[go: up one dir, main page]

WO2025075855A1 - Vecteur génétique inverse et méthode d'utilisation associée - Google Patents

Vecteur génétique inverse et méthode d'utilisation associée Download PDF

Info

Publication number
WO2025075855A1
WO2025075855A1 PCT/US2024/048515 US2024048515W WO2025075855A1 WO 2025075855 A1 WO2025075855 A1 WO 2025075855A1 US 2024048515 W US2024048515 W US 2024048515W WO 2025075855 A1 WO2025075855 A1 WO 2025075855A1
Authority
WO
WIPO (PCT)
Prior art keywords
vector
promoter
virus
phw2000
influenza
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
PCT/US2024/048515
Other languages
English (en)
Inventor
Christopher Patton
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
St Jude Childrens Research Hospital
Original Assignee
St Jude Childrens Research Hospital
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 St Jude Childrens Research Hospital filed Critical St Jude Childrens Research Hospital
Publication of WO2025075855A1 publication Critical patent/WO2025075855A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • A61K39/12—Viral antigens
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16151—Methods of production or purification of viral material
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription

Definitions

  • the influenza A virus reverse genetics (RG) system (Hoffman et al. ( (2000) Proc. Natl. Acad. Sci. USA 97 (11) : 6108-6113; US 6, 951,754 B2; FIG. 1) provides a system for studying point mutations and the identification of phenotypes conferred by a single nucleotide or amino acid substitution.
  • This system uses the bidirectional expression vector, pHW2000, which has a human pol I promoter with a murine terminator, a human cytomegalovirus (CMV) pol II promoter with a bovine growth hormone polyadenylation signal, and a T7 promoter downstream of the CMV promoter.
  • CMV cytomegalovirus
  • the pHW2000 vector encodes (3-lactamase (AmpR) as the antibiotic selection marker.
  • the orientation of the pol I and CMV pol II promoters allows the respective synthesis of negative-sense viral RNA and positive-sense mRNA from one viral cDNA template.
  • cDNAs from all eight influenza gene segments are cloned into pHW2000 using BsmBI or Bsal restriction sites resulting in eight plasmids. All eight plasmids are then transfected into human embryonic kidney (HEK) 293T cells or a coculture of HEK 293T cells and Madin Darby Canine Kidney (MDCK) cells. This dual promoter system expresses the necessary gene products for generating RG viruses.
  • HEK human embryonic kidney
  • MDCK Madin Darby Canine Kidney
  • US 2016/0030547 Al discloses a similar type of vector as pHW2000, wherein the human Pol I promoter has been replaced with the Porcine Pol I promoter.
  • This vector includes a CMV promoter, a murine polymerase I terminator sequence, a cloning site for genes to be transcribed, the swine polymerase I promoter sequence, and a bovine growth hormone polyadenylation signal.
  • pAD3000 is described by Hoffman et al. ( (2002) PNAS 99 (17) : 11411-11416) as being a variant of pHW2000, wherein the bovine growth hormone ( BGH) polyadenylation signal is replaced with the SV40 late mRNA polyadenylation signal .
  • BGH bovine growth hormone
  • This invention provides a vector including a multiple cloning site flanked by a human polymerase I promoter and a human cytomegalovirus polymerase I I promoter, wherein the vector further includes an inactive T7 promoter operably linked to the multiple cloning site .
  • the vector is a variant of pHW2000 .
  • the vector further includes an influenza virus transgene in the multiple cloning site .
  • the influenza virus transgene encodes an influenza PB2 , FBI , PA, HA, NP, NA, M or NS protein .
  • a method for producing an influenza virus vaccine using the vector is also provided .
  • FIG . 1 is a schematic representation of a method for the construction of the eight expression plasmids using the conventional pHW2000 vector .
  • Viral RNA is extracted from virus particles .
  • RT-PCR is performed with primers containing segment-specific nucleotides and sequences for the type I l s restriction endonucleases BsmBI (and its isoschizomer Bsp3 l ) or Bsal .
  • the eight viral PCR fragments are digested with BsmBI or Bsal and inserted into pHW2000 (linearized with BsmBI ) .
