WO2019143631A1 - Vaccins antiviraux universels - Google Patents

Vaccins antiviraux universels Download PDF

Info

Publication number
WO2019143631A1
WO2019143631A1 PCT/US2019/013692 US2019013692W WO2019143631A1 WO 2019143631 A1 WO2019143631 A1 WO 2019143631A1 US 2019013692 W US2019013692 W US 2019013692W WO 2019143631 A1 WO2019143631 A1 WO 2019143631A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
vuv
ira
dna
viral
Prior art date
Application number
PCT/US2019/013692
Other languages
English (en)
Inventor
Marek Malecki
Bianka Kathryn SAETRE
Original Assignee
Phoenix Biomolecular Engineering Foundation
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 Phoenix Biomolecular Engineering Foundation filed Critical Phoenix Biomolecular Engineering Foundation
Publication of WO2019143631A1 publication Critical patent/WO2019143631A1/fr

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • a Viral Universal Vaccine (VUV) composition is provided and comprises at least two domains, (1) Viral-attaching-molecule displayed by a cell (VAM), wherein the VAM is recombinant, soluble; and (2) an immunity redirecting antigen (IRA).
  • VAM Viral-attaching-molecule displayed by a cell
  • IRA immunity redirecting antigen
  • the IRA is recombinant and soluble.
  • the VAM is associated to the IRA.
  • a method of manufacturing the VUV composition can include synthesizing DNA sequences for joined VAM and IRA DNA coding sequences by the extension overlap with overhangs for Nhel and BamHI coded by the DNA sequences specified as SEQ ID NO 31-34; annealing them into ds DNA molecule coding VAM and IRA with insertion overhangs; cutting open the plasmid ds cc DNA pCMV- INS-SV40 SEQ ID No 30 with Nhel and BamHI; removing the DNA fragment INS from the plasmid pCMV-INS-SV40 specified as SEQ ID NO: 030; inserting the synthesized VAM-IRA DNA sequences coding the joined VAM and IRA directly or intercalated with the DNA merger specified as SEQ ID NO 80 into pCMV-opening-SV40 DNA vector to create a new vector pCMV-VAM-IRA-SV40; transfecting human myeloma cells
  • a method of manufacturing the VUV composition comprises synthesis and amplification of the DNA sequences encoding the VAMs specified as the DNA SEQ ID NO 1-29 with the DNA sequences encoding the overhangs specified as the DNA SEQ ID NO 31-34, synthesis and amplification of the DNA sequences encoding the IRA specified as SEQ ID NO 35-50 with the DNA sequences encoding the overhangs specified as DNA as SEQ ID NO 31-34, cloning the synthesized DNA sequences coding VAM per insertion into the pCMV-INS-SV40 DNA vector SEQ ID NO 30 and FIG.
  • a use of the composition provided herein for the treatment of a disease related to a virus occurring in a patient’s body is provided.
  • the virus is selected from the group consisting of: Corona Virus (CoV), Chickenpox Virus aka Varicella Zoster Virus aka HHV3 (VZV), Dengue Virus (DENV), Ebola Virus (EBOV), Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Herpes Virus 1/2 aka Herpes Simplex Virus aka HSV 1/2 (HHV1/2), Herpes Virus 4 aka EBV (HHV4), Herpes Virus 5 aka CMV (HHV5), Human G Virus aka HHV6 (HGV), Human Immunodeficiency Virus (HIV), Human Papilloma Viral (HPV), Influenza Viral (IV), RoseoloViral aka RosV (HHV7), Kaposi Sar
  • a method of treating a subject comprises providing a subject to be treated; and administering at least one of the compositions provided herein to the subject infected with a virus in an amount sufficient to allow binding of the VUV in the composition to the virus, that is determined by the diagnostic process assessing a Viral count of a patient.
  • a method of treatment comprises administering the composition provided herein to to a patient, by at least one of: A. intra venous (i.v.) infusion; B intra-lymphatic (i.l.) infusion; C sub-cutaneous (s.c.) injection; D intra-muscular (i.m.) injection; or E intra-cerebrospinal (i.c.s.) fluid injection.
  • A. intra venous (i.v.) infusion i.v.) infusion
  • B intra-lymphatic (i.l.) infusion C sub-cutaneous (s.c.) injection
  • D intra-muscular (i.m.) injection intra-muscular injection
  • E intra-cerebrospinal (i.c.s.) fluid injection i.c.s.
  • FIG. 1 Polymerase chain reaction (PCR) - based HIV counts as the function of time in blood of the HIV+ patient with no treatment versus treatment with Human Immunodeficiency Virus Viral Universal Vaccine (HIV-VUV) also known as the complex, artificial composition comprising: (1) virus entry receptor (VER); (2) vaccine engineered construct (VEC): that is having the specific composition: (1) Cluster of Differentiation 4 (CD4) - (2) Hepatitis B surface Antigen (HBsAg); thus collectively and specifically named CD4- HBsAg. It demonstrates rapid eradication of HIV by our UVV: CD4-HBsAg.
  • VER virus entry receptor
  • VEC vaccine engineered construct
  • CD4 Cluster of Differentiation 4
  • HBsAg Hepatitis B surface Antigen
  • FIG. 2 - Flow cytometry (FCM) - based CD4+ cells’ counts as the function of time in blood of the HIV+ patient with no treatment versus treatment with the complex composition of Human Immunodeficiency Virus - Viral Universal Vaccine (HIV-VUV) comprising: (1) virus attachment molecule (VAM) also known as virus entry receptor (VER) specifically being CD4; (2) immunogen redirecting antigen (IRA) also known as virus entry receptor (VEC), specifically being HBsAg; thus constituting HIV-VUV: CD4-HBsAg It demonstrates solid protection of the CD4+ cells’ population with HIV-VUV: CD4-HBsAg.
  • VAM virus attachment molecule
  • IRA immunogen redirecting antigen
  • VEC virus entry receptor
  • FIG. 3 The map of the ds cc DNA plasmid pCMV-INS-SV40. The sites for restriction enzymes are indicated. The unique, single cut sites for Nhel and BamHI flank the short ds DNA sequence INS, which is replaced by the DNA coding sequence of choice; in particular the DNA coding sequence for UVV.
  • UVVs e.g., recombinant, soluble CD4 locking HIV
  • the infecting viruses e.g., recombinant, soluble CD4 locking HIV
  • bringing upon them all the elements of the immune system either conditioned by previously administered vaccine (e.g., immunity induced by HBV vaccine) or induced by previously occurred natural infection, but now redirected toward these newly infecting viruses (e.g., HIV).
  • VUV Viral universal vaccines
  • VUV are composite, complex, polyfunctional, multidomain, recombinant genomically engineered entities.
  • the VUV comprise at least two main varieties, including: (1) comprise (la) virus attaching molecule (VAM) (e.g., CD4 for HIV) being proteins, glycoproteins, lipoproteins, that are naturally expressed from the DNA coding sequences postranslationally modified, often glycosylated or liposylated, while displayed by cells as cell surface proteins, lipoproteins, glycoproteins, which are often involved in the formation of the complexes promoting the viruses’ entry into the cells (e.g., CD4 with CXCR4,5), thus often collectively called virus entry receptors (VER); but for the purpose of this work, VAMs (also known as VERs) are transgenically expressed from the artificial DNA coding sequences specified herein as SEQ ID NO: 1-29, 51-79, 81-109,
  • prophylactic vaccines A problem with prophylactic vaccines are that new strains evading vaccination acquired immunity emerge; thus making those prophylactic vaccines ineffective (e.g., Influenza Virus Vaccine). For many deadly viruses, there are no prophylactic vaccines approved by health authorities at all (e.g., Human Immunodeficiency Virus aka HIV).
  • chemotherapeutics include: virus entry inhibitors (e.g., enfuvirtide or maraviroc), reverse transcriptase inhibitors (e.g., zidovudine or tenofovir), integrase inhibitors (e.g., elvitegravir), or maturation protease inhibitors (e.g., darunavir). They interfere with viruses’ propagation mechanisms.
  • virus entry inhibitors e.g., enfuvirtide or maraviroc
  • reverse transcriptase inhibitors e.g., zidovudine or tenofovir
  • integrase inhibitors e.g., elvitegravir
  • maturation protease inhibitors e.g., darunavir
  • the main problems with these methods of therapy are: mutations, mutagenesis, and adverse effects.
  • chemical small molecules repress the viruses’ replication by docking into the domains with enzymes of host cells to lock them.
  • nucleoside and nucleotide analogs prevent incorporation or do incorporate false nucleotides, which if erroneously repaired, become the sources of resistance.
  • all chemotherapeutics have very serious adverse effects. [7-11]
  • VAM virus attaching molecules
  • Anti-gpl20 antibodies served as alternative routes for blocking entry or guiding ADCC and CDC. [16, 17] They were often evaded due to HIV mutations. Moreover, they might even promote enhanced infections. As those trials were failing, the hybrid molecules comprising of CD4 and IgG were engineered, but did not attain therapeutic efficacy. [18-20] Additionally, the HIV-at-risk or -infected patients, who are exhausted by the disease and weakened by metabolism inhibiting drugs, have compromised ability to develop a de novo response. None of those attempts led to an effective therapy. [21, 22]
