EP1345622A2 - Vaccin chimere contre le virus de l'anemie infectieuse equine et diagnostic - Google Patents

Vaccin chimere contre le virus de l'anemie infectieuse equine et diagnostic

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Publication number
EP1345622A2
EP1345622A2 EP01966602A EP01966602A EP1345622A2 EP 1345622 A2 EP1345622 A2 EP 1345622A2 EP 01966602 A EP01966602 A EP 01966602A EP 01966602 A EP01966602 A EP 01966602A EP 1345622 A2 EP1345622 A2 EP 1345622A2
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EP
European Patent Office
Prior art keywords
gene
eiav
vaccine
lentivirus
deleted
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.)
Withdrawn
Application number
EP01966602A
Other languages
German (de)
English (en)
Inventor
Ronald C. Montelaro
Jodi Craigo
Charles Issel
Bridget Puffer
Kristina J. Hennessey
Karen K. Brown
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.)
Intervet International BV
Original Assignee
Akzo Nobel NV
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Filing date
Publication date
Application filed by Akzo Nobel NV filed Critical Akzo Nobel NV
Publication of EP1345622A2 publication Critical patent/EP1345622A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • 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
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention pertains to a vaccine composition which provides immunity from clinical disease signs and/or infections caused by lentivirus. More specifically, the invention relates to an Equine Infectious Anemia Virus (EIAV) vaccine composition which provides immunity from clinical disease signs and/or infection with EIAV, and which composition allows diagnostic differentiation between vaccinated and non-vaccinated but exposed or diseased mammals, and which allows the vaccinated animal to test negative using a Coggins test or other similar test that detects p26 ⁇ specific antibodies.
  • EIAV Equine Infectious Anemia Virus
  • Lentiviruses are a subfamily of retroviruses that cause persistent infection and chronic disease in numerous types of mammals including humans (HIV), equines (EIA), felines (FIV), bovines (BIV) and monkeys (SIV). All of the diseases are spread by blood transmission. EIAV causes persistent infection and chronic disease in horses, worldwide. With EIAV, . the blood transmission occurs by biting flies and other insects carrying virus particles from one horse to another. The first cycle of disease (clinical episode) in an infected horse usually occurs within 42 days after exposure to the virus. This first cycle is usually referred to as the acute stage of EIA and is characterized by pyrexia, thrombocytopenia, anorexia, depression and high plasma viremia levels.
  • Anemia is not usually detected at this stage. Resolution of this first febrile episode is normally observed after 1 to 5 days and occurs concomitantly with a dramatic drop in the amount of plasma-associated virus. Following the acute stage, some animals may remain clinically normal while others go on to experience multiple bouts of illness in which severe anemia may accompany pyrexia, thrombocytopenia, edema, and dramatic weight loss, and death. In instances of persistent infection by a lentivirus, as illustrated by EIAV, nucleotide sequence data has revealed a high mutation rate of the lentivirus genome as reported by Payne et al, Virology, 1987: 161 , p 321-331 , which is incorporated herein by reference.
  • EIAV infections it is generally thought that neutralizing antibodies aid in the selection of new antigenic virus variants during persistent infections. Also, with EIAV infections, serologically distinct variants of EIAV emerge possibly through immune selection pressure operating on random viral genome mutations. Without being bound to any particular theory, it is believed that horses that show no further clinical signs of disease have developed a mature immune response that can protect against the virus and its known mutations.
  • EIAV is useful as a model for the pathogen icity, immunology, vaccinology, treatment and prevention of HIV.
  • the disease is significant in its own right because horses that demonstrate exposure to EIAV as measured by testing for anitbodies in the blood (Coggins Test or similar p26 detecting test) are either required to be destroyed or strictly quarantined.
  • the Coggins Test is used broadly throughout the world, especially in testing performance horses that are transferred into and out of the United States. Therefore, it is critical that an effective EIAV vaccine not seroconvert horses to a positive Coggins Test or to any other test that detects p26.
  • the gag gene encodes the core proteins of the virus designated as Matrix Antigen (MA), Capsid Antigen (CA), Nucleocapsid (NC) and a protein identified as p9.
  • the env gene encodes the viral envelope proteins (gp90 and gp45).
  • the pol gene encodes the enzymes that replicate the viral genome, designated as Deoxy UTPase (DU), Reverse Transcriptase (RT) and Integrase (IN).
  • the S1 open reading frame (ORF) encodes the viral Tat protein, a transcription trans activator that acts on the viral long-terminal- repeat (LTR) promoter element to stimulate expression of all viral genes.
  • LTR long-terminal- repeat
  • the S3 ORF encodes the Rev protein, a post-transcriptional activator that acts by interacting with its target RNA sequence, named the Rev-responsive element (RRE), to regulate viral structural gene expression.
  • the S2 gene is located in the pol-env intergenic region immediately following the second exon of Tat and overlapping the amino terminus of the Env protein (see Figure 1). It encodes a 65 amino acid protein with a calculated molecular mass of 7.2 kDa.
  • S2 appears to be synthesized in the late phase of the viral replication cycle by ribosomal leaky scanning of a tricistronic mRNA encoding Tat, S2 protein, and Env protein, respectively.
  • the gag-encoded Capsid Antigen (CA) or p26 protein comprises the capsid shell of the virion that is enclosed in the viral envelope and that contains the viral RNA genome.
  • CA proteins are present in HIV, FIV, BIV and SIV and are also encoded by the respective gag genes.
  • detection of antibodies to the p26 antigen is the basis for the Coggins Test and certain other commercial tests used to diagnose EIA in horses. To be compatible with current regulatory guidelines, it is critical that an EIAV vaccine should not cause seroconversion in these diagnostic assays based on detection of serum antibodies to EIAV p26.
  • the p26 antigen is highly antigenic in that extremely small amounts of its presence in a vaccine can stimulate antibody responses and seroconversion in diagnostic assays. Attempts to extract or delete p26 antigen from a pool of EIAV have not been practical for vaccine production. Therefore, it would seem that one could eliminate it by deletion of the gag gene, a segment of the gag gene that interferes with the expression of p26 or deletion or inactivation of a control gene that regulates the expression of p26. However, it has been determined by the inventors that deletion of the gag gene or segments thereof produces an EIAV particle that is unable to replicate in vitro (tissue culture) or in vivo. Therefore, simply deleting or blocking expression of p26 makes growth of EIAV for vaccine production impractical if not impossible.
  • envelope proteins are considered the proteins of choice, as these proteins are the predominant immune targets during infection.
  • protection from disease is meant that a mammal exposed to the virus does not demonstrate clinical signs (fever, lethargy, anemia, death, etc.), but does carry virus particles in its blood, which particles are detectable by a reverse transcriptase polymerase chain reaction test (RT-PCR).
  • RT-PCR reverse transcriptase polymerase chain reaction test
  • protection from infection is meant that a mammal exposed to the virus does not demonstrate clinical signs of EIA and does not contain RT-PCR- detectable virus particles in blood.
  • the major envelope proteins of EIAV are gp90 and gp45. These are considered the protective antigens or protective components of EIAV.
  • protective components antigens that produce either protection from disease or protection from infection as indicated above. It is therefore important that any effective lentivirus vaccine contain amounts of the lentiviral Env proteins (such as, gp 120, gp90 or gp45) effective to protect mammals from disease caused by the lentivirus.
  • the protective components from EIAV include but are not limited to gp90 and gp45.
  • a donkey virus vaccine has been in use by the Chinese for more than 20 years.
  • This vaccine was developed by using total EIAV genetic material from donkey leukocyte attenuated EIAV infected cells and ribonucleic acid from virus in peripheral blood of donkey-adapted EIAV from infected donkeys (see Xinhua News Agency, May 6, 1999).
  • this vaccine produces a p26 positive response (Coggin's Test positive) in vaccinated horses or other vaccinated equids.
