly, Val, lie, Leu, Met, Phe, Pro, Trp, Val, etc.
  • a basic amino acid substituted for another basic amino acid Lys, Arg, etc.
  • the functional variants can comprise the amino acid sequence of the parent fusion proteins, polypeptide, or protein with at least one non conservative amino acid substitution.
  • the non-conservative amino acid substitution it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant.
  • the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent fusion proteins, polypeptide, or protein.
  • fusion polypeptides, and/or proteins of the invention can be obtained by methods known in the art. Suitable methods of de novo synthesizing polypeptides and proteins are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000; and U.S. Patent No. 5,449,752.
  • polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.
  • fusion proteins, polypeptides, and proteins of the invention can be isolated and/or purified from a source, such as a plant, a bacterium, an insect, a mammal, e.g., a rat, a human, etc. Methods of isolation and purification are well-known in the art.
  • a source such as a plant, a bacterium, an insect, a mammal, e.g., a rat, a human, etc. Methods of isolation and purification are well-known in the art.
  • the fusion proteins, polypeptides, and/or proteins described herein can be commercially synthesized by companies, such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA).
  • the inventive fusion proteins, polypeptides, and proteins can be synthetic, recombinant, isolated, and/or purified.
  • the present invention provides a pharmaceutical composition comprising the DNA vaccine compositions described herein.
  • the present invention provides a pharmaceutical composition comprising the synthetic polypeptide compositions described herein for use as a vaccine.
  • the present invention provides the use of a pharmaceutical composition comprising vaccine, and a pharmaceutically acceptable carrier, as a medicament, preferably as a medicament for the treatment of a SARS coronavirus infection in a subject.
  • the present invention provides the use of a pharmaceutical composition comprising the DNA vaccine compositions and/or the synthetic polypeptide compositions described herein, and a pharmaceutically acceptable carrier, as a medicament, preferably as a medicament for the treatment of COVID 19-related disease or SARS-CoV2 infection in a subject.
  • the present invention provides a method for treating a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions and/or the synthetic polypeptide compositions described herein.
  • the invention provides a method for post-exposure prophylaxis or treatment of a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions and/or the synthetic polypeptide compositions described herein.
  • the present invention provides the use of a pharmaceutical composition comprising vaccine, and a pharmaceutically acceptable carrier, as a medicament, preferably as a medicament for the treatment of a SARS-CoV, MERS-CoV or SARS-CoV2 coronavirus infection in a subject.
  • the present invention provides a method for treating a a SARS-CoV, MERS-CoV or SARS-CoV2 infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions and/or the synthetic polypeptide compositions described herein.
  • the invention provides a method for post-exposure prophylaxis or treatment of a SARS-CoV, MERS-CoV or SARS-CoV2infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions and/or the synthetic polypeptide compositions described herein.
  • the term "administering" means that the compositions of the present invention are introduced into a subject, preferably a subject receiving treatment for an infectious disease, and the compounds are allowed to come in contact with the one or more disease related cells or population of cells in vivo.
  • the DNA vaccine compositions described herein can be administered in a regimen where there is a first or priming dose of vaccine composition administered to the subject, then after a period of 5 to 50 days, a second, third or more boost dose of vaccine is then administered to the subject.
  • the boost dose is administered 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 up to 50 days apart.
  • the present invention provides a method for treating a SARS-CoV, MERS-CoV or SARS-CoV2 infection in a subject in need thereof comprising: a) administering to the subject an effective amount of a first or priming dose of the DNA vaccine compositions and/or the synthetic polypeptide compositions; b) administering to the subject after an interval of between about 5 to 180 days an effective amount of a second or boost dose of the DNA vaccine compositions and/or the synthetic polypeptide compositions; and c) optionally repeating step b).