  • virus attaching/absorbing molecule e.g., CD4
  • VER virus-entry receptor
  • IRA immune response redirecting antigen
  • VEC vaccine engineered constructs
  • VUV Viral Universal Vaccines
  • UVV Universal Viral Vaccines
  • VAM-IRA Virus Attaching Molecule - Immunity Redirecting Antigen
  • VER- VEC Virus Entry Receptor - Vaccine Engineered Construct
  • AAVA- VEC anti-virus antibody - vaccine engineered construct
  • UVVs universal viral vaccines
  • They comprise of the two main domains: (1) de novo infecting virus docking domain and (2) microbial vaccine domain, which are conjugated by linkers or integrated as fusion proteins by genetic engineering.
  • UVV ultraviolet virus
  • a Viral Universal Vaccine (VUV) (also known as Universal Viral Vaccine (UVV)) composition
  • VAM Viral-attaching-molecule
  • IRA immunity redirecting antigen
  • UVV always comprises (1) VAM; and (2) IRA.
  • the VAM is fused to the IRA to create a new, composite fusion protein by genomic engineering, which is herein identified by the DNA coding sequences specified as SEQ ID NO: 51-79 and 81-109.
  • the VAM which in some embodiments is identified herein as the DNA coding sequences specified as SEQ ID NO 1-29, is conjugated to the IRA, which in some embodiments is identified herein as the DNA coding sequences specified as SEQ ID NO 35-50, to create a composite protein conjugate by click chemistry facilitated by bifunctional linkers, such as, but not limited to:l-Ethyl-3-(3- dimcthylaminopropyljcarbodiimidc (EDC), Pentynoic acid (PA), thiol-polyethylene-glycol- azide (TPA), Poly-triethyleneacylhydrazide-dithiol (PTAD), Poly-ethylene-acylhydrazide- dithiol (PEAD), amino-propyl-tri-ethoxy- silane (APTES), N-succinimidyl-(2— 66-pyridyl- dithiol-propionate) (SPDP), and succinimid
  • the VAM composition is selected from the group of DNA coding sequences listed (SEQ ID NO: 1 - SEQ ID NO 29).
  • the VAM is the protein, or a portion of glycoprotein or lipoprotein, thereof, that is encoded by (SEQ ID NO: 1 - SEQ ID NO 29).
  • the UVV composition contains VAM as one of its two main domains, that is selected from the group of DNA coding sequences listed (SEQ ID NO: 051 - SEQ ID NO 79 and SEQ ID NO: 081 - SEQ ID NO: 109).
  • VAM domain resulting from the expression, splicing, variation, modification of the above aforementioned DNA coding sequences in the specific expression system as the protein, glycoprotein, or lipoprotein, and alike having identical immunogenic and virus harboring features.
  • the VUV composition is attained by genomic engineering and is accomplished by cloning and transgenic expression relying upon the plasmid pCMV-INS-SV40 identified by the DNA coding sequence specified as SEQ ID NO: 30) while employing the engineered constructs’ the DNA plasmids’ and DNA inserts’ unique overhangs; that are created to facilitate the unique insertion sites by identified as the DNA coding sequences unique for restriction enzymes Nhel and BamHI specified as SEQ ID NO: 31-34).
  • the VUV composition further comprises a chemical linker that conjugates the VAM to the IRA, and the chemical linker is selected from the group comprising at least one of: l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), Pentynoic acid (PA), thiol-polyethylene-glycol-azide (TPA), Poly-triethyleneacylhydrazide-dithiol (PTAD), Poly-ethylene-acylhydrazide-dithiol (PEAD), amino-propyl-tri-ethoxy-silane (APTES), N-succinimidyl-(2— 66-pyridyl-dithiol-propionate) (SPDP), and succinimidyl 4- (Nmaleimidomethyl) cyclohexane- l-carboxylate) (SMCC).
  • EDC l-Ethyl-3-(3-dimethylaminopropyl
  • the VUV is expressed as a fusion protein comprising coding sequences for VAM and IRA (SEQ ID NO: 51-79 and 81-109) and the engineered DNA constructs that are encoding them employ sequence overhangs (SEQ ID NO: 31-34) that facilitated insertions into the plasmid pCMV-INS-SV40 (SEQ ID NO: 30).
  • the IRA is selected from the group of the DNA sequences listed for HBV L, M, S - HBsAg , HPV Ll, Ll-2, their fragments, or other viral antigens (e.g., SEQ ID NO: 35 - SEQ ID NO 50), or a protein encoded thereby.
  • the VUV is expressed as a fusion protein.
  • the VUV is transgenically expressed in Human myelomas.
  • the VUVs suitable for therapy of the specific virus is selected by pairing of the unique, specific VAM domain of the VUV with the specific virus.
  • Such unique VUV are identified by its unique VUV’s VAM domain, which is identified by the DNA coding sequences specified as (SEQ ID NO: 051 - SEQ ID NO: 079 and SEQ ID NO: 081- SEQ ID NO: 109)
  • the pairing system of the virus to be treated with the appropriate VUV containing the unique for attaching to that virus VAM domain, that is identified by the unique coding sequence of DNA for VAM, that is a part of the entire coding sequence of DNA for VUV, is summarized below.
  • the Viral Universal Vaccine comprises a virus absorbing molecule displayed by cells (VAM) domain: DPP4 or a binding fragment thereof and the immunity redirecting antigen (IRA) domain: Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 051).
  • the Viral Universal Vaccine comprises HS or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 052).
  • HBsAg Hepatitis B Surface Antigen
  • the Viral Universal Vaccine comprises C) the VUV comprises NK1R or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 053).
  • HBsAg Hepatitis B Surface Antigen
  • the Viral Universal Vaccine comprises TIM1 or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 054).
  • HBsAg Hepatitis B Surface Antigen
  • the Viral Universal Vaccine comprises HAVCR1 or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 055).
  • HBsAg Hepatitis B Surface Antigen
  • the Viral Universal Vaccine comprises NTCP or a binding fragment thereof, and the IRA domain comprises Human Papilloma Viral 16 Antigen (exceptionally HBsAg substituted with HPVl6Ll-2Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 056).
  • the Viral Universal Vaccine comprises the VUV comprises CLEC4M or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 057).
  • HBsAg Hepatitis B Surface Antigen
  • the Viral Universal Vaccine comprises CR1 or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 058).
  • HBsAg Hepatitis B Surface Antigen
  • the Viral Universal Vaccine (VUV) comprises CD4 or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 059- 064).
  • the Viral Universal Vaccine (VUV) comprises EphA2R or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 065).
  • the Viral Universal Vaccine comprises CD81 or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 066- 067).
  • HBsAg Hepatitis B Surface Antigen
  • the Viral Universal Vaccine comprises ITGA6 or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 068-071).
  • HBsAg Hepatitis B Surface Antigen
  • the Viral Universal Vaccine comprises nectin or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 072).
  • the Viral Universal Vaccine comprises sialylated glycan (SG) or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 073).
  • SG sialylated glycan
  • HBsAg Hepatitis B Surface Antigen
  • the Viral Universal Vaccine comprises CD 155 or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 074-075).
  • HBsAg Hepatitis B Surface Antigen
  • the Viral Universal Vaccine comprises a2b1- ITG or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 076).
  • HBsAg Hepatitis B Surface Antigen
  • the Viral Universal Vaccine (VUV) comprises AchR or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 077- 078).
  • the Viral Universal Vaccine (VUV) comprises CS or a binding fragment thereof, and the IRA domain comprises Hepatitis B Surface Antigen (HBsAg) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 079).
  • the Viral Universal Vaccine comprises a virus attaching/absorbing molecule displayed by cells (VAM) domain: DPP4 or a binding fragment thereof and the immunity redirecting antigen (IRA) domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 081).
  • the Viral Universal Vaccine comprises HS or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 082).
  • the Viral Universal Vaccine comprises NK1R or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 083).
  • the Viral Universal Vaccine comprises TIM1 or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 084).
  • the Viral Universal Vaccine comprises HAVCR1 or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 085).
  • the Viral Universal Vaccine comprises NTCP or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 086).
  • the Viral Universal Vaccine comprises CLEC4M or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 087).
  • the Viral Universal Vaccine comprises CR1 or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 088).
  • the Viral Universal Vaccine (VUV) comprises CD4 or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 089-094).
  • the Viral Universal Vaccine (VUV) comprises EphA2R or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 095).
  • the Viral Universal Vaccine (VUV) comprises CD81 or a