  • Such a vaccine is not acceptable in those countries where equids are tested by Coggins assays or other p26-specific antibody tests.
  • numerous countries will not accept live vaccines for veterinary applications.
  • the vaccine of this invention provides a successful vaccine composition that effectively and safely immunizes mammals from diseases caused by lentiviruses.
  • the vaccine of the present invention protects equines from EIA wherein vaccinated equines can be differentiated from wild-type infected equines, which does not convert said equines to Coggins Test positive and which does not replicate in vivo. It is fully envisioned that the vaccines taught by the present invention can be used for production of any lentivirus vaccines, including vaccines for HIV, FIV, BIV and SIV.
  • FIGURE 1 is a schematic representation of EIAV designated EIAV UK .
  • Figure 2 is a circular map of infectious clone EIAV UK .
  • Figure 3a is a linear schematic of the molecular clone EIAV UK .
  • Figure 3b is a linear schematic of molecular clone EIAV UK with the CMV promoter.
  • Figure 3c is a linear schematic of molecular clone pCMVEIAV UK with the CA gene deleted.
  • Figure 3d is a linear schematic of molecular clone pCMVEIAV u ⁇ ⁇ CA with the Amp Resistance gene (Amp r ) replaced by the Kanamycin Resistance gene (Kan r ).
  • Figure 3e is a linear schematic of the p26-deleted Proviral Clone pCMV. ⁇ CA.neo.
  • Figure 4 is a circular map of the p26-deleted Proviral Clone pCMV. ⁇ CA.neo.
  • Figure 5a is a linear schematic of the EIAV UK molecular clone.
  • Figure 5b is a linear schematic representation of the El AV UK clone with the CMV promoter insert (CMVElAV UK ).
  • Figure 5c is a linear schematic representation of the pCMVEIAV UK .vis2.
  • Figure 5d is a linear schematic representation of the Proviral Clone containing the Kanamycin Resistance Marker.
  • Figure 5e is a linear schematic representation of the final pCMVEIAV UK .Vis2.neo Proviral Construct.
  • Figure 6 is a Circular map of the final pCMVEIAV UK .Vis2.neo
  • Figure 7 is the nucleotide and amino acid map of the CA gene/EIAV p26.
  • Figure 8 is the nucleotide and amino acid map of the CA gene/Visna p30.
  • Figure 9 is a comparison of the homology between p26 of EIAV and p30 of Visna virus.
  • Figure 10a is a Western Blot of p26-deleted clones, Visna chimeric clones & subclones of EIAV using gp90 & p26 monoclonal antibodies as the detector.
  • Figure 10b is a Western Blot of several p26-deleted clones, Visna chimeric clones & subclones of EIAV using p30 monoclonal antibody as the detector.
  • Figure 11 is a graph demonstrating the Reverse Transcriptase Activity of various subclones of ED cells transfected with pCMVEIAVUK.Vis2neo Proviral Construct.
  • This invention provides a vaccine composition that produces immunity to mammals from disease and/or infection caused by a lentivirus, said composition comprising a deletion in a gene that blocks replication of the virus in vivo. More specifically, this invention describes a vaccine comprising a deletion that produces a lack of ability of the lentivirus to replicate in vivo but retains the lentivirus protective components. Said vaccine allows differentiation between vaccinated and non-vaccinated, but exposed, mammals and provides safety and immunity when administered as a vaccine to mammals. Preferably said vaccine encompasses at least one deletion in a lentivirus which allows mammals to be safely vaccinated and provides protection from exposure to wild-type lentiviruses.
  • the invention further encompasses a lentivirus with a deletion in the gag gene, specifically a deletion that results in an inability of the lentivirus to express the Capsid Antigen (CA protein) in vivo or in vitro.
  • the lentivirus are equine infectious anemia virus, human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) or simian immunodeficiency virus (SIV).
  • HIV human immunodeficiency virus
  • FMV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • SIV simian immunodeficiency virus
  • safe is meant that vaccination with of mammals with vaccines of the present invention does not produce infection, disease or any other adverse reaction in the vaccinated mammals.
  • the invention also encompasses a marker vaccine in which a foreign gene is inserted into the gene-deleted region, said inserted gene providing a diagnostic tool for use in vaccinated mammals and, potentially, protection from infection from a foreign disease.
  • the invention encompasses a vaccine for effectively and safely immunizing mammals from EIA, said composition comprising a gene-deleted EIAV construct wherein said gene-deleted construct interrupts virus replication in vivo and blocks the expression of p26 in vivo while retaining the EIAV protective components.
  • vaccinated equines would be protected from disease caused by EIAV and not convert to a seropositive status on the Coggin's Test or any other test that measures p26 antibodies.
  • EIA refers to the disease Equine Infectious Anemia
  • EIAV refers to the Equine Infectious Anemia Virus that causes the disease. Additionally, said EIAV vaccine cannot cause clinical disease in mammals or spread or shed to other mammals including equines.
  • this invention encompasses a marker vaccine in which vaccinated equines that are safely and effectively protected from disease caused by EIAVcan be distinguished from non- vaccinated equines by detection of a foreign gene expressed in said vaccine and which produces antibodies in the vaccinated animals. A diagnostic test to detect this foreign gene or gene product is also described.
  • a more specific embodiment of the invention is a vaccine wherein the lack of ability to express p26 antigen results from one or more gene deletions within the gag gene, one or more deletions within a gene having a regulatory effect on gag CA production, an insertion of one or more stop codons into the gag CA gene or a gene regulating CA production, or insertion of a foreign gene into the gag CA gene or a gene regulating CA production.
  • insertion of a foreign gene is meant that the gene being inserted is not a gene associated with EIAV. Said foreign gene is obtained from a non-EIAV organism.
  • the EIAV in the vaccine contained multiple deletions including but not limited to a deletion in the gag gene affecting the expression of p26.
  • said gene deletions could serve as potential points for insertion of foreign genes to produce a multiple protective vaccine.
  • a single vaccination with the EIA vaccine carrying a foreign gene e.g., influenza hemagglutin gene
  • influenza hemagglutin gene could protect the mammal from both the lentivirus disease (e.g., HIV or EIA) and the disease associated with the foreign gene insert (e.g., human or equine influenza).
  • This invention encompasses a vaccine for effectively and safely immunizing mammals against diseases caused by lentiviruses selected from the group consisting of EIAV, HIV, FIV, BIV and SIV, said composition comprising a gene-deleted lentivirus construct.
  • This invention encompasses a vaccine comprising a deletion that produces a lack of ability of the lentivirus to replicate in vivo and retains the lentivirus protective components.
  • lentivirus protective components is meant the protective antigens associated with the envelope, said antigens including but not limited to gp120, gp90 and gp45.
  • the invention encompasses a lentivirus that is unable to express the Capsid Antigen (CA protein) in vivo.
  • a deletion can be produced in the lentivirus genome by using specific restriction endonucleases to remove all or part of one or more genes.
  • a preferred gene for removal is the gene encoding the Capsid Antigen (CA).
  • CA Capsid Antigen
  • Such gene deletion can be accomplished by using PCR, ligation and PCR cloning; to delete the selected gene sequence.
  • Restriction endonucleases can also be used to remove specific portions of genes once the gene sequence of the lentivirus and the gene sequence of the gene to be excised are known. Using specific restriction endonucleases, the gag gene can be removed in whole or part.
  • a stop codon can be inserted into the gene, preferably at the 5' end wherein the stop codon causes the gene not to express its CA protein.
  • a foreign gene from another lentivirus or an unrelated virus can be inserted into the gene-deleted region producing a multiply protective vaccine.
  • the invention describes the deletion of a region of the EIAV genome large enough to insert a gene expressing a protective antigen from a non-EIAV organism, preferably a virus. Therefore, the hemagglutinin (HA) gene from equine influenza A2 or A1 can be inserted into the gag CA region allowing expression of gp90 and gp45 of EIAV as well as HA of A1 and A2 equine influenza.