  • the carrier is preferably a pharmaceutically acceptable carrier.
  • the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration ⁇
  • the pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.
  • the choice of carrier will be determined in part by the chemical properties of the vaccines as well as by the particular method used to administer the vaccines. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention.
  • the following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and intraperitoneal administration are exemplary and are in no way limiting. More than one route can be used to administer the first and second vaccine, and in certain instances, a particular route can provide an immediate and more effective response than another route.
  • the vaccine is administered intra-muscularly. In another embodiment, and the vaccine is administered intra-dermally.
  • Injectable formulations are in accordance with the present invention.
  • the vaccines of the present invention can be administered other ways known in the art.
  • the vaccines can be administered via use of electroporation techniques. Suitable electroporation techniques are disclosed in U.S. Pat. Nos. 6,010,613, 6,603,998, and 6,713,291, all of which are incorporated herein by reference.
  • Other physical approaches can include gene gun, biojector, ultrasound, and hydrodynamic delivery, all of which employ a physical force that permeates the cell membrane and facilitates intracellular gene transfer.
  • Chemical vaccination approaches typically use synthetic or naturally occurring compounds (e.g. cationic lipids, cationic polymers, lipid-polymer hybrid systems) as carriers to deliver the nucleic acid into the cells.
  • intramuscular administration of the vaccines of the present invention may be achieved by the use of a needless injection device to administer a virus or plasmid DNA suspension (using, e.g., BiojectorTM) or a freeze-dried powder containing the vaccine (e.g., in accordance with techniques and products of Powderject).
  • a needless injection device to administer a virus or plasmid DNA suspension (using, e.g., BiojectorTM) or a freeze-dried powder containing the vaccine (e.g., in accordance with techniques and products of Powderject).
  • the amount or dose of the vaccine administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject over a reasonable time frame. The dose will be determined by the efficacy of the first and second vaccine and the condition of a human, as well as the body weight of a human to be treated.
  • the attending physician will decide the dosage of first and second vaccine with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, to be administered, route of administration, and the severity of the condition being treated.
  • the dose of the vaccine is about 1 to 10,000 ⁇ g of vaccine to the subject being treated.
  • the dosage range of the vaccine is about 500 ⁇ g-6,000 ⁇ g of vaccine.
  • the dosage of the vaccine is about 3,000 Pg ⁇
  • the present invention provides a pharmaceutical composition comprising the DNA vaccine compositions and/or the synthetic polypeptide compositions described herein in combination with at least one additional biologically active agent.
  • an “active agent” and a “biologically active agent” are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and/or physiological effect, wherein the effect may be prophylactic or therapeutic.
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs and the like.
  • active agent pharmaceutically active agent
  • drug drug
  • the invention includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs etc.
  • the active agent can be a biological entity, such as a virus or cell, whether naturally occurring or manipulated, such as transformed.
  • the biologically active agent may vary widely with the intended purpose for the composition.
  • active is art-recognized and refers to any moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject.
  • biologically active agents that may be referred to as “drugs”, are described in well-known literature references such as the Merck Index, the Physicians’ Desk Reference, and The Pharmacological Basis of Therapeutics, and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.
  • Non- limiting examples of biologically active agents include following: anti inflammatory agents such as steroids, non-steroidal anti-inflammatory agents, anti-pyretic and analgesic agents, antigenic materials, and anti-viral drugs.