  • binding fragment thereof, and the IRA domain HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 096-097).
  • the Viral Universal Vaccine comprises ITGA6 or a binding fragment thereof, and the IRA domain: exceptionally HPV Ll Antigen substituted with HBsAg (wherein the DNA coding sequence for the entire VUV is SEQ ID NO 098-101).
  • the Viral Universal Vaccine comprises nectin or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 102).
  • the Viral Universal Vaccine comprises SG or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 103).
  • the Viral Universal Vaccine comprises CD155 or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 104-105).
  • the Viral Universal Vaccine comprises a2b1- ITG or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 106).
  • the Viral Universal Vaccine comprises AchR or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 107-108).
  • the Viral Universal Vaccine comprises CS or a binding fragment thereof, and the IRA domain: HPV Ll Antigen (Ag) (wherein the DNA coding sequence for this VUV is specified as SEQ ID NO 109).
  • any of the VUV or its components can employ any one or more of the sequences specified by a SEQ ID NO: provided herein.
  • the sequences can be a variant of the sequence specified herein, and can be at least 80, 85, 90, 95, 96, 97, 98, 99, 99.9 or greater identical to the sequence specified in the SEQ ID NO:.
  • any adequate percent identical sequence can be used, as long as the VUV, IRA, and VAM function as described herein; therefore despite the alteration in process of making they deliver identical immunological and molecules effects.
  • the Viral Universal Vaccine is manufactured by any of a variety of biotechnology manufacturing techniques.
  • the method comprises one or more of the steps of synthesizing DNA sequences for joined VAM and IRA as in SEQ ID NO 51-109 by the extension overlap with overhangs for Nhel and BamHI (SEQ ID NO 31-34) into the cc DS DNA construct pCMV-INS-SV40 (SEQ ID NO: 30), transfecting Human Myeloma cells, and transgenic expression; the VUV composition attaining methods that involve: synthesis of the DNA constructs or reverse transcription and amplification of cds, annealing them into ds DNA molecule coding VAM and IRA with insertion overhangs; cutting open the plasmid pCMV-INS-SV40 SEQ ID No 30 with Nhel and BamHI; removing the DNA fragment INS from the plasmid pCMV-INS-SV40; inserting the synthesized V
  • cloning is achieved by synthesis of DNA fragments for joined VAM and IRA wherein the DNA coding sequence is specified as SEQ ID NO 051- 079 and 081-109 by extension overlap with overhangs wherein the DNA coding sequence is specified as SEQ ID NO: 031 - SEQ ID NO: 034 and inserting into the open plasmid wherein the DNA coding sequence is specified as SEQ ID NO 030.
  • the method of manufacturing the VUV composition comprises one or more of the following steps: synthesis and amplification of the DNA sequences encoding the VAM specified as SEQ ID NO 001-029 with overhangs specified as the DNA coding sequences SEQ ID NO 031-034, synthesis and amplification of the DNA sequences encoding the IRA specified as the DNA coding sequences specified as SEQ ID NO 035-050 with overhangs as the DNA coding sequences specified as SEQ ID NO 031-034, cloning the synthesized DNA sequences coding VAM per insertion into the pCMV-INS-SV40 DNA vector specified as SEQ ID NO 030 to create a new vector pCMV-VAM-SV40; cloning the synthesized DNA sequences coding IRA per insertion into the pCMV-INS-SV40 DNA vector SEQ ID NO 30 to create a new vector pCMV-IRA-SV40; transfecting human myeloma cells with the pCMV-
  • SMCC maleimidomethyl cyclohexane- l-carboxylate
  • functionalization of any of IRA with any of these linkers and click chemistry linking any of the VAM expressed from the DNA coding sequences specified as SEQ ID NO 001-029 with the IRA expressed from the DNA coding sequences specified as SEQ ID NO 035-050 using any combination of the linkers provided herein.
  • click chemistry linking any of the VAM expressed from the DNA coding sequences specified as SEQ ID NO 001-029 with the IRA expressed from the DNA coding sequences specified as SEQ ID NO 035-050 using any combination of the linkers provided herein.
  • Other methods of making the constructs can also be employed.
  • the VAM or their fragments is selected from the group consisting of at least one of: DPP4, HS, NK1R, TIM1, HAVCR1, NTCP, CLEC4M, CR1/2, CD4, EphA2R, CD81, ITGA6, nectin, SG, CD155, a2pl-ITG, AchR, and CS.
  • the VAM is selected from the group consisting of: dipeptidyl peptidase 4 (DPP4), heparan sulfate (HS), neurokinin 1 receptor (NK1R), T-cell immunoglobulin and mucin domain (TIM1), Hepatitis A Viral cellular receptor 1 (HAVCR1), S odium- taurocholate co-transporting polypeptide (NTCP), C-type lectin domain family 4 (CLEC4M), complement receptor 1 (CR1), cluster of differentiation 4 (CD4), ephrin A2 receptor (EphA2R), cluster of differentiation 81 (CD81), integrin alpha-6 ( ITGA6), nectin (Nec), sialylated glycans (SG), cluster of differentiation 155 (CD155), integrin alpha-beta (a.2b 1 -ITO), acetylcholine receptor (AchR), and chondroitin sulfate (CS).
  • DPP4 dipeptidyl
  • the composition comprises chemical bifunctional linkers: l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), Pentynoic acid (PA), thiolpolyethylene-glycol-azide (TPA), Poly-triethylene-acylhydrazide-dithiol (PTAD), Poly- ethylene-acylhydrazide-dithiol (PEAD), amino-propyl-triethoxy-silane (APTES), N succinimidyl 3 (2-pyridyldithio) propionate, N-succinimidyl-(2-pyridyl-dithiol-propionate) (SPDP), (succinimidyl 4-(Nmaleimidomethyl) cyclohexane- l-carboxylate) (SMCC).
  • EDC l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • PA
  • the method further comprises making a functionalized VAM (isolated by biochemical methods or transgenically expressed from the DNA coding sequence for VAM) wherein the DNA coding sequence for the selected VAM is specified as SEQ ID NO: 001-029 and a functionalized IRA wherein the DNA coding sequence for the selected IRA is specified as SEQ ID NO 035-050 the method employing a chemical linker provided herein followed by click chemistry conjugating covalently functionalized VAM (wherein the DNA coding sequence for the selected VAM is specified as SEQ ID NO: 001-029) and functionalized IRA (wherein the DNA coding sequence for the selected IRA is specified as SEQ ID NO 035-050); thus creating monomeric VUV composition.
  • a functionalized VAM isolated by biochemical methods or transgenically expressed from the DNA coding sequence for VAM
  • the composition includes at least one of: l-Ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC), Pentynoic acid (PA), thiolpolyethylene-glycol- azide (TPA), Poly-triethylene-acylhydrazide-dithiol (PTAD), Polyethylene-acylhydrazide- dithiol (PEAD), amino-propyl-tri-ethoxy- silane (APTES), SPDP N succinimidyl 3 (2- pyridyldithio)propionate, N-succinimidyl-(2-pyridyl-dithiolpropionate) (SPDP), (and/or succinimidyl 4-(Nmaleimidomethyl) cyclohexane- l-carboxylate) (SMCC).
  • EDC Pentynoic acid
  • TPA thiolpolyethylene-glycol- azide
  • PTAD Poly-triethylene
  • the method of making further comprises sterilizing the composition in a sealed vial.
  • any of the compositions provided herein can be used in the treatment of a disease.
  • a use of the composition can be for the treatment of a disease related to a virus occurring in a patient’s body.
  • the virus is selected from the group consisting of: Corona Virus (CoV), Chickenpox Virus aka Varicella Zoster Virus aka HHV3 (VZV), Dengue Virus (DENV), Ebola Virus (EBOV), Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Herpes Virus 1/2 aka Herpes Simplex Virus aka HSV 1/2 (HHV1/2), Herpes Virus 4 aka EBV (HHV4), Herpes Virus 5 aka CMV (HHV5), Human G Virus aka HHV6 (HGV), Human Immunodeficiency Virus (HIV), Human Papilloma Virus (HPV), Influenza Virus
  • Corona Virus Corona Virus
  • the use includes at least one of the following pairings of any of the infecting Virus and any of the Virus attaching/absorbing molecule (VAM) domain of the specific VUV is necessary for pursuing their therapeutic utility: DPP4 (wherein the DNA coding sequence for the VUV containing the required pairing VAM domain is specified as SEQ ID NO: 051 and 081) for CoV, heparan sulfate (HS)/neurokinin receptor (NK1R) (wherein the DNA coding sequence for the VUV containing the required pairing VAM domain is specified as SEQ ID NO: 052-053 and 082-083) for VZV, TIM-l (wherein the DNA coding sequence for the VUV containing the required pairing VAM domain is specified as SEQ ID NO: 054 and 084) for DENV, TIM-l (wherein the DNA coding sequence for the VUV containing the required pairing VAM domain is specified as SEQ ID NO: 004 and 084) for EBOV, TIM1/
  • a method of treating a subject comprises providing a subject to be treated and administering at least one of the compositions provided herein to the subject infected with a virus in an amount sufficient to allow binding of the VUV in the composition to the virus, that is determined by the diagnostic process assessing a Viral count of a patient.