  • FIG. 2A shows the point mutation in the T7 promoter of the pHW2000 vector resulting in the modified pPW2000 vector .
  • FIG. 2B shows the genetic elements controlling dual expression in pHW2000 and pPW2000 vectors.
  • FIG. 3 is a schematic representation of cloning an influenza A transgene segment into pPW2000 and pHW2000.
  • FIGS. 4A-4F show the measured differences comparing transformation efficiency between pPW2000 and pHW2000. Comparison of transformation efficiency of DH5-ot and HB101 bacteria when transformed with supercoiled vector lacking cDNA insert (FIG. 4A) . Transformation efficiencies from ligation reactions of Penn/1370 matrix (M) gene (FIG. 4B) or hemagglutinin (HA) gene (FIG. 4C) in pPW2000 or pHW2000 vectors. To ensure that subtle differences in vector quantity were not a contributing factor, increased amounts of vector were included in ligation reactions (FIG. 4D) .
  • M Penn/1370 matrix
  • HA hemagglutinin
  • FIGS. 5A-5B show PGR results of RNA samples, treated with DNase I treated or subjected to mRNA pull-down, amplifying p-lactamase (FIG. 5A) , indicating that the mRNA samples were void of contaminating plasmid DNA.
  • RT-qPCR was used to compare leaky expression in HB101 bacteria harboring either pHW2000 or pPW2000 vectors ( FIG . 5B ) .
  • Plasmid-free mRNA template was then subj ected to real-Time qPCR to quantify differences in leaky expression of M gene transcripts using the method .
  • p-lactamase was used to normalize mRNA input . Differences in fold-change are relative to M gene expression in the pPW2000 vector .
  • P values were calculated using a two- tailed Mann-Whitney U test . **, P ⁇ 0 . 01 .
  • This disclosure provides a modified pHW2000 vector of the reverse genetics system described in US 6, 951 , 754 B2 , which is incorporated herein by reference in its entirety .
  • the pHW2000 vector described provides a dual promoter for the production of negative strand segmented viruses (e . g . , influenza A, influenza B , Bunyaviridae) . Because the vector uses a single viral cDNA template for both protein and viral RNA synthesis , this vector reduces the total number of plasmids required for virus production and allows the development of vaccines quickly and cheaply .
  • the pHW2000 vector has been modified to knockout the T7 promoter .
  • pPW2000 This modified vector, referred to herein as pPW2000, allows for easier cloning of viral transgenes .
  • the pPW2000 vector provides for reduced selective pressure of E. coli to recombine or introduce mutations into the viral transgene inserted into the pPW2000 vector and reduced expression of gene products that are deleterious to virus rescue .
  • the invention provides a bidirectional vector and expression system including the same .
  • vector, " "cloning vector” and "expression vector” mean the vehicle by which a DNA or RNA sequence (e . g . , a foreign gene or transgene) can be introduced into a host cell , so as to transform the host and promote expression (e . g . , transcription and translation) of the introduced sequence .
  • Plasmids are preferred vectors of the invention .
  • a viral transgene is inserted between an RNA polymerase I (pol I ) promoter and terminator sequences ( inner transcription unit ) .
  • This entire pol I transcription unit is flanked by an RNA polymerase I I (pol II ) promoter and a polyadenylation site (outer transcription unit ) .
  • the pol I and pol II promoters are on opposite sides of a multiple cloning site for introducing a viral transgene ( e . g . , a cDNA) into the vector, wherein an upstream pol II promoter produces positive-sense capped mRNA and a downstream pol I promoter produces negative-sense uncapped viral RNA (vRNA) .
  • This pol I-pol I I system starts with the initiation of transcription of the two cellular RNA polymerase enzymes from their own promoters .
  • the pol I promoter and pol I terminator sequence in the bidirectional vector may be referred to as including a "downstream-to-upstream orientation" whereas the pol I I promoter and polyadenylation signal in the bidirectional vector may be referred to as including an "upstream-to- downstream orientation" .
  • a promoter , terminator or polyadenylation signal is "upstream" of a transgene if it is proximal to the start of the transgene ( e . g . , the first codon) and distal to the end of the transgene (e . g . , the termination codon ) .