  • biologically active agents include, without limitation, such forms as uncharged molecules, molecular complexes, salts, ethers, esters, amides, prodrug forms and the like, which are biologically activated when implanted, injected or otherwise placed into a subject.
  • the present invention provides a method for treating a SARS-CoV, MERS-CoV or SARS-CoV2infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions and/or the synthetic polypeptide compositions in combination with one or more additional biologically active agents.
  • the invention provides a method for post-exposure prophylaxis or treatment of a SARS-CoV, MERS-CoV or SARS-CoV2 infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions and/or the synthetic polypeptide compositions in combination with an effective amount of one or more additional biologically active agents.
  • the methods of treatment using an effective amount of the DNA vaccine compositions in combination with an effective amount of one or more additional biologically active agents can occur either simultaneously or serially with at least one other.
  • the dosing regimens of the above methods can also comprise a first dose of vaccine an additional biologically active agent, followed by a second or more dose of vaccine and optionally an additional biologically active agent as needed.
  • the term "subject” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
  • mammals of the order Rodentia such as mice and hamsters
  • mammals of the order Logomorpha such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is
  • the present invention provides a cell or population of cells expressing the synthetic polypeptide compositions described herein. It will be understood that the cells or population of cells expressing the synthetic polypeptide compositions were in contact with the DNA vaccine compositions and/or the synthetic polypeptide compositions in vitro or in vivo.
  • the term "contacting" means that the one or more compounds of the present invention are introduced into a sample having at least one cancer cell and appropriate enzymes or reagents, in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to permit binding and uptake of the at least one compound to the cancer cell.
  • Methods for contacting the samples with the compounds, and other specific binding components are known to those skilled in the art, and may be selected depending on the type of assay protocol to be ran. Incubation methods are also standard and are known to those skilled in the art.
  • kits that contain the compositions or pharmaceutical compositions used with the inventive methods, as described above, to practice the methods of the invention.
  • the kits can contain various combinations of vaccines and the like.
  • the kit can contain instructional material teaching methodologies, e.g., means to administer the compositions used to practice the invention, means to inject or infect cells, patients or animals with vaccines of the invention, means to monitor the resultant immune response and assess the reaction of the individual to which the compositions have been administered, and the like.
  • the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ⁇ 100% in some embodiments ⁇ 50%, in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
  • the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth.
  • the recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1 , and the like) and any range within that range.
  • human albumin (Hal) was first amplified via PCR using the cDNA template of human albumin (GenBank: AAA98797.1, TransOMIC Technologies, Huntsville, AL) and the following set of primers: 5'- A A ACTCG AGGCC ACC ATGA AGTGGGT A ACCTTT- 3 ' (SEQ ID NO: 7) and 5'- TTTGAATTCTAAGCCTAAGGCAGCTTG-3 ' (SEQ ID NO: 8). The amplified human albumin product was then cloned into the Xho/EcoRI sites of a pcDNA3 vector (Invitrogen Corp., Carlsbad, California).
  • RBD was amplified via PCR using the cDNA template of RBD (GenBank QHD43416.1) from Sino Biological (Wayne, PA 19087) and the following primers: 5'- AAAGAATTCATGAGGGTCCAACCAACAGAG-3' (SEQ ID NO: 9) and 5'- TTTGGATCCTC AGAAGTTC ACAC ACTTGTT-3 ' (SEQ ID NO: 10).
  • the amplified product was then cloned into the EcoRI/BamHI sites of pcDNA3 -Albumin creating pcDNA3- Hal-RBD.
  • the amplified cDNA RBD product was cloned into the EcoRI/BamHI sites of pcDNA3 with no albumin product.
  • albumin-RBD cDNA was synthesized by Thermofisher and cloned into Xbal/BamHI of pNGVL4a vector.