  • a method of treatment comprises administering the composition as provided herein to a patient, by at least one of: A. intra- venous (i.v.) infusion; B intra-lymphatic (i.l.) infusion; C sub-cutaneous (s.c.) injection; D intra-muscular (i.m.) injection; or E intra-cerebrospinal (i.c.s.) fluid injection.
  • the subject to be treated is identified as having a viral infection
  • the virus is selected from the group consisting of: Corona Virus (CoV), Chickenpox Virus aka Varicella Zoster Virus aka HHV3 (VZV), Dengue Virus (DENV), Ebola Virus (EBOV), Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Herpes Virus 1/2 aka Herpes Simplex Virus aka HSV 1/2 (HHV1/2), Herpes Virus 4 aka EBV (HHV4), Herpes Virus 5 aka CMV (HHV5), Human G Virus aka HHV6 (HGV), Human Immunodeficiency Virus (HIV), Human Papilloma Virus (HPV), Influenza Virus (IV), RoseoloVirus aka RosV (HHV7), Kaposi Sarcoma Associated aka KSAV (HHV8), M
  • the VUV comprises a VAM-IRA as chemical conjugates of the functionalized VAM and the functionalized IRA. This can be used for one of the denoted Viral infections.
  • the specific VUV is selected for the specific virus infection based upon the selection of the VAM being capable of harboring the specific virus. This selection is based upon pairing of the VAM domain of the VUV with the specific virus as outlined below.
  • the pairing VAM is DPP4 (encoded by the DNA sequence SEQ ID NO: 001), or a binding fragment thereof, for CoV.
  • the pairing VAM is (HS)/neurokinin receptor (NK1R) (encoded by the DNA sequence SEQ ID NO: 2/3), or a binding fragment thereof, for VZV.
  • the pairing VAM is TIM-l(encoded by the DNA sequence SEQ ID NO: 004), or a binding fragment thereof, for DENV.
  • the pairing VAM is TIM-l(encoded by the DNA sequence SEQ ID NO: 004), or a binding fragment thereof, for EBOV.
  • the pairing VAM isTIMl/HAVCRl(encoded by the DNA sequence SEQ ID NO: 004/005), or a binding fragment thereof, for HAV.
  • the pairing VAM is: NTCP (encoded by the DNA sequence SEQ ID NO: 006), or a binding fragment thereof, for HBV.
  • the pairing VAM is: TIMl(encoded by the DNA sequence SEQ ID NO: 004), or a binding fragment thereof, for HCV.
  • the pairing VAM is: heparan sulfate (HS)/CLEC4M (encoded by the DNA sequence SEQ ID NO: 002/007), or a binding fragment thereof, for HHV4.
  • the pairing VAM is: CRl(encoded by the DNA sequence SEQ ID NO: 008), or a binding fragment thereof, for HHV5.
  • the pairing VAM is: CD4 (encoded by the DNA sequence SEQ ID NO: 009-014), or a binding fragment thereof, for HHV7.
  • the pairing VAM is : EphA2R(encoded by the DNA sequence SEQ ID NO: 015), or a binding fragment thereof, for HHV8.
  • the pairingVAM is: CD81 (encoded by the DNA sequence SEQ ID NO: 016-017), or a binding fragment thereof, for HGV.
  • the pairing VAM is: CD4 (encoded by the DNA sequence SEQ ID NO: 009-014), or a binding fragment thereof, for HIV.
  • the pairing VAM is: ITGA6 (encoded by the DNA sequence SEQ ID NO: 018-021), or a binding fragment thereof, for HPV.
  • the pairing VAM is: nectin (encoded by the DNA sequence SEQ ID NO: 022), or a binding fragment thereof, for HSV1/2.
  • the pairing VAM is: sialylated glycans (SG) (encoded by the DNA sequence SEQ ID NO: 023), or a binding fragment thereof, for IV.
  • the pairing VAM is: TIM- 1 (encoded by the DNA sequence SEQ ID NO: 004), or a binding fragment thereof, for MuV.
  • the pairing VAM is: TIM-l (encoded by the DNA sequence SEQ ID NO: 004), or a binding fragment thereof, for MeaV.
  • the pairing VAM is: CD 155 (encoded by the DNA sequence SEQ ID NO: 024-025), or a binding fragment thereof, for PV.
  • the pairing VAM is: a2p i-intcgrin (encoded by the DNA sequence SEQ ID NO: 026), or a binding fragment thereof, for RoV. [0109] In some embodiments, the pairing VAM is: AchR (encoded by the DNA sequence SEQ ID NO: 027-028), or a binding fragment thereof, for RaV.
  • the pairing VAM is: CD4 (encoded by the DNA sequence SEQ ID NO: 009-014), or a binding fragment thereof, for RuV HIV.
  • the pairing VAM is: chondroitin sulfate (CS) (encoded by the DNA sequence SEQ ID NO: 029), or a binding fragment thereof, for VARV.
  • CS chondroitin sulfate
  • the pairing VAM is: TIM 1 (encoded by the DNA sequence SEQ ID NO: 004), or a binding fragment thereof, for YFV.
  • the pairing VAM is: TIM-l (encoded by the DNA sequence SEQ ID NO: 004), or a binding fragment thereof, for ZikV.
  • the VUV: VAM-IRA is a fusion protein that can be expressed transgenically from a synthetic construct, that can be used for one of the denoted Viral infections
  • the chosen VUV is selected by pairing of the specific VAM domain within the VUV composite molecule is: DPP4 (wherein the entire VUV is encoded by the DNA sequence SEQ ID NO: 051 and 081), or a binding fragment thereof, for CoV, heparan sulfate (HS)/neurokinin receptor (NK1R) (wherein the entire VUV is encoded by the DNA sequence SEQ ID NO: 052-53 and 082-083), or a binding fragment thereof, for VZV, TIM-l(wherein the entire VUV is encoded by the DNA sequence SEQ ID NO: 054 and 084), or a binding fragment thereof, for DENV, TIM-l (wherein the entire VUV is encoded by the DNA sequence SEQ ID NO: 054 and 084), or a binding fragment thereof
  • the VUV comprises an antibody or its fragments Fab, (Fab)2, scFv, dcFv, and alike against an envelope or capsid molecule of a virus in the subject (anti-virus antibody aka AVA) where the AVA is substituting VAM in VUV composition, in it the AVA binds to the Viral antigen on viruses selected from the group consisting of: 1. California Encephalitis Virus, 2. Chickengunya Virus, 3. Corona Virus, 4. Dengue Virus, 5. Ebola Virus, 6. Hanta Virus, 7. Hepatitis Virus, 8. Hepatitis B Virus, 9. Hepatitis C Virus, 10. Hepatitis D Virus, 11. Human Herpes Virus, 12.
  • viruses selected from the group consisting of: 1. California Encephalitis Virus, 2. Chickengunya Virus, 3. Corona Virus, 4. Dengue Virus, 5. Ebola Virus, 6. Hanta Virus, 7. Hepatitis Virus, 8. Hepatitis B Virus, 9. Hepati
  • Human Herpes Virus 13. Human Herpes Virus 3 - Chickenpox - Varicella, 14. Human Herpes Virus 4 - EBV, 15. Human Herpes Virus 5 - CMV, 16. Human Herpes Viral 6, 17. Human Herpes Virus 7, 18. Human Herpes Virus 8, 19. Human Immunodeficiency Virus, 20. Human Parainflunza Virus type 1, 21. Human Parainflunza Virus type 2, 22. Human Parainflunza Virus type 3, 23. Human Parainflunza Virus type 4, 24. Human Respiratory Syncytial Virus - HRSV, 25. Influenza A Virus, 26. Influenza B Virus, 27. Influenza C Virus, 28.
  • Lymphocytic Choriomeningitis Virus 29. Marburg Virus, 30. Mumps Virus, 31. Measles Virus, 32. Rabies Virus, 33. Rubella Virus, 34. Smallpox Virus, 35. West Nile Virus, 36. Yellow Fever Virus, or 37. Zika Virus.
  • the VUV comprises a VAM that binds to one or more of the following Viruses: 1. California Encephalitis Virus, 2. Chickengunya Virus, 3. Corona Virus,, 4. Dengue Virus, 5. Ebola Virus, 6. Hanta Virus, 7. Hepatitis Virus, 8. Hepatitis B Virus, 9. Hepatitis C Virus, 10. Hepatitis D Virus, 11. Human Herpes Virus, 12. Human Herpes Virus, 13. Human Herpes Virus 3 - Chickenpox - Varicella, 14. Human Herpes Virus 4 - EBV, 15. Human Herpes Virus 5 - CMV, 16. Human Herpes Viral 6, 17. Human Herpes Virus 7, 18.
  • Human Herpes Virus 8 19. Human Immunodeficiency Virus, 20. Human Parainflunza Virus type 1, 21. Human Parainflunza Virus type 2, 22. Human Parainflunza Virus type 3, 23. Human Parainflunza Virus type 4, 24. Human Respiratory Syncytial Virus - HRSV, 25. Influenza A Virus, 26. Influenza B Virus, 27. Influenza C Virus, 28. Lymphocytic Choriomeningitis Virus, 29. Marburg Virus, 30. Mumps Virus, 31. Measles Virus, 32. Rabies Virus, 33. Rubella Virus, 34. Smallpox Virus, 35. West Nile Virus, 36. Yellow Fever Virus, or 37. Zika Virus.
  • VAMs identified in the disclosure by the DNA sequences specified as SEQ ID NO: 001-029 could be linked to any of the IRAs identified by the DNA coding sequences specified as SEQ ID NO: 35-50 by employing the appropriate combination of linkers listed in herein
  • UVVs genomically engineered universal viral vaccines
  • the following examples related to HIV and the HIV-infected cells are presented herein.
  • the paradigm of the presented invention is easily applicable to all viruses’-induced diseases, while all the procedures described herein are easily performed by any investigators skilled in art, what surely leads to reproducing composition, as well as attaining efficacy by administration of the methods outline herein.
  • these compositions, processes, and methods are easily applicable to other viruses, other viruses’-infected cells, and therapy of patients infected by other viruses.
  • HIV Human Immunodeficiency Virus
  • HSV Herpes Simplex Virus
  • CMV Cytomegalo-virus