  • a promoter, terminator or polyadenylation signal is "downstream" of a transgene if it is proximal to the end of the transgene and distal to the start of the transgene .
  • Promoters in the vector of the invention which are functionally associated with a transgene, are oriented to promote transcription of a sense or an antisense strand of the transgene .
  • a "promoter” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a sequence .
  • a promoter that is located upstream of a transgene e . g . , cDNA
  • a transcription initiation site e . g . , cDNA
  • a promoter that is located downstream of a transgene (to express a (- ) RNA) is bounded at its 5 ' terminus by a transcription initiation site and extends downstream ( 3 ' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background .
  • the bidirectional system of the invention includes both upstream and downstream promoters . Within the promoter sequence will be found a transcription initiation site ( conveniently defined for example, by mapping with nuclease SI ) , as well as protein binding domains ( consensus sequences ) responsible for the binding of RNA polymerase .
  • a coding sequence is "under the control of” , “ functionally associated with” or “operatively associated with” transcriptional and translational control sequences (e . g . , a pol I or pol II promoter) in a cell when RNA polymerase transcribes the coding sequence into RNA, e . g . , mRNA or vRNA.
  • transcriptional and translational control sequences e . g . , a pol I or pol II promoter
  • Such viruses may include hepatitis viruses (e . g . , those of the Picornaviridae, Flaviviridae , and Hepeviridae families,) , as well as members of the Orthomyxovirus family, the Paramyxovirus family, the Filoviridae family, the Bornaviridae family, the Hantaviridae family, the Arenaviridae family, Coronaviridae family, or the Rhabdovirus family .
  • the invention advantageously provides for inserting the genes for the primary antigens from such strains into a plasmid-based system, in which the remaining viral genes have desired culture and/or attenuation characteristics , thus providing for production of quantities of virus of the appropriate antigenic background for vaccine production .
  • a "viral transgene” is preferably a cloned cDNA corresponding to a genomic RNA molecule from the RNA virus genome .
  • cDNA is preferred, any other type of nucleic acid that encodes a viral gene which is to be expressed may be used .
  • PCR-amplif ied products or restriction fragments including viral genes may be used .
  • the transgenes expressed by the vector of the invention may be fused to or tagged with other genes such as purif ication/detection tags ( e . g . , glutathione-S transferase, polyhistidine , green fluorescent protein, myc tags and FLAG tags ) .
  • the present invention also provides embodiments wherein partial gene sequences are used in the vectors herein .
  • Viral transgene sequences are introduced into the vector via a multiple cloning site .
  • a "multiple cloning site" is a nucleic sequence of the vector that includes a variety of singular restriction enzyme recognition sequences so that a suitable insertion point for a transgene is available .
  • the vector will incorporate gene segments for antigens required to produce a protective immunological response .
  • a "protective immunological response” includes a humoral (antibody) or cellular component , or both, effective to eliminate infectious virions and infected cells in an immunized (vaccinated) subj ect .
  • a protective immune response can prevent or resolve an RNA virus , e . g . , influenza virus , infection .
  • the antigens are "surface antigens, " i . e . , expressed on the surface of the virion or the surface of infected cells . More preferably, the surface antigens are glycoproteins .
  • the primary glycoprotein antigens are hemagglutinin (HA or H) and neuraminidase (NA or N) .
  • immunogenic means that an antigen is capable of eliciting a humoral or cellular immune response , and preferably both .
  • An immunogenic composition is a composition that elicits a humoral or cellular immune response , or both, when administered to an animal .