  • albumin- SARS-CoV-MERS-CoVSARS-CoV2 RBD cDNA was synthesized by Thermofisher and cloned into Xbal/BamHI of pNGVL4a vector.
  • HEK 293 cells were transfected with 10 ⁇ g of either pcDNA3-RBD or pcDNA3-Hal-RBD plasmid. After 48 hours of transfection, cell lysates were collected to complete a Western blot analysis. Blots were blocked with PBS/0.05%, Tween 20 (PBST) containing 5% nonfat milk for one hour at room temperature.
  • PBST Tween 20
  • Membranes were probed with anti-SARS-CoV Spike Antibody (Cat: 40150-T62-COV2, Sino Biological) at 1:3000 dilution in PBST containing 5% nonfat milk overnight at 4 °C, washed three times with PBST, and then incubated with goat antimouse IgG conjugated to alkaline phosphatase (Amersham, Piscataway, NJ) at 1:5000 dilution in PBST containing 5% nonfat milk. Membranes were washed three times with PBST and developed using enhanced Hyperfilm- enhanced chemiluminescence (Amersham, Piscataway, NJ). GAPDH (Cat: 60004- 1-Ig, Proteintech) was loaded as a control.
  • mice 6-8 week old female C57BL/6 mice (5 mice per group) were vaccinated. Each mouse was vaccinated with 20 ⁇ g/mouse of pcDNA3- SARS-CoV2 RBD, pcDNA3-Hal- SARS-CoV2 RBD, pNGVL4a-Hal-SARS-CoV3 RBD, and the variant pNGVL4a-Hal-SARS-CoV3 RBD-MERS-CoV RBD-SARS-CoV RBD plasmids via intramuscular injection in the right and left hind leg (10 ⁇ g/leg) at day 0. The mice were boosted with the same regimen twice, once at day 7 and once at day 14. One week after the final vaccination (day 21), sera was collected.
  • mice serum was analyzed by ELISA lug/ml of RBD protein (from Sino Biological) in PBS was coated on BRANDplates ® microplates overnight at 4 °C. The next morning, the plates were washed, blocked with eBioscienceTM ELISA/ELISPOT Diluent (Thermo Fisher Scientific), and added serial dilution of serum for two hours at room temperature. All serum used ranged from 1:100 - 1:10,240, and non- vaccinated mice serum was used as control. Goat anti-mouse IgG-HRP secondary antibody was added at 1:5000 dilution for 1 hour, followed by TMB substrate. The OD at 450 nm was determined by 800TM TS Absorbance Reader (BioTek Instruments, Inc).
  • pcDNA3 was first generated encoding human albumin (pcDNA3- Albumin).
  • the DNA sequence encoding human albumin was amplified with PCR using the cDNA template of human albumin and a set of primers framing the albumin gene.
  • the amplified product was then cloned into the Xho/EcoRI sites of the pcDNA3 vector to form pcDNA3 -Albumin.
  • RBD gene was amplified via PCR using the cDNA template of RBD and a set of primers framing the RBD gene.
  • the amplified gene product was then cloned into the EcoR I/Bam HI sites of pcDNA3- Albumin to create pcDNA3-Albumin-RBD (See Figures 1 and 2).
  • pcDNA3-Albumin-RBD See Figures 1 and 2.
  • the accuracy of the DNA constructs was confirmed by DNA sequencing.
  • pNGVL4a plasmids were substituted for the pcDNA3 plasmid and the fusion constructs for Hal-SARS-CoV-2, MERS-CoV RBD, and SARS-CoV RBD were constructed in the same manner.
  • the resultant pcDNA3-albumin-RBD or pcDNA3-RBD DNA constructs were transfected into HEK 293 cells in order to probe for resultant expressed protein.
  • HEK 293 cells were transfected with either pcDNA3-RBD or pcDNA3-albumin-RBD DNA constructs for 48 hours. A western blot was then completed to gauge protein expression, using GAPDH as a control.
  • the HEK 293 cells transfected with pcDNA3-albumin- RBD DNA construct produced the novel chimeric albumin-RBD protein with a molecular weight of around 50-75 kD.
  • HEK 293 cells transfected with the pcDNA3- RBD DNA construct produced the SARS-CoV2 RBD protein with a molecular size of around 25 kD (Fig 3B).
  • the amount of the chimeric albumin-RBD protein is significantly larger compared to that of RBD protein.
  • mice 6-8 week old female C57BL/6 mice (5 per group) were vaccinated with 20 ⁇ g/mouse of either pcDNA3-RBD or pcDNA3-Hal-RBD three times at 1-week intervals (Fig 4A).
  • mouse sera was harvested to analyze via ELISA for SARS-CoV2 RBD-specific antibody responses.
  • the mice vaccinated with pcDNA3 -Albumin-RBD had significantly higher levels of RBD-specific antibody responses compared to mice vaccinated with pcDNA3-RBD.
  • mice vaccinated with pcDNA3-RBD DNA vaccine showed no significant difference in SARS-CoV2 RBD-specific antibody levels when compared to the control group receiving no RBD DNA injection at all (Fig 4B).