  • HBsAg HBsAg was isolated from the patients suffering from acute hepatitis B: either from the blood by PEG fractionations or from the liver biopsies by CsCl gradient centrifugation. To assure exact immunogenic compatibility with the immunity induced by vaccinations with the FDA approved HBsAg, which were produced in yeast, the HBsAg in this project were also generated in yeast as originally described. Biotechnology of the recombinant HBsAg was pursued based upon the published DNA coding sequence that are specified as SEQ ID NO 35-49 and HPV16 Ll as SEQ ID NO 50.
  • Hepatitis B virus like particles were initially synthesized in yeast— Saccharomyces cerevisiae as originally described.
  • the expression plasmid pHBS- 16 included the HBsAg surface antigen (HBsAg) controlled by the yeast alcohol dehydrogenase (ADHI) promoter through introduced by EcoRI restriction sites into the DNA construct of the pBR322 plasmid. That followed by yeast replication origin, yeast trpl gene. This biotechnology was later modified to be pursued in Pichia pastoris.
  • yeast cultures of Pichia pastoris were grown at 30 °C in rich medium (YPD; 1% yeast extract, 2% bactopeptone, 2% glucose) initially and shifted either to synthetic media (YNM, 0.67% yeast nitrogen base supplemented with 0.5% (v/v) methanol) for immunoprecipitation and immunofluorescence experiments, or to mineral media (MMOT, 0.2% (v/v) oleate and 0.02% (v/v) Tween-40) for fractionation studies. All the protocols’ products— HBsAg VLPs were referenced and validated to the FDA approved and the CDC recommended Engerix B and Recombivax and the antiHBV antibody titer assays
  • Blood was drawn according to the standard clinical procedure by venipuncture into citric acid / dextran receiving buffer. It was stored at 4 deg. C until further processing.
  • the blood was depleted of fibrinogen and calcium (later re-adjusted to physiological levels).
  • Erythrocyte-free blood was prepared by magnetic apheresis aided by antibodies conjugated with magnetic beads.
  • Plasma was prepared by simple sedimentation and collecting supernatant or alternatively sampled during plasmapheresis.
  • T cell fractions were prepared by activated sheep erythrocyte resetting. B cells were removed by complement receptor activated lysis.
  • Desired cell fractions were enriched by FACS after labeling with fluorescent antibodies followed or by MACS after labeling with superparamagnetic antibodies.
  • Plasma and B cells were isolated from erythrocyte-free blood by MACS after labeling with anti-CD 19 and anti-CD20 magnetic antibodies. [29, 30].
  • Lymph was acquired according to the standard clinical procedure during surgeries on open abdomens.
  • the viruses and cells from lymph were prepared according to the protocols for erythrocyte-free blood.
  • the samples were prepared by cryo-biobanking.
  • the samples were equilibrated with 10% DMSO in the patients’ serum at 4 deg C. They were frozen according to the gradual lowering temperature down to -35 deg C, -70 deg C, -196 deg C per 24 h on each step.
  • Crystal-immobilization apparatus was constructed based upon the NSF Grant to MM). These samples were stored indefinitely without compromised quality. When needed, these samples were thawed according to the reverse-to-freezing protocol for processing. Alternatively, the total RNA was prepared and either stored as such or converted into cDNA for storing and / or shipping. [31-32]
  • HIV+ patients viremia in blood, lymph, or plasma were tested by reverse transcription and polymerase chain amplification of the sequences nested by the primers designed upon the published HIV-l sequences in PubMed / GenBank from the samples generated by isolation of total RNA.
  • GenBank: X01762.1 ; GenBank: M17451.1; GenBank: M27323.1 [GenBank: X01762.1 ; GenBank: M17451.1; GenBank: M27323.1 ]
  • the samples samples acquired from the HIV+ patients, whose HIV count were adjusted to the experimental levels were spiked with GEM-SPMs as indicated.
  • the infected samples in vials were incubated at the desired time at 37 deg. C and 5 % C02 while on the gyroscopic tables.
  • NP40 NP40
  • RIPA immunoprecipitation
  • HIV was aspirated from the peritoneal or pleural effusions of the HIV+ oncology patients, who were diagnosed with Kaposi sarcoma. Of these sterile effusions, 100 microliters containing the HIV copy number determined by RT-PCR, was injected into in the H9 culture (ATCC) and propagated strictly according to the published protocols. [27-28]
  • HIV glycoproteins gpl60 as well as its proteolytic fragments gpl20, gp4l, as well as p24, were prepared from the effusions of the HIV+ oncology patients admitted primarily for treatment of Kaposi sarcomas. Templates were generated by two ways. Total mRNA was isolated from HIV+ producing CD4+ lymphocytes of the HIV+ patients as described.
  • the gpl60, as well as it fragments for gpl20 and gp4l mRNA was converted from total mRNA into ds cDNA by reverse transcription and polymerase chain reaction aided by the primers for gpl60, gpl20, and gp4l having sequences imported from GenBank (NCBI), primers designed on 5Prime (NIH), and synthesized on oligonucleotide synthesizers (Applied Biosystems).
  • GenBank GenBank
  • NASH primers designed on 5Prime
  • oligonucleotide synthesizers Applied Biosystems.
  • the yielded amplicons were inserted into plasmids comprising CMV promoters and metal binding coding sequences or bifunctional linker binding domains as in the details described elsewhere.
  • pCMV-MBS-p24 were electroporated into the Human myeloma cells in cultures established from effusions of the oncology patients diagnosed with Multiple myelomas. The culture media were based upon RPMI1640 supplemented with effusion fluids rather than bovine sera. Alternatively, the HIV was isolated and propagated directly from the patients’ samples.
  • the plasma and B cells were selected from blood of HIV+ patients by MACS and FACS using anti-CDl9 and anti-CD20 magnetic antibodies. Total mRNA was isolated and stored. [30] After importing the human HC and LC sequences [Kabat], the primers were designed with the aid of 5Prime software [NIH] for heavy and light variable chains and synthesized. After reverse transcription, these primers primers served to create cDNA templates, which were cloned into the plasmid containing CMV promoter and metal binding domains. [29] Plasmids were propagated in Escherichia coli grown in the Luria-Bartani media in cultures maintained on the shakers at 37 deg. C.
  • the plasmids were electroporated into the cells in cultures of myelomas, which were established from the effusions of the oncology patients, who were diagnosed with Multiple myelomas.
  • the cells were cultured in RPMI1640 media, but modified on such a way that they were supplemented with the supernatants of the patients’ effusions, but not bovine sera. Later, the cells were conditioned to grow in serum- free media in roller bottles at 37 deg C and 5% C02. Therefore, supernatant could be easily used to test specificity of secreted antibodies.
  • Metal binding domains facilitated rendering them superparamagnetic and / or fluorescent, if MBS were saturated with Eu or Tb.
  • the metal binding domains were chosen to provide strong direct binding to Ni, Co, Fe, Au, Si02, as well as activated shells of core-shell superparamagnetic molecules as outlined herein.
  • Human CD4 - HIV Virus Attaching Molecule VAM
  • VERs Virus Entry Receptors
  • Human CD4 was manufactured on two ways: cell lysis immunoprecipitation - immunoblotting and genomic isolation - amplification - expression.
  • T lymphocytes from healthy volunteers were initially selected by sheep erythrocyte resetting precipitation followed by B cell complement lysis. Later, these fractions were enriched by CD4+ selection with our anti- CD4 superparamagnetic antibodies, raised from the plasma and B cells of the anti-CD4+ patient, on MACS or our anti-CD4 fluorescent antibodies by FACS.
  • the enriched fractions of the CD4+ cells were lysed with NP40.
  • the soup was mixed with our anti-CD4, superparamagnetic, genomically engineered anti-CD4 antibodies, incubated for 1 h at 4 deg. C, and inserted into magnetic field at room temperature for 15 minutes. Diamagnetic content was rinsed off, while in the field. The fraction retained by anti-CD4 was released after the magnetic field ceased. Aliquots of this fraction were electrophoresed on PAGE and transferred onto the PVDF membranes (Amersham). The immunoblots were tested by the standard OKT4 antibody produced by the hybridoma cell line (ATCC), which was initially grown in the recommended cell culture conditions (ATCC)., but which we modified so the cells were grown in sera- free media as described. [21]
  • the enriched fractions of the CD4+ cells were lysed with RIPA.
  • the lysates were electrophoresed by PAGE and validated as immune-precipitated ones.
  • CD4 mRNA main transcripts were imported from GenBank (NCBI) specified by SEQ ID NO: 009-014.
  • the primers flanking the CD4 coding sequence were designed with the aid of 5Prime software (NIH) and synthesized (Applied Biosystems). These primers guided creating the cDNA templates, which were cloned into the plasmids comprising CMV promoter and metal binding domains, and hetero-bifunctional linker domains linking chelates.