  • HA hemagglutinin
  • genes from equine herpes viruses types 1 , 2, and 4 can be inserted into the EIAV construct to provide protection against disease of equines caused by EIAV and equine herpes viruses.
  • Other equine viruses which could have genes encoding for protective antigens inserted in the EIAV include but are not limited to equine arteritis, encephalomyelitis viruses (Eastern, Western, Venezuelan and. Rift Valley Fever virus).
  • Genes encoding protective antigens from parasites (Sarcocystis neurona that causes Equine Protozoal Encephalitis or EPM, Neospora heugesi that is also possibly related to EPM,
  • Toxoplasma gondii, etc. can also be inserted into an EIAV construct to protect against these diseases.
  • genes encoding for bacterial diseases of horses including but not limited to Streptococcus equi and Clostridium tetani, can be inserted into an EIAV construct to provide multiple disease protection. It is expected that even a gene encoding for an immunostimulatory protein (immunomodulator gene) or glycoprotein can be inserted into the gene-deleted region in order to enhance the immunity provided by the virus construct.
  • a non-equine gene such as a gene from a Visna virus, can be inserted into the deleted gene region in order to produce a marker vaccine. Said marker vaccine is useful in differentiating vaccinated equines from non-vaccinated or infected equines.
  • a method for deleting a gene of a lentivirus e.g., the CA gene
  • insertion of a foreign gene utilizes the techniques of PCR, ligation, and a method of PCR cloning.
  • Primers are designed to amplify a region of a promoter-lentivirus genome upstream of the CA open reading frame (ORF). Additional primers are used to amplify the region of the promoter-lentivirus genome downstream of the CA.
  • the amplified PCR products are purified using agarose gel electrophoresis and ligated together. A final round of PCR is performed using the 5' primer of the upstream fragment, and the 3' primer of the downstream fragment, followed by gel purification. The final product would comprise a representative size of the gag gene with a deletion of the CA open reading frame.
  • the PCR product is gel purified and digested with specified restriction endonucleases such that it can be ligated with a plasmid that had been digested with the same restriction enzymes or enzymes producing the same blunt ends.
  • the ligated insert is preferably added to a lentivirus clone comprising a promoter and genes allowing for selection of clones (e.g., antibiotic resistance genes) thus producing a promotor-lentivirus clone. Then the promoter-lentivirus clone is transformed into competent bacterial cells and colonies of the bacteria are screened for insertion of the genes. Clones may be genetically sequenced to verify that the CA region had been deleted and an insert had been made.
  • a gene-defeted/gene-inserted construct (also herein designated as a chimera) could be commercially produced (produced in large scale) by transfecting susceptible tissue culture cells, harvesting the fluids and formulating the fluids with an adjuvant.
  • said harvest fluids may be inactivated with art-known inactivating agents such as formalin, binary ethyleneimine, beta-propiolactone, thimerasol and psoralen.
  • gene- deleted/gene-inserted construct is meant a lentivirus in which a deleted gene or portion thereof replaced in part or in whole by a gene from another virus, including but not limited to a lentivirus or a non-lentivirus.
  • tissue culture cells may be transfected with the construct using transfecting agents such as DEAE dextran, GenePORTERTM (Gene Therapy Systems), etc. to incorporate the necessary genomic material into the cell DNA such that the cells produce lentivirus antigens.
  • tissue culture cells are seeded into wells of tissue culture vessels (e.g., plates), exposed to the gene-deleted construct or the gene-deleted/gene-inserted construct in the presence of a transfecting agent, incubated to allow transfection and then overlayed with a selection medium.
  • Selection media is defined as any nutrient medium that contains components to kill non-transfected cells but does not inhibit growth of transfected cells.
  • gene-deleted constructs contain inserts of a resistance gene in order to allow the construct to grow in said selection media.
  • Selection media can contain antibiotics, antimicrobials and selective antibiotics.
  • Transfected cells should consistently produce the virus construct, indicating a stable transfected or producer cell.
  • stable- transfected Master Cells also referred to as persistently infected cells by various regualtory agencies
  • Working Cells are defined as those cells that are used to prepare Production Cells.
  • Production Cells are the cells used to manufacture vaccines.
  • Master Cells, Working Cells and Production Cells are all generally stored in liquid nitrogen for retaining viability and stability of the transfecting clone.
  • a vaccine comprising a gene- deleted construct lacks the ability to replicate in vivo and, possibly, in vitro.
  • this type of deletion if producing an inability to replicate or grow in vitro, requires transfection and cloning as described above.
  • CA Capsid Antigen protein
  • This invention more specifically encompasses a composition wherein the lack of ability to express p26 antigen results from one or more gene deletions within the gag gene or one or more deletions within a gene having a regulatory effect on gag CA production, or an insertion of one or more stop codons or insertion of a foreign gene.
  • a marker vaccine is a vaccine that contains a foreign gene that produces antibody in the mammal receiving a vaccination, said antibody being detected by a diagnostic test and being used to distinguish a vaccinated equid from a non-vaccinated equid and a vaccinated equid from an infected equid.
  • EIAV it is preferred to insert a CA gene from a different lentivirus that does not cross-react with p26 in the Coggins Test or equivalent tests. Therefore, insertion of the p30 gene from a different lentivirus such as a Visna virus would be expected to allow an EIAV vaccine to be used for vaccination of mammals, preferably equids. Said equids would demonstrate no p26 antibody in the Coggins Test or any other test measuring the presence of antibody to p26, and would also demonstrate antibody to p30 which could be detected by an enzyme linked immunosorbant assay (ELISA), immunodiffusion test, fluorescent antibody test (FA), or any other test that can be used to detect antibodies in mammals.
  • ELISA enzyme linked immunosorbant assay
  • FA fluorescent antibody test
  • the gag gene-deleted constructs discussed above will not grow or replicate in vitro. Therefore, in order to produce large quantities for manufacturing purposes, the cloned constructs can either be expressed by bacterial cells or by mammalian cells (tissue culture). The process of transformation has been described briefly above and is described in detail in the EXAMPLES. Production of a stable transfected tissue culture cell line (persistently infected Master Cell) is preferable and is accomplished by transfecting mammalian cells in tissue culture. A preferred technique for EIAV constructs is described in the EXAMPLES to follow.
  • the resulting p26 deleted construct can be employed in a vaccine for effectively and safely immunizing equines from EIAV, said vaccine comprising a gene-deleted EIAV construct wherein said gene deletion blocks the expression of p26 in vivo.
  • Vaccine viruses of this invention can be further treated with inactivating agents such as formalin, beta propiolactone, binary ethyleneimine, thimerasol or any other that effectively inactivates viruses.
  • inactivating agents such as formalin, beta propiolactone, binary ethyleneimine, thimerasol or any other that effectively inactivates viruses.
  • agents can be used in amounts varying from 0.00001% to 0.5%, preferably from 0.00001 % to 0.1 % and more preferably from 0.00001 % to 0.01 %.
  • adjuvants or immunomodulators/immunostimulators may be added to the vaccine to enhance the immune response produced by the vaccine.
  • Adjuvants can be selected for the group consisting of polymers such as Carbopol®-based, HAVLOGEN® and POLYGEN®, block co-polymers, oil-in-water such as EMULSIGEN® or EMULSIGEN® PLUS, water-in-oil, aluminum salts, lipid-based, lipoprotein, endotoxin- based and combinations thereof.
  • Immunomodulators and imun ⁇ - stimulators include but are not limited to Corynebacteria pyogenes and extracts or subunits thereof, parapox viruses and extracts or subunits thereof, modified live viruses that stimulate interferon production, as well as cytokines.
  • the vaccines of the present invention can be administered by any route. For instance, they can be administered intramuscularly, subcutaneously, intradermally, intranasally, orally, intravenously or intraperitoneally. It is preferable to administer the vaccines either intramuscularly, subcutaneously, orally or intranasally.