  • Fig 4B The mice vaccinated with pcDNA3-RBD DNA vaccine showed no significant difference in SARS-CoV2 RBD-specific antibody levels when compared to the control group receiving no RBD DNA injection at all.
  • EXAMPLE 4 [0156] pcDNA3-Hal-RBD vaccination generate the highest levels of SARS-CoV2 RBD- specific antibody in vaccinated mice among all of the DNA vaccines tested. Characterization of Hal-RBD DNA vaccination by ELISA.
  • Fig. 5A 6-8 week old female C57BL/6 mice (5 mice/group) were vaccinated with 20 ⁇ g/mouse of pcDNA3-Sig/RBD, pcDNA3-Sig-IgG- RBD or pcDNA3-Hal-RBD DNA by intramuscular injection. The mice were boosted with the same regimen once with a 1-week interval. One week after last vaccination, serum were collected for ELISA.
  • Fig. 5A 6-8 week old female C57BL/6 mice (5 mice/group) were vaccinated with 20 ⁇ g/mouse of pcDNA3-Sig/RBD, pcDNA3-Sig-IgG- RBD or pcDNA3-Hal-RBD DNA by
  • Antibodies specific for the human SARS-CoV-2 Spike protein were analyzed by ELISA. Briefly, 1 ⁇ g/ml of human SARS-CoV-2 Spike protein in PBS were coated on BRANDplates® microplates for overnight. The next day wells were washed, blocked with eBioscienceTM ELISA/ELISPOT Diluent, and added serial dilutions of serum (all ranging from 1:100-1:10,240) for two hours at room temperature. Non-vaccinated mice serum as control. Goat anti-mouse IgG-HRP secondary antibody was added for one hour at 1:5000 dilution, followed by TMB substrate.
  • Vaccination with DNA encoding albumin-RBD elicits antibody responses recognizing the spike protein of SARS-CoV-2 expressed on the cell surface.
  • pcDNA3-Albumin-RBD generates a higher amount of RBD-Alb protein than pcDNA3-RBD generates its respective RBD protein in DNA transfected cells, suggesting pcDNA3-Albumin-RBD may be more immunogenic.
  • the inventors have shown that pcDNA3-Albumin-RBD DNA vaccine generates a significantly higher level of SARS- CoV-2 spike protein RBD specific antibody responses compared to pcDNA3-RBD DNA vaccine in vaccinated mice. The stronger immune response elicited by pcDNA3-Albumin- RBD indicates potential opportunity of the DNA vaccine in preventing COVID-19.
  • pcDNA3-Hal-RBD and pNGVL4a-Hal RBD expresses higher levels of secreted protein in a western blot.
  • HEK 293 cells were transfected with 10 ⁇ g of each DNA construct. After 48 hours transfection, cell lysates and supernatant were collected for Western blot analysis. Lane 1: cell pellet from pcDNA3-Hal- RBD transfected cells. Lane 2: cell pellet from pNGVL4a-Hal-RBD transfected cells. Lane 3: supernatant from pcDNA3-Hal-RBD transfected cells. Lane 4: supernatant from pNGVL4a - Hal-RBD transfected cells. The blot shows that the secreted protein in the supernatant of cell culture media had a higher concentration of expressed fusion protein than the transfected cells themselves (Lig. 7).
  • mice immunized with the synthetic fusion protein constructs generated antibodies to RBD.
  • C57BL/6 mice (5 per group) were vaccinated subcutaneously with 10 ⁇ g of RBD,
  • the immune system By extending the time the body is exposed to the RBD antigen, the immune system is provided with a longer span of time to properly mount an immune response and produce antibody when compared to a shorter period of antigen exposure.
  • linking albumin to RBD also promotes drainage of the RBD protein to the lymph nodes, as albumin is noted in its ability to target the lymph nodes, which is important for triggering immune responses (Wang, Y.,
  • lymph nodes contain high levels of immune cells, meaning that when RBD is targeted to the lymph nodes, the immune system can better mount an immune response. Without albumin, less RBD is drained to the lymph nodes, which results in a less pronounced antibody response. Finally, as described above, linking albumin to RBD resulted in increased expression the chimeric protein (Fig 3).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des constructions de vaccin à ADN comprenant la liaison de polynucléotides codant pour l'albumine humaine à des polynucléotides comprenant un ou plusieurs des domaines de liaison au récepteur (RBD) de la protéine de spicule du SARS-CoV-2 et/ou du MERS-CoV et/ou du SARS-CoV. L'invention concerne également des procédés de préparation des constructions de vaccin et leur utilisation dans la prophylaxie et le traitement d'infections par un coronavirus responsable du SRAS.
PCT/US2021/026268 2020-04-09 2021-04-07 Vaccin à adn innovant contre les sars-cov, sars-cov-2 et mers-cov WO2021207420A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202063007608P 2020-04-09 2020-04-09
US63/007,608 2020-04-09
US202063042260P 2020-06-22 2020-06-22
US63/042,260 2020-06-22