  • NASH 5Prime software
  • Applied Biosystems synthesized
  • Propagated plasmids were electroporated into the human myelomas, which were established from the oncology patients diagnose with Multiple myelomas, to express the recombinant CD4.
  • the cells were conditioned to grow in the serum-free RPMI media in roller bottles at 37 deg. C and 5% C02. Therefore, supernatant could be easily used to test secreted recombinant receptors expressed from all the transcript variant versions by NMR and FCM.
  • anti-virus antibodies with superparamagnetic features tag not only HIV-infected cells through the HIV envelope molecules of budding viruses, but also the viruses themselves. Separation of HIV- infected cells from the viruses was performed by spinning the labelled samples at 1000 rpm at room temperature. The supernatant contained the HIV tagged with antibodies, while the HIV- infected cells, but not healthy cells, were collected in the pellet.
  • the enriched fractions of the CD4+ cells were lysed with NP40.
  • the soup was mixed with our anti-CD4, superparamagnetic, genomically engineered antibodies (GEA), incubated for 1 h at 4 deg. C, and inserted into magnetic field at room temperature for 15 minutes. Diamagnetic content was rinsed off, while in the field. The fraction retained by anti-CD4 GEAs was released after the magnetic field ceased. Aliquots of this fraction were electrophoresed on PAGE and transferred onto the PVDF membranes (Amersham). The immunoblots were tested by the standard OKT4 antibody produced by the cell line were initially grown in the recommended cell culture conditions, which we modified so the cells were grown in sera-free media (ATCC).
  • ATCC sera-free media
  • the enriched fractions of the CD4+ cells were lysed with RIPA.
  • the lysates were electrophoresed by PAGE and validated as immune-precipitated ones.
  • Propagated plasmids were electroporated into the human myelomas, which were established from the oncology patients diagnose with Multiple myelomas, to express the recombinant CD4.
  • the cells were conditioned to grow in the serum- free RPMI media in roller bottles at 37 deg. C and 5% C02. Therefore, supernatant could be easily used to test secreted recombinant receptors expressed from all the transcript variant versions by NMR and FCM.
  • VUV Viral Universal Vaccine
  • VAM-IRA virus entry receptor - vaccine engineered constructs
  • Genomically engineered UVV comprise the two main domains: (1) virus attaching/absorbing molecule (VAM) alaso known as virus entry receptor (VER); (2) immunity redirecting antigen (IRA) also known as vaccine engineered constructs (VECs) , which are integrated into a single biomolecule.
  • VAM virus attaching/absorbing molecule
  • IRA immunity redirecting antigen
  • VECs vaccine engineered constructs
  • An example for manufacturing of a specific VER-VEC: CD4 - HBsAg is intended as an example, but by no means is meant to limit the scope of the invention as it can be easily applied to any virus infection and any vaccine.
  • VAM-IRA aka VER-VEC: CD4 - HBsAg were prepared on two ways: chemical conjugation and genomic engineering.
  • VUV compositions were started by expressing separately transgenic, soluble, monomeric VAM from the DNA coding sequences (SEQ ID NO: 1-29) and transgenic, soluble, monomeric IRA from the DNA coding sequences (SEQ ID NO: 35-50). These components were also acquired by biochemical isolation from cells at the initial phases of this project as in details described above. That followed by functionalization of the expressed VAM and IRA separately with the bifunctional linkers listed above. That followed by click chemistry that was linking both molecules together into one monomeric VUV.
  • the first step involved functionalization of VAM: CD4 identified by its the DNA coding sequence specified as SEQ ID NO: 59.
  • CD4 identified by its the DNA coding sequence specified as SEQ ID NO: 59.
  • 100 mg of CD4 was dissolved in 10 ml of the PBS buffer consisting of 0.15M sodium chloride, 0.1 M sodium phosphate adjusted to pH 7.5 at room temperature.
  • 3 mg of SPDP was dissolved in 1 ml of DMF. While mixing on a stirrer, 200 microl of the SPDP solution in DMF was added to the 10 ml of CD4 in PBS solution and reacted at room temperature for 30 min.
  • Separation of unreacted compounds was performed by running on a desalting column equilibrated with the buffer consiting of 0.15M sodium chloride, 0.1 M sodium phosphate, 10 mM EDTA adjusted to pH 7.5 at room temperature degassed, while maintained in oxygen free environment.
  • the functionalized CD4 was brought to 10 mg / ml on centrifuged concentrator with 10 kDa cutoff as verified by the spectrophotometer.
  • the second step involved functionalization of IRA: HBsAg identified by its the DNA sequence specified as SEQ ID NO: 35.
  • 100 mg of HBsAg was dissolved in 10 ml of the buffer consiting of 0.15M sodium chloride, 0.1 M sodium phosphate adjusted to pH 7.5 at room temperature.
  • 3 mg of SPDP was dissolved in 1 ml of DMF. While mixing on a stirrer, 240 microl of the SPDP solution in DMF was added to the 10 ml of CD4 in PBS solution and reacted at room temperature for 30 min.
  • Separation of unreacted compounds was performed by running on a desalting column equilibrated with the buffer consisting of 0.15 M sodium chloride, 0.1 M sodium phosphate, 10 mM EDTA adjusted to pH 7.5 at room temperature degassed, while maintained in oxygen free environment.
  • the functionalized HBsAg was brought to 10 mg / ml on centrifuged concentrator with 10 kDa cutoff as verified by the spectrophotometer. 17.2 mg of DTT was added to 1 ml of double distilled water and 500 microl of this DTT solution in water was added promptly to the functionalized HBsAg containing solution, while mixing on the stirrer. The reaction continued for 30 min at room temperature at the oxygen free environment.
  • HBsAg Separation of unreacted compounds was performed by running on a desalting column equilibrated with the buffer consisting of 0.15 M sodium chloride, 0.1 M sodium phosphate, 10 mM EDTA adjusted to pH 7.5 at room temperature degassed, while maintained in oxygen free environment.
  • the functionalized HBsAg was brought to 10 mg / ml on centrifuged concentrator with 10 kDa cutoff as verified by the spectrophotometer.
  • UVV Universal Viral Vaccine
  • the very first step in manufacturing of the UVV by genomic engineering was initiated with synthesis on of the one sequence of the DNA, which comprised the DNA coding sequence for VAM and the DNA coding sequence for IRA integrated into one sequence specified as SEQ ID NO 59. It was accomplished on the DNA synthesizer. It was also synthesized commercially at the initial phases of this project. Later, the long planned as the complementary overlapping fragments of the the DNA coding sequences for CD4 merged with the DNA coding sequences for HBsAg were manufactured by Gibson assembly; thus creating the DNA coding sequences for VUV : CD4 - HBsAg as specified SEQ IF NO 059. Importantly, for inserting into the plasmid the DNA coding sequences for this UVV had terminal overhangs compatible with the DNA sequences unique for Nhel and BamHI restriction enzymes as specified SEQ ID NO: 031-035.
  • the first step in this procedure in preparation for cloning the plasmid DNA, 10 microg of the pCMV-INS-SV40 was dissolved in the buffer consisting of 50 mM Potassium acetate 20 mM Tris-acetate 10 mM Magnesium acetate 100 pg/ml BSA pH 7.9 at room temperature of 25 deg C.
  • the plasmid carried the single, unique restriction enzymes sites for Nhel and BamHI, which flanked short the DNA coding sequence INS sequence as identified on the map in FIG. 4 and specified within SEQ ID NO 030.
  • Adding these two restriction enzymes to the plasmid DNA containing solution opened the cc ds DNA plasmid.
  • the reaction was performed at 37 deg C for 1 h.
  • the release of these high fidelity restriction enzymes from the cut DNA was accomplished by adding SDS to a final concentration of 0.1% - 0.5%.
  • the reaction was terminated by a stop solution consisting of 2.5% Ficoll 400, lOmM EDTA, 3.3mM Tris-Hcl, 0.08% SDS, 0.02% pH 8.0 at 25°C (NEB).
  • a stop solution consisting of 2.5% Ficoll 400, lOmM EDTA, 3.3mM Tris-Hcl, 0.08% SDS, 0.02% pH 8.0 at 25°C (NEB).
  • To test efficacy of the cuts the plasmid was electrophoresed in 1% agarose gel.
  • the released ds DNA INS fragment was removed by magnetic separation using core-shell superparamagnetic particles tagged probes in the field of 1.5 - 3T as described below.
  • the synthesized DNA coding sequence for VUV: CD4-HBsAg as specified SEQ ID NO: 059 was added to the solution containing the open plasmid pCMV- -SV40 having exposed the overhangs capable for accepting for complement overhangs specified as SEQ ID NO 031- 034 were flanking the terminals of the DNA coding sequence of the DNA coding sequence of the VUV specified as SEQ ID NO 059. After sealing into cc ds DNA, it was electroporated into Multiple myeloma cells. The Myeloma cells were grown in the serum free media. The pure CD4 - HBsAg was recovered through HPLC.