  • this invention encompasses an EIAV construct combined with antigens from the group selected from equine influenza (A1 and A2), equine herpes virus (subtypes 1 , 2, 3 or 4), equine arteritis virus, eastern equine encephalomyelitis, western equine encephalomyelitis, Venezuelan equine encephalitis, Rift Valley Fever
  • Clostridium botulinum Clostridium tetani, Clostridium difficile or any other equine disease-producing agent.
  • the Clostridium botulinum can include types A, B, C, D, E, and/or F.
  • a diagnostic test can be used to differentiate vaccinated equines from non-vaccinated and/or infected equines by measuring the presence or absence of antibodies to the deleted gene protein, to the inserted gene protein or to both proteins.
  • a PCR-based diagnostic test could be used to detect the presence or absence of the genes or gene sequences in body fluids or tissues from the equine and, thus, detect whether an equine had been infected with EIAV or vaccinated with the composition of this invention.
  • the diagnostics of choice measure the presence or absence of p26 antibodies in an equine.
  • an inserted gene from a non- equine organism such as a Visna virus
  • the protein product of the marker vaccine can be measured.
  • An example described herein includes the insertion of the p30 gene from Visna virus wherein the p30 can be detected in vaccinated equines but is not present in non-vaccinated or EIAV infected equines.
  • Diagnostic differentiation can be measured by developing an immunoassay, an antibody-detecting assay (e.g. indirect fluorescent antibody, immunodiffusion, agar diffusion, electrophoresis) or a PCR- based assay known to the art.
  • An example of an immunoassay is an enzyme linked immunosorbant assay (ELISA) that detects and/or quantitates antibodies to specific proteins in serum, blood or tissues.
  • ELISA technology could also be used to detect the presence or absence of virus-associated antigens in the blood, serum or tissues.
  • virus-associated antigens is meant the presence or absence of a gene expression product such as the p26 protein of EIAV or p30 protein of Visna virus or in the case of the p26 or p30 genes, respectively.
  • PCR- based assays have been used to measure the presence or absence of genes or gene sequences in the blood, serum or tissues of an equine, thus indicating that a horse had been infected or vaccinated, as the case may be.
  • an ELISA would detect the presence of antibodies to the p26 or p30 proteins. If p26 antibodies were present in horses that were tested it would indicate that the horse had been infected with EIAV. Horses that had been vaccinated with a gene- mutated EIAV construct containing a non-functional p26 gene would not contain p26 antibodies in their serum.
  • Horses that had been vaccinated with a gene-mutated EIAV construct containing a p30 gene insertion would contain p30 antibodies in their serum. Thus, vaccinated horses could be differentiated from infected horses.
  • the PCR-based assays would be used to detect the presence or absence of gene sequences within the horse. For instance, if a horse had been infected with a wild- type EIAV, it would contain the gene sequence for wild-type p26. However, equines immunized with vaccines comprising a gene-mutated EIAV, particularly one wherein the p26 gene comprised deletions or specific mutations would not contain the gene sequence for wild-type p26. Alternatively, horses that had been vaccinated with a gene deleted EIAV construct containing a p30 gene insertion would contain the p30 gene sequence in their serum.
  • the procedure for the construction of the p26 deletion mutant proviral clone was as follows. First, the CMV promoter was inserted into the 5' LTR region through a process of PCR, ligation, and PCR cloning. Primers CMV3'Blunt (SEQ ID No. 1 ) and 5'CMVBssH (SEQ ID No. 2) were used to amplify the CMV promoter from the plasmid pRC/CMV (InVitrogen).
  • PCR conditions were set up as follows in thin- walled 0.5ml PCR tubes (PGC Scientific): 40.6 ⁇ l dH 2 0, 5 ⁇ l cloned Pfu DNA Polymerase 10X reaction buffer, 0.8 ⁇ l 25mM Deoxy- A,C,G,T
  • the two PCR products (50 ⁇ l) were gel purified on a 0.8% agarose gel with GeneClean (Bio101). The two purified PCR products were set up in individual kinase reactions as follows: 5 ⁇ l DNA, 2 ⁇ l ATP, 2 ⁇ l 10X Protein Kinase buffer (New England Biolabs), 10 ⁇ l dH 2 O, and 1 ⁇ l Protein Kinase. The reaction product was incubated a 37°C 2 hours.
  • the resulting kinased products were purified through chloroform extraction and ethanol precipitated.
  • the resultant products (3 ⁇ l) were ligated together overnight (16°C) at their individual blunt ends with T4 ligase (New England Biolabs) in the following reaction mixture: 1 ⁇ l 10X T4 ligase buffer, 2 ⁇ l dH 2 O, and 1 ⁇ l T4 ligase.
  • a second round of PCR using the primers CMV ⁇ 'BssH (SEQ ID 2) and MA3Tth (SEQ ID 4) amplified the final product to be cloned into the EIAV UK clone.
  • the reaction conditions were as stated above using 1 ⁇ l of the ligation reaction.
  • This final PCR product (50 ⁇ l) was gel purified again on a 0.8% agarose gel.
  • the purified PCR product was digested with the restriction enzymes BssHII and Tth111 l in the following manner: 17 ⁇ l PCR product, 2 ⁇ l BssHII 10X buffer(NEB), and 2 ⁇ l BssHII (NEB), incubated at 50°C for 2 hours, chloroform extracted and ethanol precipitated.
  • the digestion was completed as follows: 16 ⁇ l DNA (BssHII digested), 2 ⁇ l 10X reaction buffer #4 (NEB), 2 ⁇ l Tth1111, incubated at 65°C for 3 hours.
  • the EIAV UK clone (500ng) was partially digested with Mlul (New England Biolabs).
  • reaction mixture 1 ⁇ l 10X # reaction buffer, 1 ⁇ l of restriction enzyme, 2 ⁇ l of dH 2 O and immediate submersion on ice followed by gel purification.
  • the appropriate size band was then completely digested with Tth1111 in a reaction mixture consisting of 1 ⁇ l 10X # 4 reaction buffer (NEB), 1 ⁇ l of restriction enzyme and 2 ⁇ l of dH 2 O.
  • the resulting fragment was gel purified on a 0.8% agarose gel.
  • the promoter fragment (3 ⁇ l) was ligated into the EIAV UK clone (3 ⁇ l) with T4 ligase in a mixture of 1 ⁇ l 10X T4 ligase buffer, 2 ⁇ l dH 2 O, and 1 ⁇ l T4 ligase.
  • the resulting ligation product (4 ⁇ l) was transformed into competent DH5 ⁇ bacterial cells (100 ⁇ l).
  • the transformation procedure consisted of: incubation on ice for 30 minutes, heat shock at 42°C for 45 seconds, incubation on ice for 2 minutes, addition of 900 ⁇ l SOC borth (a media supplement containing 20% bacto- tryptone, 5% bacto-yeast , 0.5% NaCI, 2.5mM KCI, 10 mM magnesium chloride and 20 mM glucose), incubation at 37°C for 1 hour, and 200I plated on LBAmp plates. Clones were sequenced to verify correct promoter arrangement as schematically represented in Figure 3b
  • PCR, ligation, PCR method of cloning was used to delete the Capsid Antigen (CA) sequence.
  • Primers gag441 (SEQ ID No. 5) and MAT (SEQ ID No. 6) were used to amplify a 398bp region of the molecularly- modified EIAV designated as CMVEIAV UK genome upstream of the CA open reading frame. PCR conditions were set up as follows in PGC
  • PCR products 50 ⁇ l were gel purified on a 0.8% agarose gel with GeneClean (Bio 101 ).
  • the two purified PCR products (3 ⁇ l) were ligated together overnight (16°C) with T4 ligase (New England Biolabs) in the following reaction mixture: 1 ⁇ l 10X T4 ligase buffer, 2 ⁇ l dH 2 O, and 1 ⁇ l T4 ligase.