  • a molecule is "antigenic” when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin ( antibody) or T cell antigen receptor .
  • An antigenic polypeptide contains an epitope of at least about five, and preferably at least about 10, amino acids .
  • An antigenic portion of a polypeptide can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conj ugating the antigenic portion to a carrier polypeptide for immunization .
  • a molecule that is antigenic need not be itself immunogenic, i . e . , capable of eliciting an immune response without a carrier .
  • the vector and system of this invention is used in the design and recovery of reassortment viruses .
  • a "reassortment" virus is a virus in which gene segments encoding antigenic proteins from a pathogenic virus are combined with gene segments encoding viral polymerase complex or other similar genes (e . g . , non-glycoprotein genes , including M genes and NS genes ) from viruses adapted for growth in culture (or attenuated viruses ) .
  • the reassortment virus thus carries the desired antigenic characteristics in a background that permits efficient production in a host cell .
  • Such a reassortment virus is a desirable "virus seed" for production of virions to produce vaccine .
  • the invention permits creating cross-species reassortments , e . g . , an influenza B antigen in an influenza A background .
  • RNA virions preferably negative strand segmented RNA virions
  • exemplary host cells include , but are not limited to, Madin-Darby Canine Kidney (MDCK) cells, VERO cells , CV1 cells, COS-1 and COS-7 cells, and BHK-1 cells , for example and not by way of limitation .
  • MDCK Madin-Darby Canine Kidney
  • VERO VERO cells
  • CV1 cell
  • COS-1 and COS-7 cells a transient co-culture is preferred for producing virions .
  • Co-culturing permits ef ficient transfection of a receptive cell , such as a 293T cell, with subsequent infection of a permissive cell for viral growth, such as an MDCK cell .
  • a receptive cell such as a 293T cell
  • a permissive cell for viral growth such as an MDCK cell .
  • cell culture or "co-culture” as used herein refers to an acceptable method for propagating virus for vaccine production, e . g . , embryonated hens ' eggs , and cell culture .
  • the present vector and associated system provides an efficient and economic strategy for production of vaccines for treating or preventing RNA viral infections, preferably, negative strand RNA virus infections .
  • this invention provides a method for using the pPW2000 vector to prepare a vaccine , in particular an influenza virus vaccine against seasonal influenza strains , pandemic strains , and highly pathogenic avian influenza strains .
  • the vector of the invention eliminates the need for selection and provides close control of reassortment viruses .
  • six plasmids containing the non-glycoprotein segments e . g .
  • PB1 , PB2 , PA, NP, M and NS from a high yield strain can be cotransfected with two expression plasmids encoding HA and NA from the candidate vaccine virus . Since no helper virus is required, the generated virus is an influenza virus with the desired gene constellation .
  • An analogous approach may be used to produce any variety of inactivated, reassortment RNA virus for use in a vaccine .
  • Expression vectors harboring viral gene segments for a target virus e . g . , nonglycoprotein segments
  • Virus produced in accordance with the invention can be used in traditional or new approaches to vaccination, particularly in the development of live , attenuated vaccines .
  • Three types of inactivated influenza vaccines are conventionally available : whole virus, split-product , and surface antigen vaccines . Because the present vector permits the rapid development of a desired reassortment virus with acceptable growth characteristics in culture, it advantageously positions a vaccine manufacturer to generate a sufficient quantity of vaccine to meet public health needs and ensure standardi zation, which is an important requirement currently mitigated by the need to produce clinical quantities of vaccine, usually an 8 to 9 month period .
  • a “vaccine” is a composition that can elicit protective immunity when administered to a subj ect .