  • DNA sequences for Example 3 can be easily reproduced in any appropriate combination of the DNA coding sequences for VAM with the DNA coding sequences for IRA as specified SEQ ID NO: 51-79 and SEQ ID NO: 81-109.
  • tgCD4 The soluble, recombinant transgenically expressed CD4 (tgCD4) was dissolved from lyophilized from PBS powder. It was dialyzed against phosphate buffer solution (PBS) pH 7.0 24 deg. C in lOkDa cutoff bags. The concentration was adjusted to 20 microM. An aliquot of 12 ml was sampled from that solution. Meanwhile, 1 ml of 100 mM stock solution of 4-pentynoic acid (PA) in 50 % of THF in PBS was prepared. While on the stirrer, 67 mirol of the PA stock solution was added to 12 ml of tgCD4 in PBS, while mixing at room temperature of around 24 deg C.
  • PBS phosphate buffer solution
  • PA 4-pentynoic acid
  • superparamagnetic particles (SPM) at 2.8 nM concentration in double distilled water was provided.
  • SPM superparamagnetic particles
  • TPA thiol-PEG-azide
  • the CD4-SPM conjugates were cleared from the non-reacted reagents by spinning down at l5,000g at 4 deg C and resuspending in PBS at least 5 times. Upon completion of this procedure the tgCD4-SPM conjugates were effectively binding HIV.
  • transgenically expressed soluble CD4 (tgCD4) with SPM.
  • 10 ml of 0.15 M NaCl, 0.01 M sodium phosphate solution transgenically expressed soluble CD4 up was dissolved up to 10 mg/ml concentration.
  • 0.088 M NaI04 solution in water in darkness at room temperature of 24 deg C was prepared. Having both solutions ready, 1 ml of NaI04 solution was added to the tgCD4 solution in the darkness at room temperature for 15 min.
  • 1 ml of glycerol was added and running through the desalting column, while collecting the fractions determined on the spectrophotometer on the fraction collector at 280 nm.
  • the fractions were pooled together and adjusted to 10 mg/ml and stored at 4 deg C. Conjugation process was pursued by the preparing stock solution of 10 mM polyetheleglycoldithiolacylhydrazide (PEAD) or polytriethyleneglycol-acylhydrazide-dithiol (PTAD) in PBS. Having both solutions ready in darkness, 10 mM PTAD was added to the tgCD4 solution to attain equimolar concentration of tgCD4 and PTAD, while stirred at 4 deg C for 1 h. The reaction was completed by dialysis at 4 deg C against PBS in dialysis bags at 10 kDa cutoff for 1 h, while changing the soaking solution 5 times.
  • PEAD polyetheleglycoldithiolacylhydrazide
  • PTAD polytriethyleneglycol-acylhydrazide-dithiol
  • Superparamagnetic molecules are prepared to comprise solid homogenous or core-shell architecture. The choice of the supermagnets is driven by future applications. The solid superparamagnetic particles are used for in vitro diagnosis and research.
  • the core-shell particles are manufactured for in vivo, in patients therapy. Their inner core provides superparamagnetic properties.
  • the outer layer - shells comprise biologically inert elements to protect the patients from potentially leaking, toxic magnetic material and to offer interfacing layer to link them with antibodies and recombinant receptors.
  • the core-shell particles comprise Fe304 or Ni cores and Au or Si02 shells engineered according to classical protocols adopted in this project.
  • the cores are synthesized by mixing aqueous solutions of FeQ3 x6H20 / FeCl2x4H20 in 1 ⁇ 2 molar ratio, followed by adding lm NaOH and stirring initially at room temperature, that was gradually increased to 90 deg. C for lh. The process is completed by multiple cycles of rinsing with water. The superparamagnetic particles are then retained by magnets and dispersed in water as ferrofluid.
  • the gold shells are prepared according to the modification of the classical Turkevich procedure.
  • the cells were labeled with the superparamagnetic genomically engineered antibodies (GEA-SPMs) as described in details elsewhere. [29, 23, 24] Briefly, the AVA-VEC and VER-VEC were dissolved and all washing steps carried in phenol-free, Ca-i- / Mg+- free, PIPES buffered saline solution, supplemented with 20 mM glucose, 10% human serum. The aliquots were dispensed into the magnetism-free NMR tubes (Shigemi). The relaxation times Tl and T2 were measured in resonance to the applied pulse sequences on the NMR spectrometers: DMX 400 WB or AVANCE II NMR (Bruker, Billerica, MA) or the Signa clinical scanners (General Electric).
  • DMX 400 WB or AVANCE II NMR Bruker, Billerica, MA
  • Signa clinical scanners General Electric
  • the superparamagnetic AVA-VEC and VER-VEC were used to isolate the labeled molecules and/or cells from the solution.
  • the labeled cells rendered superparamagnetic properties, which facilitated their isolation on the magnetic, activated cell sorter (MACS) operated at 0.5 T - 1.5 T (NSF grant support to MM) and / or clinical MRI instruments operating at 0.5 T - 3 T and / or NMR scanners operating at 0.5 T- 7 T (Bruker).
  • MCS magnetic, activated cell sorter
  • FCM Flow cytometry
  • the cells were labeled with the fluorescent antibodies as described in details elsewhere. [23, 24] They were sorted on the Calibur, Vantage SE, or Aria (Becton-Dickinson). The antibodies were dissolved and all washing steps carried in phenol-free, Ca-i- / Mg+- free, PIPES buffered saline solution, supplemented with 20 mM glucose, 10% human serum. Sorting was performed on Aria, Calibur, Vantage SE (Becton-Dickinson) with the sheath pressure set at 20 pounds per square inch pressure and low count rate. The sorted batches were analyzed on Calibur or Aria using FACSDiva software or on the FC500 (Beckman-Coulter).
  • the fluorescently labeled cells or tissues were imaged with the Axiovert (Zeiss) equipped with the Enterprise argon ion (457 nm, 488 nm, 529 nm lines) and ultraviolet (UV) (364 nm line) lasers; Odyssey XL digital high- sensitivity with instant deconvolution confocal laser scanning imaging system operated up to 240 frames/s (Noran), and the Diaphot (Nikon) equipped with the diode-pumped Nd:YLF solid state laser (1048 nm line) (Microlase).
  • XRFS X-ray reflection fluorescence spectroscopy
  • Elemental analyses were pursued by EDXS, EELS, and XRFS as described earlier.
  • the field emission, scanning transmission, electron microscope FESTEM HB501 (Vacuum Generators) was equipped with the energy dispersive x-ray spectrometer (EDXS) (Noran) and post-column electron energy loss spectrometer (EELS) (Gatan).
  • the cryo-energy filtering transmission electron microscope 912 Omega was equipped with the in column, electron energy loss spectrometer (EELS) and the energy dispersive x-ray spectrometer (EDXS) (Zeiss).
  • the cryoenergy filtering transmission electron microscopes 410 and 430 Phillips were equipped with the post-column, electron energy loss spectrometers (EELS) and the energy dispersive x-ray spectrometer (EDXS) (Noran).
  • the field emission, scanning electron microscope SEM1530 (Zeiss) was equipped with the energy dispersive x- ray spectrometer (EDXS) (Noran).
  • the field emission, scanning electron microscope 3400 was equipped with the energy dispersive x-ray spectrometer (EDXS) (Hitachi).
  • the S2 Picofox XRFS spectrometer was equipped with a molybdenum (Mo) X-ray target and the Peltier cooled Xflash Silicon Drift Detector (Bruker AXS). Scan times ranged up to 1000 seconds.
  • the ICP standard of 1000 mg/l of mono-element Gallium or Gadolinium (CPI International) was added to 500 microL of each sample to the final concentration of 10 mg/l. Instrument control, data collection, and analysis were under the SPECTRA 7 software (Bruker AXS).
  • CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 1983; 312:763-765.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Il y a un manque de vaccins prophylactiques pour protéger des personnes contre une infection par de nombreux virus mortels. Il y a un manque de vaccins thérapeutiques pour soigner des personnes infectées par de nombreux virus mortels. Les problèmes avec tous les agents chimiothérapeutiques approuvés sont : ils deviennent inefficaces après les mutations de virus, introduisent des mutations de virus, et infligent des effets indésirables très sérieux aux patients. La présente invention concerne, en tant que solution à ces problèmes, des compositions, des procédés de fabrication et des procédés d'utilisation pour traiter des patients infectés par des virus avec des vaccins antiviraux universels (VUV aka UVV). Elles comprennent des molécules de fixation de virus exprimées de façon transgénique, solubles, présentées sur des cellules (VAM) et des antigènes de redirection d'immunité ou des domaines de vaccin (IRA). Le procédé de VUV repose sur leur administration aux patients présentant un risque et/ou infectés par des virus.
PCT/US2019/013692 2018-01-16 2019-01-15 Vaccins antiviraux universels WO2019143631A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201862617606P 2018-01-16 2018-01-16
US62/617,606 2018-01-16