  • T4 ligase New England Biolabs
  • a final round of PCR was performed using the gag441 primer (SEQ ID 5) and p9f3' primer (SEQ ID 8). The ligated sequence when in the correct orientation would yield a PCR product of approximately 755bp.
  • the PCR product was gel purified on a 0.8%) agarose gel with GeneClean.
  • the purified fragment was digested with Tth1111 and BsrGI in the following manner: 151 PCR product, 21 BSA, 21 10X buffer #2 (NEB), and 2 ⁇ l BsrGI (NEB), incubated at 37°C for 3 hours, chloroform extracted and ethanol precipitated.
  • the digestion was completed as follows: 16 ⁇ l DNA(BsrGI digested), 2 ⁇ l 10X reaction buffer #4 (NEB), 2 ⁇ l Tth1111, incubated at 65°C for 3 hours, and gel purified in the same manner previously mentioned.
  • the CMVEIAV UK clone was digested with the same restriction enzymes and gel purified in a similar format.
  • the two fragments (3 ⁇ l each) were ligated together with T4 ligase in a mixture of 1 ⁇ l 10X T4 ligase buffer, 2 ⁇ l dH 2 O, and 1 ⁇ l T4 ligase, and transformed into competent DH5 ⁇ bacterial cells (100 ⁇ l).
  • the transformation procedure consisted of: incubation on ice for 30 minutes, heat shock at 42°C for 45 seconds, incubation on ice for 2 minutes, addition of 900 ⁇ l SOC broth, incubation at 37°C for 1 hour, and 200 ⁇ l plated on LBAn ⁇ p plates. Individual clones were screened for insert. Clones were sequenced to verify that the CA region had indeed been deleted as schematically diagrammed in Figure 3c. The ⁇ identifies the deletion.
  • the original proviral DNA carried an ampicillin resistance marker (Amp r ). Because this would not be the ideal marker for a vaccine used in mammals, it was replaced with a Kanamycin resistant marker (Kan r ) using the following procedure.
  • the proviral DNA was subcloned into a kanamycin-resistant vector designated as pLG339/SPORT (Cunningham et al. Gene, 124: 93-98, 1993). The vector was digested with the restriction enzymes Mlul and EcoRI (New England Biolabs). The proviral clones were also digested fully with EcoRI and partially digested with Mlul.
  • the plasmids (500ng) were each partially digested individually through incubation at 37°C for 5 minutes in the following reaction mixture: 2 ⁇ l 10X # 1 reaction buffer, 1 ⁇ l of restriction enzyme (Mlul), 12 ⁇ l of dH 2 O and immediate submersion on ice followed by gel purification.
  • the appropriate size band was then completely digested with EcoRI in a reaction mixture consisting of 1 ⁇ l 10X #2 reaction buffer, 1 ⁇ l of restriction enzyme and 2 ⁇ l of dH 2 O.
  • the desired fragments were gel purified on a 0.8% agarose gel with GeneClean.
  • the proviral DNA (4 ⁇ l) and vector (2 ⁇ l) were ligated together overnight (16°C) with T4 ligase (New England Biolabs) in the following reaction mixture: 1 ⁇ l 10X T4 ligase buffer, 2 ⁇ l dH 2 0, and 1 ⁇ l T4 ligase.
  • the ligation product (4 ⁇ l) was transformed into competent DH5 ⁇ bacterial cells (100 ⁇ l).
  • the transformation procedure consisted of: incubation on ice for 30 minutes, heat shock at 42°C for 45 seconds, incubation on ice for 2 minutes, addition of 900 ⁇ l SOC broth, incubation at 37°C for 1 hour, and 200 ⁇ l plated on LBKan plates. Individual clones were screened for insert into the proper Mlul site.
  • Figure 3d shows a schematic representation of this construct demonstrating the Amp'resistance marker being replace by the Kan' resistance marker.
  • a neomycin resistance marker was added in order to allow selection of clones in eukaryotic cells.
  • the neomycin resistance marker was excised from the commercial vector pRC/CMV (InVitrogen) using the restriction enzymes EcoRI and Xhol (New England Biolabs).
  • the area excised from the pRC/CMV encompassed the entire neomycin open reading frame as well as the SV40 promoter, origin of replication, and SV40 poly A recognition sequence.
  • the digestion was executed at 37°C in a reaction mixture which consisted of 500ng pRC/CMV plasmid DNA, 2 ⁇ l 10X #2 reaction buffer, 2 ⁇ l BSA, 2 ⁇ l dH 2 O, and 1 ⁇ l each of the restriction enzymes.
  • the resulting kanamycin-resistant proviral clone was digested with the restriction enzymes EcoRI and Sail (GIBCO BRL). Sail digested ends can ligate into Xhol digested ends.
  • the digestion was carried out in the following reaction mixture: 1 ⁇ g proviral DNA, 2 ⁇ l 10X REACT 6 buffer, 2 ⁇ l BSA, 2 ⁇ l H 2 O and 1 ⁇ l each restriction enzyme.
  • the digested neomycin fragment and proviral clone were gel purified on a 0.8% agarose gel with GeneClean, and ligated together at 16°C overnight with T4 ligase in the following reaction mixture: 4 ⁇ l purified proviral DNA, 3 ⁇ l purified neomycin insert DNA, 1.5 ⁇ l 10X T4 ligase buffer, 5.5 ⁇ l dH 2 O and 1 ⁇ l T4 ligase.
  • the ligated DNA (6 ⁇ l) was transformed into competent DH5 bacterial cells (100 ⁇ l).
  • Visna Chimera Proviral Clone designated as pCMV.Vis2.neo In order to substitute a foreign gene into the Capsid Antigen region (CA) of the gag gene and perhaps, to produce a replicating Proviral Clone with a p26 deletion, it was decided to insert the p30 gene from a Visna virus, another lentivirus which does not produce a positive response on the Coggin's Test. If the p30 could be adapted to replace the mechanism for p26 of the EIAV, then a replicating proviral clone could be produced.
  • CA Capsid Antigen region
  • the procedure for preparation of this EIAV construct was as follows: The CMV promoter was inserted into the 5' LTR region of EIAV UK through a process of PCR, ligation, PCR cloning as referenced previously. Primers CMV3'Blunt (SEQ ID No.1) and 5'CMVBssH (SEQ ID No.2) were used to amplify the CMV promoter from the plasmid pRC/CMV (InVitrogen).
  • PCR conditions were set up as follows in PGC thin-walled 0.5ml PCR tubes: 40.6 ⁇ l dH 2 O, 5 ⁇ l cloned Pfu DNA Polymerase 10X reaction buffer, 0.8 ⁇ l 25mM dNTP mixture, 2.5 ⁇ l each primer (100ng/ ⁇ l), 1 ⁇ l template DNA (10ng/ ⁇ l) 2.0 ⁇ l cloned Pfu DNA Polymerase (2.5U/ ⁇ l- Stratagene). Amplification was performed in a Hybaid thermocycler and consisted of 30 cycles of: 94°C-20seconds, 60°C-20 seconds, 72°C-1 minute. Primers LTRBIunt ⁇ ' (SEQ ID No. 3) and MA3Tth (SEQ ID NO.
  • PCR products (50 ⁇ l) were gel purified on a 0.8% agarose gel with GeneClean (Bio 101 ). The two purified PCR products were, set up in individual kinase reactions as follows: 5 ⁇ l DNA, 2 ⁇ l ATP, 2 ⁇ l 10X Protein Kinase buffer (New England Biolabs), 10 ⁇ l dH 2 O, and 1 ⁇ l Protein Kinase. The reaction was incubated a 37°C 2 hours. The kinased products were purified through chloroform extraction and ethanol precipitated.
  • the resultant products (3 ⁇ l) were ligated together overnight (16°C) at their individual blunt ends with T4 ligase (New England Biolabs) in the following reaction mixture: 1 ⁇ l 10X T4 ligase buffer, 2 ⁇ l dH 2 O, and 1 ⁇ l T4 ligase.