  • the term “vaccine” refers to a composition containing virus, inactivated virus, attenuated virus, split virus, or viral protein, i.e. , a surface antigen, that can be used to elicit protective immunity in a recipient. It should be noted that to be effective, a vaccine can elicit immunity in a portion of the population, as some individuals may fail to mount a robust or protective immune response, or, in some cases, any immune response.
  • This inability may stem from the individual's genetic background or because of an immunodeficiency condition (either acquired or congenital) or immunosuppression (e.g., treatment with immunosuppressive drugs to prevent organ rejection or suppress an autoimmune condition) .
  • Efficacy can be established in animal models.
  • a "protective dose" of a vaccine is an amount, alone or in conjunction with an adjuvant, effective to elicit a protective immune response in a recipient subject. Protection can also depend on the route of administration, e.g. , intramuscular (preferred for an inactivated vaccine) or intranasal (preferred for an attenuated vaccine) .
  • adjuvant refers to a compound or mixture that enhances the immune response to an antigen.
  • An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specif ically enhances the immune response.
  • Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Coryn eba ct er i um parvum, An example of a preferred synthetic adjuvant is QS-21.
  • immunostimulatory proteins can be provided as an adjuvant or to increase the immune response to a vaccine.
  • the adjuvant is pharmaceutically acceptable.
  • Vaccination effectiveness may be enhanced by coadministration of an immunostimulatory molecule such as an immunostimulatory, immunopotentiating, or pro-inflammatory cytokine, lymphokine, or chemokine with the vaccine, particularly with a vector vaccine.
  • an immunostimulatory molecule such as an immunostimulatory, immunopotentiating, or pro-inflammatory cytokine, lymphokine, or chemokine
  • cytokines or cytokine genes such as interleukin IL-1, IL-2, IL-3, IL'S, IL-12, IL-13, granulocyte-macrophage (GM) -colony stimulating factor (CSF) and other colony stimulating factors, macrophage inflammatory factor, Flt3 ligand, as well as some key costimulatory molecules or their genes (e.g. , B7.1, B7.2) can be used.
  • cytokines or cytokine genes such as interleukin IL-1, IL-2, IL-3, IL'S
  • mucosal immunization strategies include encapsulating the virus in microcapsules (See, e.g., US 5,075,109, US 5,820,883, US 5,853,763) and using an immunopotentiating membranous carrier (WO 98/0558) .
  • Immunogenicity of orally administered immunogens can be enhanced by using red blood cells (RBC) or RBC ghosts (US 5, 643,577) , or by using blue tongue antigen (US 5,690,938) .
  • HEK human embryonic kidney
  • MEM opti- minimum essential media
  • Plasmid Isolation Plasmid DNA was isolated from 2 mL of transformed bacteria in logarithmic growth phase using the QIAprep® spin miniprep kit. Plasmid stocks were made by lysing bacteria and isolating plasmid DNA from 120 mL of transformed bacteria grown in the presence of ampicillin (100 pg/mL) . Both Mini preps and Maxi preps were performed according to the manufacturer's recommendation.
  • the cDNA from all eight genomic segments from A/Chicken/Pennsylvania/1370/1983 (H5N2) (Penn/1370) and A/Puerto Rico/8/1934 (H1N1) (PR/8) were cloned into the influenza A virus RG expression vectors pHW2000 and pPW2000 .
  • RG viruses were generated as 7+1 viruses where HA, NA and internals (excluding M) originated from PR/8 and the M was derived from Penn/1370 .
  • All of the PR/8 genomic segments were cloned into pHW2000 while the M from Penn/1370 was cloned into both pHW2000 and pPW20000 as previously described (Hof fman et al . (2000 ) Proc . Natl .
  • plasmids encoding cDNA of all eight viral RNA segments were transfected into human embryonic kidney (HEK) 293T cells using the transfection reaction sold under the tradename LIPOFECTAMINE® 3000 reagent (Thermo Fisher) .
  • LIPOFECTAMINE® 3000 reagent Thermo Fisher
  • cell supernatant was harvested and 200 pL of cell supernatant was inj ected into 10-day old embryonated chicken eggs to propagate virus and incubated at 35 °C .
  • Virus rescue was considered successful with a positive hemagglutinin agglutination titer >2 following egg propagation .