Publications (1)

Publication Number Publication Date
WO2021207420A1 true WO2021207420A1 (fr) 2021-10-14

Family

ID=78023934

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/026268 WO2021207420A1 (fr) 2020-04-09 2021-04-07 Vaccin à adn innovant contre les sars-cov, sars-cov-2 et mers-cov

Country Status (1)

Country Link
WO (1) WO2021207420A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023079528A1 (fr) * 2021-11-05 2023-05-11 King Abdullah University Of Science And Technology Compositions appropriées pour une utilisation dans un procédé pour déclencher une immunité protectrice croisée contre des coronavirus

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7572437B2 (en) * 2002-07-01 2009-08-11 Yan Fu Long acting human interferon analogs

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7572437B2 (en) * 2002-07-01 2009-08-11 Yan Fu Long acting human interferon analogs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KIM, D. GAMBHIRA, R. KARANAM, B. MONIE, A. HUNG, C.F. RODEN, R. WU, T.C.: "Generation and characterization of a preventive and therapeutic HPV DNA vaccine", VACCINE, ELSEVIER, AMSTERDAM, NL, vol. 26, no. 3, 21 December 2007 (2007-12-21), AMSTERDAM, NL , pages 351 - 360, XP022419014, ISSN: 0264-410X *
ZHANG HAIBO ET AL.: "Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target", INTENSIVE CARE MED., vol. 46, no. 4, 3 March 2020 (2020-03-03), pages 586 - 590, XP037077565, DOI: 10.1007/s00134-020- 05985-9 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023079528A1 (fr) * 2021-11-05 2023-05-11 King Abdullah University Of Science And Technology Compositions appropriées pour une utilisation dans un procédé pour déclencher une immunité protectrice croisée contre des coronavirus

Similar Documents

Publication Publication Date Title
KR101983989B1 (ko) 인플루엔자 바이러스 백신 및 이의 용도
TW202206444A (zh) 用於預防或治療SARS-CoV-2感染的疫苗組成物
EP2343081A1 (fr) Analogues d'interféron
US20230348880A1 (en) Soluble ace2 and fusion protein, and applications thereof
CN115246874A (zh) 一种重组新型冠状病毒s-rbd三聚体蛋白、其制备方法和应用
WO2022110099A1 (fr) Vaccins à coronavirus et leurs utilisations
McNee et al. Establishment of a pig influenza challenge model for evaluation of monoclonal antibody delivery platforms
US20230212231A1 (en) Severe acute respiratory syndrome coronavirus 2 (sars-cov-2) polypeptides and uses thereof for vaccine purposes
AU2014305658A1 (en) An antibody therapy for amyloid beta disease
WO2021262672A1 (fr) Constructions de rbd contre le sars-cov-2
ES2302774T3 (es) Anticuerpos contra el receptor de il-8 y sus usos terapeuticos.
WO2021207420A1 (fr) Vaccin à adn innovant contre les sars-cov, sars-cov-2 et mers-cov
KR20230084478A (ko) 면역원성 코로나 바이러스 융합 단백질 및 관련 방법
JP7009625B2 (ja) Ｈ３ｎ２亜型インフルエンザウイルスのヘマグルチニンタンパク質の突然変異体及びその使用
CN109422816B (zh) 一种靶向次级淋巴组织的肝癌疫苗
AU2021287508B2 (en) Fusion protein of pentamer and gB of cytomegalovirus, and vaccine containing said fusion protein
JP2018052953A (ja) インフルエンザウイルスワクチンおよびその使用
CN112574298B (zh) 一种抗西尼罗河病毒的人源中和性抗体
EP4232084A1 (fr) Vaccin
JP2024502783A (ja) コロナウイルスワクチン
KR101570453B1 (ko) 돼지 유행성 설사 바이러스의 재조합 스파이크 1 단백질을 발현하는 재조합 세포
JP2003528614A (ja) 肝炎ウイルスのｏｒｆ２のｎ末端領域に由来するプロセシング成分および抗原性ポリペプチドをコードする核酸構築物
CN105814077A (zh) 能够中和狂犬病毒的结合分子
WO2022148374A1 (fr) Anticorps 76e1 neutralisant entièrement humain à large spectre contre le coronavirus, et utilisation associée
KR102297440B1 (ko) 표적 세포 특이적으로 결합하여 다중 면역기능이 강화된 키메라항원 및 이의용도

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21784824

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21784824

Country of ref document: EP

Kind code of ref document: A1