  • the reaction conditions were as stated above using 1 ⁇ l of the ligation reaction.
  • This final PCR product (50 ⁇ l) was gel purified again on a 0.8% agarose gel.
  • the purified PCR product was digested with the restriction enzymes BssHII and Tth111 l in the following manner: 17 ⁇ l PCR product, 2 ⁇ l BssHII 10X buffer(NEB), and 2 ⁇ l BssHII (NEB), incubated at 50°C for 2 hours, chloroform extracted and ethanol precipitated.
  • the digestion was completed as follows: 16 ⁇ l DNA (BssHII digested), 2 ⁇ l 10X reaction buffer #4 (NEB), 2 ⁇ l Tth1111, incubated at 65°C for 3 hours.
  • the EIAV UK clone (500ng) was partially digested with Mlul (New England Biolabs).
  • reaction mixture 1 ⁇ l 10X # reaction buffer, 1 ⁇ l of restriction enzyme, 2 ⁇ l of dH 2 O and immediate submersion on ice followed by gel purification.
  • the appropriate size band was then completely digested with Tth1111 in a reaction mixture consisting of 1 ⁇ l 10X # reaction buffer, 1 ⁇ l of restriction enzyme and 2 ⁇ l of dH 2 O. The fragment was gel purified on a 0.8% agarose gel.
  • the promoter (3 ⁇ l) was ligated into the EIAV UK clone (3 ⁇ l) with T4 ligase in a mixture of 1 ⁇ l 10X T4 ligase buffer, 2 ⁇ l dH 2 O, and 1 ⁇ l T4 ligase.
  • the ligation product (4 ⁇ ) was transformed into competent DH5 ⁇ bacterial cells (100 ⁇ l).
  • the transformation procedure consisted of: incubation on ice for 30 minutes, heat shock at 42°C for 45 seconds, incubation on ice for 2 minutes, addition of 900 ⁇ l SOC broth, incubation at 37°C for 1 hour, and 200 ⁇ l plated on LBAmp plates. Clones were sequenced to verify correct promoter arrangement.
  • Figure 5b is a schematic representation of the EIAV UK clone with the CMV promoter insert (CMVEIAV UK ).
  • the source of the Visna p30 capsid sequence was the pVisna clone puc9-4.9V2 (Braun, MJ et al, Journal of Virology, 61 (12): 4046- 4054, 1987).
  • the Visna p30 (7 ⁇ l containing 1 ⁇ g) was excised out of the clone using the restriction enzymes Apal and Tth1111 in the following reaction: 4 ⁇ l dH 2 O, 1.5 ⁇ l BSA, 1.5 ⁇ l 10X #4 reaction buffer (NEB), .5 ⁇ l Apal and Tth1111 (NEB), incubated at 65°C for 2 hours; 0.5 ⁇ l more of Apal added to the reaction mixture and incubated at room temperature (25°C) overnight.
  • the desired fragment was gel purified in a 0.8% agarose gel with GeneClean.
  • the CMVEIAV UK clone (5 ⁇ l containing 1 ⁇ g) was digested with Blpl (NEB enzyme for Bpu1102l) and Tth 1111 (NEB) in the following reaction mixture: 1.5 ⁇ l 10X buffer #4 (NEB), and 1 ⁇ l BsrGI (NEB), 7.5 ⁇ l dH 2 O, incubated at 37°C for 3 hours, chloroform extracted and ethanol precipitated.
  • the digestion was completed as follows: 15 ⁇ l DNA(Blpl digested), 2 ⁇ l 10X reaction buffer #4 (NEB), 1 ⁇ l Tth1111, 2 ⁇ l dH 2 O, incubated at 65°C for 3 hours.
  • the digested proviral DNA was gel purified on a 0.8% agarose gel with GeneClean.
  • the two fragments were ligated with T4 ligase in the following mixture: DNA fragments (3 ⁇ l each) were ligated together with T4 ligase in a mixture of 1 ⁇ l 10X T4 ligase buffer, 2 ⁇ l dH 2 O, and 1 ⁇ l T4 ligase.
  • the ligation product (4 ⁇ l) was transformed into competent DH5 ⁇ bacterial cells (100 ⁇ l).
  • the proviral DNA was subcloned into a kanamycin-resistant vector designated as pLG339/SPORT (Cunningham et al. Gene, 124: 93-98,
  • the vector was digested partially with Mlul and fully with EcoRI (New England Biolabs).
  • the proviral clones were also digested fully with EcoRI and partially digested with Mlul.
  • the plasmids (500ng) were each partially digested individually through incubation at 37°C for 5 minutes in the following reaction mixture: 2 ⁇ l 10X #2 reaction buffer, 1 ⁇ l of restriction enzyme, 12 ⁇ l of dH 2 O and immediate submersion on ice followed by gel purification.
  • the appropriate size band was then completely digested with EcoRI in a reaction mixture consisting of 1 ⁇ l 10X #2 reaction buffer, 1 ⁇ l of restriction enzyme and 2 ⁇ l of dH 2 O.
  • the desired fragments were gel purified on a 0.8% agarose gel with 5 GeneClean.
  • the proviral DNA (4 ⁇ l) and vector (2 ⁇ l) were ligated together overnight (16°C) with T4 ligase (New England Biolabs) in the following reaction mixture: 1 ⁇ l 10X T4 ligase buffer, 2 ⁇ l dH 2 O, and 1 ⁇ l T4 ligase.
  • the ligation product (4 ⁇ l) was transformed into competent DH5 ⁇ bacterial cells (100 ⁇ l).
  • the neomycin resistance marker was excised from the commercial vector pRC/CMV (InVitrogen) using the restriction enzymes EcoRI and Xhol. This encompassed the entire 0 neomycin open reading frame as well as the SV40 promoter (SEQ ID No. 9), origin of replication (SEQ ID. No. 10), and SV40 poly A recognition sequence (SEQ ID. No. 11 ).
  • the digestion was executed at 37°C in a reaction mixture that consisted of ⁇ OOng pRC/CMV plasmid DNA, 2 ⁇ l 10X #2 reaction buffer, 2 ⁇ l BSA, 2 ⁇ l dH 2 O, and 1 ⁇ l each of the restriction ⁇ enzymes.
  • the new kanamycin-resistant proviral clone was digested with the restriction enzymes EcoRI and Sail (GIBCO BRL). Sail digested ends can ligate into Xhol digested ends. The digestion was carried out in the following reaction mixture: 1 ⁇ g proviral DNA, 2 ⁇ l 10X REACT 6 buffer, 2 ⁇ l BSA, 2 ⁇ l H 2 O and 1 ⁇ l each restriction enzyme.
  • the digested neomycin 0 fragment and proviral clone were gel purified on a 0.8% agarose gel with GeneClean, and ligated together at 16°C overnight with T4 ligase in the following reaction mixture: 4 ⁇ l purified proviral DNA, 3 ⁇ l purified neomycin insert DNA, 1.5 ⁇ l 10X T4 ligase buffer, 5.5 ⁇ l dH 2 O and 1 ⁇ l T4 ligase.
  • the ligated DNA (6 ⁇ l) was transformed into competent DH5 ⁇ bacterial cells (100 ⁇ l).
  • FIG. 5e shows a schematic drawing of the final pCMVEIAV UK .Vis2.neo proviral construct (hereinafter designated pCMV.Vis2.neo) and Figure 6 shows the final circular map of this construct.
  • the pCMV.Vis2.neo proviral construct was tested for its ability to replicate in vitro by using the standard replication assay as described in EXAMPLE 1. As with the Proviral Clone pCMV. ⁇ CA.neo, this pCMV.Vis2.neo proviral construct did not replicate in vitro and would not be expected to replicate in vivo. It was therefore decided to develop a transfected cell line (persistently-infected cell line).