  • Transformation Approximately 30 pL of competent bacteria were mixed with 2 pL of ligation reaction . Bacteria were heat-shocked for 90 seconds, allowed to undergo one round of replication, spread on LB agar supplemented with 100 pg/mL of ampicillin (Amp + ) , and grown overnight at 37 ° C . Transformation efficiency was determined by transforming bacteria with pHW2000 or pPW2000 lacking cDNA insert and determining the number of CFU/pg of DNA . Transformation efficiency ( % ) of ligation reactions was determined by dividing the number of transformed CFUs by the total number of CFUs in the bacterial stock .
  • RNA Extraction Total RNA was extracted by adding 750 pL of TrizolTM reagent to 250 pL of bacteria in exponential growth phase harboring either pHW2000 or pPW2000 . One-hundred and fi fty pL of chloroform was added to the TrizolTM reagent for RNA isolation by phase separation . RNA was recovered from the aqueous phase and then precipitated with isopropanol and then washed two times with 75% ethanol . RNA pellets were air dried and then resuspended in 50 pL of RNase-free water . RNA quantity and quality was determined by Nanodrop® UV-Vis spectrophotometry .
  • RNA Isola tion Total RNA isolated via the Trizol®/ chloroform method was subj ected to the RNeasy pure mRNA bead kit (Qiagen) . Briefly, 5 pg of total RNA was suspended in 250 pL of nuclease-free water and mixed with 275 pL of polyA-oligo-dT beads . RNA was incubated for 2 minutes at 70 °C to break RNA secondary structures . RNA and polyA- oligo-dT mixture were then incubated for 10 minutes at room temperature to allow hybridi zation of the mRNA poly A tail and oligo-dT beads .
  • mRNA-oligo-dT was then centrifuged and resuspend in washing buffer and added to a spin column that did not allow oligo-dT beads to pass through .
  • mRNA- oligo-dT was washed, eluted by adding 20 pL of elution buffer pre-warmed to 65 °C to denature the mRNA-oligo-dT hybrid, and centrifuged to collect isolated mRNA. This was done two times to increase mRNA yield in a total elution volume of approximately 40 pL . Three pL of purified mRNA was then subj ected to RT-qPCR to quantify M gene transcripts .
  • Virus Propagation Viruses were propagated in 10-day old embryonated chicken eggs . RG viruses were made on the backbone of PR/8 and propagated in chicken eggs for 72 hours and then chilled overnight at 4 °C . After chilling, approximately 10 mL of virus was harvested from each egg and virus was subj ected to HA assay for titer determination . Viruses with the highest HA titer were pooled and aliquoted for storage at -80 °C .
  • pHW2000 had one recombination event between the cDNA insert and the bacterial chromosome .
  • RT-qPCR real-time quantitative PCR
  • Total RNA was extracted from bacteria, and mRNA was purified using an mRNA-pull down.
  • a DNase I digestion and a mRNA-pulldown were both carried out (FIG. 5A) .
  • the purified mRNA was then subjected to conventional PCR using only high-fidelity DNA polymerase, which would only allow amplification of contaminating plasmid.
  • HB101 bacteria transformed with pPW2000 were used as a positive control.
  • the mRNA pull-down samples were subsequently used for transcript expression analysis by real time qPCR (FIG. 5B) .
  • a dose-dependent response was observed in the real-time PCR assay, which was utilized as a standard curve.
  • the amount of template added to the PCR reactions were normalized to [3- lactamase expression, which was also encoded on the RG plasmids pHW2000 and pPW2000. It was found that pHW2000 had increased expression of Ml transcripts compared to pPW2000 (p ⁇ 0.01) .
  • influenza A virus RG vector pHW2000
  • the improved vector pPW2000 had significantly reduced expression of cloned gene segments compared to pHW2000. Loss of T7 promoter function increased cloning and transformation efficiencies, which has pertinent applications for generating future influenza vaccines .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un vecteur pHW2000 modifié comprenant un promoteur T7 inactif, ainsi qu'une méthode de préparation d'un vaccin contre le virus de la grippe à l'aide du vecteur modifié.
PCT/US2024/048515 2023-10-03 2024-09-26 Vecteur génétique inverse et méthode d'utilisation associée Pending WO2025075855A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363587504P 2023-10-03 2023-10-03
US63/587,504 2023-10-03