Publications (1)

Publication Number Publication Date
WO2019143631A1 true WO2019143631A1 (fr) 2019-07-25

Family

ID=67302451

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/013692 WO2019143631A1 (fr) 2018-01-16 2019-01-15 Vaccins antiviraux universels

Country Status (1)

Country Link
WO (1) WO2019143631A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4608251A (en) * 1984-11-09 1986-08-26 Pitman-Moore, Inc. LHRH analogues useful in stimulating anti-LHRH antibodies and vaccines containing such analogues
WO2001064752A2 (fr) * 2000-03-02 2001-09-07 New York University Procedes d'utilisation d'un element facilitant l'entree retrovirale dans les cellules
WO2013003555A1 (fr) * 2011-06-28 2013-01-03 Whitehead Institute For Biomedical Research Utilisation de sortases pour installer des attaches de chimie click pour la ligature de protéine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4608251A (en) * 1984-11-09 1986-08-26 Pitman-Moore, Inc. LHRH analogues useful in stimulating anti-LHRH antibodies and vaccines containing such analogues
WO2001064752A2 (fr) * 2000-03-02 2001-09-07 New York University Procedes d'utilisation d'un element facilitant l'entree retrovirale dans les cellules
WO2013003555A1 (fr) * 2011-06-28 2013-01-03 Whitehead Institute For Biomedical Research Utilisation de sortases pour installer des attaches de chimie click pour la ligature de protéine

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ARTHOS J. ET AL.: "Biochemical and biological characterization of a dodecameric CD 4-Ig fusion protein: implications for therapeutic and vaccine strategies", J. BIOL. CHEM. 2002, vol. 277, no. 13, 22 January 2002 (2002-01-22), pages 11456 - 11464, XP002339484, doi:10.1074/jbc.M111191200 *
BERKOWER I. ET AL.: "Assembly, structure, and antigenic properties of virus-like particles rich in HIV-1 envelope gp120", VIROLOGY, vol. 321, no. 1, 2004, pages 75 - 86, XP004497045, doi:10.1016/j.virol.2003.12.017 *
CHEN X. ET AL.: "The delivery of HBcAg via Tat-PTD enhances specific immune response and inhibits Hepatitis B virus replication in transgenic mice", VACCINE, vol. 28, 2010, pages 3913 - 3919, XP027059427 *
FANG N. ET AL.: "Differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system", BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY, vol. 32, no. 1, 2000, pages 27 - 33 *
HINES J. F. ET AL.: "The Expressed L1 Proteins of HPV-1 HPV-6, and HPV-11 Display Type-Specific Epitopes with Native Conformation and Reactivity with Neutralizing and i Nonneutralizing Antibodies", PATHOBIOLOGY, vol. 62, 1994, pages 165 - 171 *
LOWRIE, DOUGLAS B. ET AL.: "DNA vaccines: methods and protocols", METHODS IN MOLECULAR MEDICINE, vol. 528, 2000, pages 81, 279, 280 *

Similar Documents

Publication Publication Date Title
Holl et al. Nonneutralizing antibodies are able to inhibit human immunodeficiency virus type 1 replication in macrophages and immature dendritic cells
US20220241399A1 (en) Hiv rna vaccines
CN105602992B (zh) 一种基于复制缺陷性重组慢病毒的car-t转基因载体及其构建方法和应用
CN105777911B (zh) 抗bcma嵌合抗原受体、编码基因、重组表达载体及其构建方法和应用
CN108676091B (zh) Hiv-1的中和抗体及其用途
Forthal et al. Rhesus macaque polyclonal and monoclonal antibodies inhibit simian immunodeficiency virus in the presence of human or autologous rhesus effector cells
KR20210038886A (ko) 개인화된 암 백신 에피토프 선택
US11268099B2 (en) High temperature selection of nucleotide-supported carbohydrate vaccines and resulting glycosylated oligonucleotides
JPH06508024A (ja) Ｃ型肝炎診断試薬およびワクチン
NZ525752A (en) HIV peptides, antigens, vaccine compositions, immunoassay kit and a method of detecting antibodies induced by HIV
JPH07278189A (ja) Ｈｉｖグループに属するレトロウイルス由来のペプチド及びそれらの使用
CA2178341A1 (fr) Detection et traitement des cancers du sein et des cancers gynecologiques
WO2019126768A1 (fr) Molécules modifiées par ingénierie génomique (gem) composites multidomaines
JP2013505201A (ja) 広域反応性中和抗ｈｉｖ抗体を誘導するための配合物
Nakamura et al. Lack of infectivity of HIV-1 integrase zinc finger-like domain mutant with morphologically normal maturation
Li et al. Identification of an N-linked glycosylation in the C4 region of HIV-1 envelope gp120 that is critical for recognition of neighboring CD4 T cell epitopes
EP2588211A2 (fr) Immunogènes trimères d'env
EP3494984A1 (fr) Traitement et diagnostic du cancer ou de maladies infectieuses à l'aide du mécanisme de reconnaissance du récepteur des cellules t
CN104628848A (zh) 单克隆抗体mers-27及其编码基因和应用
Gonelli et al. Immunogenicity of HIV-1-based virus-like particles with increased incorporation and stability of membrane-bound Env
CN104628849B (zh) 单克隆抗体mers‑4及其编码基因和应用
WO2019143631A1 (fr) Vaccins antiviraux universels
Provine et al. The infectious molecular clone and pseudotyped virus models of human immunodeficiency virus type 1 exhibit significant differences in virion composition with only moderate differences in infectivity and inhibition sensitivity
Malecki et al. HIV Universal Vaccine
McIntosh et al. An engineered non-toxic superantigen increases cross presentation of hepatitis B virus nucleocapsids by human dendritic cells

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: 19740799

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC, EPO FORM 1205A DATED 21.10.2020

122 Ep: pct application non-entry in european phase

Ref document number: 19740799

Country of ref document: EP

Kind code of ref document: A1