  • the p26-deleted Proviral Clone pCMV. ⁇ CA.neo and proviral construct pCMV.Vis2.neo were used to evaluate their ability to transfect cells in a manner similar to the wild-type EIAV UK .
  • the procedure used was as follows. One microgram of proviral clone or proviral construct DNA was used to transfect an Equine Dermal (ED) cell line (ATCC CRL 6288).
  • ED Equine Dermal
  • the ED cell line was grown in 6 well tissue culture plates seeded with between 2 and 4 x 10 5 ED cells per well in 2 mL of the complete growth Minimum Essential Media with Earles salts (EMEM) plus 10% fetal calf serum, 100 units/mL of penicillin, 100 ⁇ g/mL of streptomycin (Gibco BRL 1 ⁇ 140-122) and 2 mm L-glutamine (Gibco BRL 25030-081 ). The plates were incubated at 37° C in a CO 2 incubator approximately 16 to 24 hours until the cells are between 50 and 80% confluent.
  • EMEM Minimum Essential Media with Earles salts
  • 0.8 mL of OPTI- 0 MEM I RSM was added to the tube containing the DNA-liposome complexes, the tube was mixed gently and the contents were overlayed onto the rinsed cells. No antibiotics were added during transfection.
  • the DNA-liposome/tissue cultures were incubated for ⁇ hours at 37°C in a C0 2 incubator. Following incubation, 1 mL of complete growth MEM containing ⁇ twice the normal concentration of serum was added to the cell culture without removing the transfection mixture. Twenty four hours following the start of transfection the medium was replaced with fresh complete growth medium (EMEM).
  • tissue culture supematants were taken at periodic intervals 0 and analyzed by using a standard reverse transcriptase (RT) assay as a measure of virus production. Supematants resulting in RT activity were titrated in an infectivity assay based on cell-ELISA readings as described by Lichtenstein et al, 1995. Neither the Proviral Clone pCMV. ⁇ CA.neo nor the proviral construct pCMV.Vis2.neo replicated in tissue culture. The 5 RT levels were less than or equal to those of the negative control in tissue culture cells normally capable of being infected with EIAV, that were exposed to the culture medium from the transfected cells.
  • RT reverse transcriptase
  • Virus particles were produced using Proviral Clone pCMV. ⁇ CA.neo and the proviral construct pCMV.Vis2.neo transfected in the monkey cell line COS-1 (ATCC_CRL 1650). Cells were plated at approximately 50% confluency into 60mm plates (Falcon) 24 hours prior to transfection. Approximately 1 ⁇ g of proviral clone DNA (pCMV. ⁇ CA.neo or pCMV.Vis2.neo ) was transfected into the cells using DEAE Dextran methodology.
  • a 50mg/ml solution of DEAE dextran was diluted 1 :50 (1 mg/ml final concentration) in Tris-buffered saline (TBS) with DNA and added to the cells in serum-free media (DMEM).
  • TBS Tris-buffered saline
  • DMEM serum-free media
  • the DNA solution was incubated on the cells for 1 hour at 37°C in the presence of 5% C0 2 with rocking every 1 ⁇ minutes. Regular growth medium was replaced at this point.
  • Forty-eight hours post-transfection the supematants were assayed for RT activity. The RT activity was detected in cell-free supernatant samples using the micro reverse transcriptase assay (Lichtenstein et al., ibid). Protein content was detected using a Western Blot Anlaysis procedure.
  • virus particles were pelleted from 10mls of cell-free supernatant over a 20% glycerol cushion in an ultracentrifuge (Beckman SW41Ti rotor) at ⁇ 0,000 x g for 45 min. Pellets were lysed in 100 ⁇ l of lysis solution containing 10mM NaCI, 1 % ' Deoxycholic acid (DOC), 0.1 % Sodium Dodecyl Sulfate (SDS), 2 ⁇ mM Tris-HCI and 1 % TritonX-100 and transferred to .5ml eppendorf tubes. After lysis, the samples were boiled in 20 ⁇ l of 6X SDS gel loading buffer and loaded onto a 12% SDS-polyacrylamide gel.
  • DOC Deoxycholic acid
  • SDS 0.1 % Sodium Dodecyl Sulfate
  • EIA PV Gradient purified EIA PV (1 ⁇ g) was also loaded onto the gel to serve as a marker for viral proteins. Electrophoresis was carried out at approximately 10mA overnight with cooling. Proteins were transferred onto Millipore membranes using BioRad's protein transfer cell system in a buffer containing 2 ⁇ mM Tris, 192mM glycine, 20% methanol and 0.0 ⁇ % SDS. Transfer was completed after 3 hours at 400mA with cooling. EIAV proteins were detected using monoclonal antibodies. Prior to antibody incubation the blot was blocked in 5% blotto (5% drymilk, 5% FBS and 0.2 ⁇ % Tween-20 in 1X PBS) for 1 ⁇ hour at room temperature.
  • 5% blotto 5% drymilk, 5% FBS and 0.2 ⁇ % Tween-20 in 1X PBS
  • Mouse monoclonal ⁇ -gp90 and -p26 were used together in 5% blotto for 1 hour at room temperature.
  • Secondary antibody ⁇ -mouse IgG conjugated with horse-radish-peroxidase (Sigma lot # 115H899 ⁇ ) was incubated at room temperature for one hour.
  • the blot was washed for 3-5 minute periods in 1XPBS/0.025% Tween-20 between 0 primary and secondary antibody incubations.
  • Stable Transfections in CHO, C-33A & ED-MCS Cell Lines 0 Stable production of virus particles was attempted in three cell lines; a human cell line C-33A (ATCC HTB-31), a Chinese hamster ovary cell, CHO (ATCC CRL-9618), and an equine cell line ED-MCS. Transfections were all done in duplicate. Cells were consistently maintained in an incubator at 37°C with ⁇ %C0 2 . Cell lines were seeded ⁇ onto 10mm plates manufactured by Sarstedt and Falcon 24 hours prior to transfection at the following densities: CHO & C-33A 1X 10 6 cells/plate, ED-MCS 3.5 X 10 5 cells/plate.
  • Proviral clones, pCMV.Vis2.neo and pCMV. ⁇ CA.neo (20 ⁇ g/plate) were transfected into the cells using 55 ⁇ l of the reagent GenePORTERTM (Gene Therapy Systems) in serum-free 0 DMEM (Gibco). Manufacturers' instructions were followed. Twenty-four hours post-transfection media was changed from transfection media to selection media (DMEM) which contained 800g/ml G-418 (Geneticin, Gibco BRL) and 10% FBS (Hyclone). A plate that was not transfected was carried as a control for selection in the same media.
  • DMEM selection media
  • G-418 Geneticin, Gibco BRL
  • FBS Hyclone
  • RT activity initially indicated highest production in the human and mouse cell lines.
  • the equine dermal cell line proved to develop the most stable construct during long-term production, producing continuously the highest levels out to post-selection day 150.
  • tissue culture cells can be transfected by the p26-deleted clone as well as by the chimera wherein a foreign gene from a Visna virus (p30) was inserted into the p26 region.
  • Reverse trascriptase activity from these transfected cells reached levels as high as 10,000 CPM/10 ⁇ l of tissue culture fluid. This is equivalent to RT activity produced by wild-type EIAV when transfected into tissue culture.
  • Western Blot analysis was conducted as described previously except that a second western blot was done in the same format as before, re-probing the membrane with goat ⁇ -Visna p30 to detect the Visna chimera proteins.
  • Virus particles were pelleted from 10mls of cell-free supernatant over a 20% glycerol cushion in the ultracentrifuge SW41Ti rotor (Beckman). Pellets were lysed in 100 ⁇ l of lysis solution containing 10mM sodium chloride (NaCI), 1% DOC, 0.1 % Sodium Dodecyl Sulafte (SDS), 25mM Tris-HCI and 1 % TritonX-100 and transferred to 1.5ml eppendorf tubes. After lysis, the samples were boiled in 20 ⁇ l of 6X SDS buffer gel loading buffer and loaded onto a 12% SDS-polyacrylamide gel.