Publications (1)

Publication Number Publication Date
WO2025075855A1 true WO2025075855A1 (fr) 2025-04-10

Family

ID=95283984

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/048515 Pending WO2025075855A1 (fr) 2023-10-03 2024-09-26 Vecteur génétique inverse et méthode d'utilisation associée

Country Status (1)

Country Link
WO (1) WO2025075855A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5385834A (en) * 1993-08-13 1995-01-31 Georgia Tech Research Corporation Mutant T7 RNA polymerase GP1(lys222) exhibiting altered promoter recognition
US20020164770A1 (en) * 2000-04-28 2002-11-07 St. Jude Children's Research Hospital DNA transfection system for the generation of infectious influenza virus
CN112680477A (zh) * 2020-12-30 2021-04-20 华农（肇庆）生物产业技术研究院有限公司 一种基于无缝克隆技术的h9n2亚型禽流感病毒的拯救方法
WO2022112790A1 (fr) * 2020-11-30 2022-06-02 University Of Dundee Particules de type viral et procédés de production associés

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5385834A (en) * 1993-08-13 1995-01-31 Georgia Tech Research Corporation Mutant T7 RNA polymerase GP1(lys222) exhibiting altered promoter recognition
US20020164770A1 (en) * 2000-04-28 2002-11-07 St. Jude Children's Research Hospital DNA transfection system for the generation of infectious influenza virus
WO2022112790A1 (fr) * 2020-11-30 2022-06-02 University Of Dundee Particules de type viral et procédés de production associés
CN112680477A (zh) * 2020-12-30 2021-04-20 华农（肇庆）生物产业技术研究院有限公司 一种基于无缝克隆技术的h9n2亚型禽流感病毒的拯救方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MOSTAFA AHMED, KANRAI PUMAREE, ZIEBUHR JOHN, PLESCHKA STEPHAN: "Improved dual promotor-driven reverse genetics system for influenza viruses", JOURNAL OF VIROLOGICAL METHODS, ELSEVIER BV, NL, vol. 193, no. 2, 1 November 2013 (2013-11-01), NL , pages 603 - 610, XP093302953, ISSN: 0166-0934, DOI: 10.1016/j.jviromet.2013.07.021 *

Similar Documents

Publication Publication Date Title
AU2008203186B2 (en) Signal for packaging of influenza virus vectors
US6974686B2 (en) Recombinant tryptophan mutants of influenza
CN103540568A (zh) 制备流感病毒的多质粒系统
KR19990022335A (ko) 인플루엔자의 신규한 재조합 감온성 돌연변이체
JP2004531232A (ja) 高められた転写および複製能力をもつインフルエンザウイルス
CA2816242C (fr) Signal d'incorporation de vecteurs de virus de la grippe
CN113631187A (zh) 嵌合RSV和hMPV F蛋白、免疫原性组合物和使用方法
US20230310575A1 (en) Hemagglutinin Modifications for Improved Influenza Vaccine Production
JP2002513575A (ja) 弱毒インフルエンザウイルス
CN117205309B (zh) 一种流感免疫原组合物和制备方法及其用途
CN116284261B (zh) 一种非洲猪瘟病毒结构蛋白组合物及其制备的疫苗
WO2025075855A1 (fr) Vecteur génétique inverse et méthode d'utilisation associée
WO2025149032A1 (fr) Compositions de vaccin contre le virus respiratoire syncytial et leur utilisation
JP2004508814A (ja) タンデム配置された２つの遺伝子をコードする２シストロンｖＲＮＡを担持する組換えインフルエンザウイルス
WO2002083896A2 (fr) Virus recombinant segmente a polarite negative contenant un segment d'arnv bicistronique avec duplication de sa sequence laterale 3' non codante, et vaccins et compositions therapeutiques le contenant
Cai Live-attenuated vaccines against influenza viruses and role of NS1 in host response
HK1133442A1 (en) Influenza b viruses having alterations in the hemaglutinin polypeptide

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: 24875177

Country of ref document: EP

Kind code of ref document: A1