  • NaCI sodium chloride
  • SDS 0.1 % Sodium Dodecyl Sulafte
  • EIAV PV One microgram of gradient purified pony virus EIAV PV was also loaded onto the gel to serve as a marker for viral proteins. Electrophoresis was carried out ⁇ at approximately 10mA overnight with cooling. Proteins were transferred onto Millipore membranes using BioRad's protein transfer cell system in a buffer containing 2 ⁇ mM Tris, 192mM glycine, 20% methanol and 0.0 ⁇ % SDS. Transfer was completed after 3 hours at 400mA with cooling. EIAV proteins were detected using monoclonal antibodies. Prior to antibody 0 incubation the blot was blocked in 5% blotto ( ⁇ % drymilk, 5% FBS and 0.2 ⁇ % Tween-20 in 1X PBS) for 1 hour at room temperature.
  • Mouse monoclonal ⁇ -gp90 and ⁇ -p26 were used together in ⁇ % blotto for 1 hour at room temperature.
  • Secondary antibody -mouse IgG conjugated with horse-radish-peroxidase (Sigma lot # 11 ⁇ H899 ⁇ ) was incubated at room ⁇ temperature for one hour.
  • the blot was washed for 3- ⁇ minute periods in 1XPBS/0.02 ⁇ % Tween-20 between primary and secondary antibody incubations.
  • the second western was done in the same format as before, re-probing the membrane with goat ⁇ -Visna p30 to detect the Visna chimera proteins.
  • Secondary antibody was ⁇ -goat IgG whole molecule-HRP (Sigma lot# 117H4831 ).
  • the Visna p30 protein was 0 detected in the Visna chimeric proviral constructs (pCMV.Vis2.neo) see Figure 10b.
  • the presence of gp90 indicates that these p26-deleted constructs produce the protective antigen.
  • the fact that the chimera (pCMV.Vis2.neo) also produces the Visna p30 antigen confirms that vaccines prepared from the latter can be defined as Marker vaccines, ⁇ Not only do they lack the ability to produce p26 antibodies in animals but they also cause the animals vaccinated with them to produce antibodies to p30.
  • the presence of p30 in an equine will indicate that the horse has been vaccinated.
  • An assay to detect the presence of this p30 antibody can be developed in order to differentiate horses that are vaccinated with 0 the vaccines of this invention from horses that have not been vaccinated or horses that have been infected with wild-type EIAV.
  • a diagnostic that detects all or part of the p30 gene sequence or the p30 protein can be used similarly as a diagnostic tool.
  • Stablely-selected Visna (pCMV.Vis2.neo) transfected ED-MCS cells which had been frozen back at day 40 of selection were thawed at 37°C and seeded into a T7 ⁇ flask in normal growth medium (no G-418).
  • Cells 0 were grown at 37°C with ⁇ % CO 2 in G-418-negative medium for 48-hours prior to plating for cloning.
  • Cells were trypsonized from the T7 ⁇ flasks, counted, an plated onto 100mm Falcon plates at densities of approximately 100 cells per plate. The cells were selected in medium containing 800 ⁇ g/ml G-418. Media was changed approximately every four ⁇ days and cells were grown in the plates until visible colonies had formed.
  • the mixture of supernatant (sample) and reaction mixture are mixed together and incubated at 37°C for 1.5hr-2.0hr.
  • the total volume is ( ⁇ 60 ⁇ l) pipetted onto DEAE coated filter paper and allowed to dry completely.
  • the filters are then washed 3X for 1 ⁇ minute each in 1X SSC and again allowed to dry completely.
  • the filters are then immersed in scintillation fluid and the incorparated activity measured.
  • the 12 "subclones" with the highest RT activity were trypsonized and passaged into 6-well plates (Falcon), still selecting in 800 ⁇ g/ml G-418. Supematants were analyzed for RT activity after 4 days of selection in the 6-well plates.
  • the 8 subclones with the highest RT activity were trypsonized and passaged into T7 ⁇ flasks (Falcon) still selecting in 800 ⁇ g/ml G-418. Supematants were analyzed for RT activity after 7 days of selection in the flasks. The amount of G-418 was reduced at this passage point to 600 ⁇ g/ml. Selection was carried out for 4 more days, RT activity analyzed, and the level of G-418 lowered again to 400 ⁇ g/ml. After 7 days of selection another RT assay was performed on the 8 subclones to monitor selection. Following 7 more days of selection, another RT assay was performed. The 4 highest producing cell lines were passaged again, lowering the level of G-418 to 200 ⁇ g/ml (the other 4 were frozen back).
  • the highest-producing subclone, F-1V2.23 (also designated ⁇ V2.23), was producing a high level of RT activity (between 4000 and ⁇ 0,000 CPM per 10 ⁇ l of tissues culture fluid as shown in Figure 11.
  • Figure 12 shows an electron micrograph of this subclone of ED MCS cells transfected with the pCMV.vis2.neo Proviral Construct producing virus-like particles.
  • the top EM demonstrates a released virus-like particle whereas 0 the bottom EM shows the virus-like particle budding from the cells. This result indicates that the constructs of this invention produce virus-like particles that bud from a cell and appear like a typical lentivirus.

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Abstract

L'invention concerne un vaccin chimère contre un lentivirus, un diagnostic servant à détecter ce lentivirus et des procédés de préparation et d'utilisation de ce vaccin qu'on peut mettre en application en toute sécurité et de façon efficace afin d'immuniser des mammifères contre une infection et/ou une maladie provoquées par un lentivirus, tel que le virus de l'anémie infectieuse équine (EIAV), ce vaccin permettant une différentiation entre des mammifères vaccinés et des mammifères non vaccinés mais exposés, en particulier, des chevaux.
EP01966602A 2000-09-09 2001-09-06 Vaccin chimere contre le virus de l'anemie infectieuse equine et diagnostic Withdrawn EP1345622A2 (fr)

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US3873690A (en) * 1974-01-23 1975-03-25 Iii James H Rand Equine infectious anemia vaccine
US5614404A (en) * 1988-06-10 1997-03-25 Theriod Biologics, Incorporated Self-assembled, defective, non-self-propagating lentivirus particles
US5955342A (en) * 1994-08-15 1999-09-21 Connaught Laboratories Limited Non-infectious, replication-defective, self-assembling HIV-1 viral particles containing antigenic markers in the gag coding region
JP2001513643A (ja) * 1997-03-06 2001-09-04 ウーベルラ、クラウス レンチウイルスをベースとするベクター及びベクター系
NZ500740A (en) * 1997-05-13 2001-02-23 Univ North Carolina Recombinant lentivirus-based gene transfer vectors comprising 3 vectors from Equine Infectious Anemia Virus (EIAV)
PT1895010E (pt) * 1997-12-22 2012-01-25 Oxford Biomedica Ltd Vectores com base no vírus da anemia infecciosa equina (eiav)
CN1173036C (zh) * 1999-04-21 2004-10-27 卫生部艾滋病预防与控制中心 马传染性贫血病毒驴白细胞弱毒疫苗株的全长基因序列

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HAGA T. ET AL: "A new approach to AIDS research and prevention: the use of gene-mutated HIV-1/SIV chimeric viruses for anti-HIV-1 live attenuated vaccines", MICRBIOL. IMMUNOL., vol. 42, no. 4, 1998, pages 245 - 251 *
WANG C.-T.; BARKLIS E.: "Assembly, processing and infectivity of human immunodeficiency virus type 1 gag mutants", JOURNAL OF VIROLOGY, vol. 67, no. 7, July 1993 (1993-07-01), pages 4264 - 4273 *

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BR0113745A (pt) 2004-03-02
WO2002020049A3 (fr) 2003-07-03

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