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EP1080217A1 - Systeme d'expression de cytomegalovirus de recombinaison ciblant la region du gene precoce immediat - Google Patents

Systeme d'expression de cytomegalovirus de recombinaison ciblant la region du gene precoce immediat

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Publication number
EP1080217A1
EP1080217A1 EP99924573A EP99924573A EP1080217A1 EP 1080217 A1 EP1080217 A1 EP 1080217A1 EP 99924573 A EP99924573 A EP 99924573A EP 99924573 A EP99924573 A EP 99924573A EP 1080217 A1 EP1080217 A1 EP 1080217A1
Authority
EP
European Patent Office
Prior art keywords
recombinant
virus
cytomegalovirus
promoter
sequence
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Withdrawn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
EP99924573A
Other languages
German (de)
English (en)
Inventor
Malcolm Andrew Lawson
Serani Lea Henri Van Dommelen
Megan Louise Lloyd
Geoffrey Randolph Shellam
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.)
Agricultural Protection Board Of Western Australi
Executive Director Of Department Of Conservat
Australian National University
Commonwealth Scientific and Industrial Research Organization CSIRO
University of Western Australia
Grains Research and Development Corp
Original Assignee
Agricultural Protection Board of Western Australia
EXECUTIVE DIRECTOR OF DEPARTMENT OF CONSERVATION AND LAND MANAGEMENT
Australian National University
Commonwealth Scientific and Industrial Research Organization CSIRO
University of Western Australia
Grains Research and Development Corp
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
Priority claimed from AUPP3656A external-priority patent/AUPP365698A0/en
Priority claimed from AUPP9584A external-priority patent/AUPP958499A0/en
Application filed by Agricultural Protection Board of Western Australia, EXECUTIVE DIRECTOR OF DEPARTMENT OF CONSERVATION AND LAND MANAGEMENT, Australian National University, Commonwealth Scientific and Industrial Research Organization CSIRO, University of Western Australia, Grains Research and Development Corp filed Critical Agricultural Protection Board of Western Australia
Publication of EP1080217A1 publication Critical patent/EP1080217A1/fr
Withdrawn legal-status Critical Current

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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
    • C12N15/86—Viral vectors
    • 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
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16741—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16743—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element

Definitions

  • the present invention relates to a recombinant cytomegalovirus (CMV) vector and in particular to a recombinant CMV which includes at least a heterologous DNA sequence within the genomic region encoding for immediate-early proteins.
  • CMV cytomegalovirus
  • derived from shall be taken to indicate that a specific integer may be obtained from a particular source albeit not necessarily directly from that source.
  • Cytomegaloviruses are a subfamily of Herpesviridae (commonly known as the herpes viruses). They exhibit a narrow host range, there is a long duration in their replication cycle frequently resulting in slowly progressing lytic foci in cell culture, and they often produce enlarged cells (cytomegalia) both in vitro and in vivo.
  • CMVs have a genomic organisation that is distinct from other herpes virus groups.
  • the 230-kb dsDNA genome of human CMV (HCMV) was sequenced in about 1990 and has at least 200 open reading frames (ORFs).
  • ORFs open reading frames
  • the function of some HCMV proteins are known or predicted due to their homology with other viral and cellular proteins. However, for the majority of the HCMV ORFs, the function(s) of the proteins they encode is unknown.
  • Murine CMV (MCMV) has also been sequenced and is closely related to HCMV. Seventy-eight of the 170 MCMV ORF exhibits sequence similarity with HCMV.
  • Viral delivery systems have been used for many years, beginning with vaccinia virus recombinant studies. In those studies vaccinia virus was genetically manipulated to express heterologous antigens. While the concept of using viruses as a delivery system for heterologous genes and or antigens became obvious with those studies, what was not obvious was the answer to a more practical question of which are the best candidate virus vectors. In answering this question, details of the pathogenicity of the virus, its site of replication, the kind of immune response it elicited, the potential for it to express foreign antigens and its suitability for genetic engineering, are all factors in the selection. For example, a viral vector carrying a therapeutic agent needs to target the correct cell type to deliver the therapeutic agent. One viral subfamily that has received little attention as a possible vector system are cytomegaloviruses.
  • the present invention seeks to provide a CMV vector that ameliorates some or all of the problems associated with the prior art. Summary of the Invention
  • the present invention consists in a recombinant cytomegalovirus comprising: a cytomegalovirus genome including within the immediate-early (IE) gene region of the virus genome, a genetic cassette including at least a promoter adjacent to at least a DNA sequence and a polyadenylation stop sequence, wherein (1 ) the promoter is adapted to drive the expression of the DNA sequence, and (2) the promoter, DNA sequence and the polyadenylation stop sequence is heterologous to the virus in which the cassette is inserted.
  • IE immediate-early
  • the invention consists of a recombinant cytomegalovirus comprising: a cytomegalovirus genome including within the immediate-early (IE) gene region of the virus genome, a genetic cassette including at least a HCMV /E-1 promoter adjacent to at least a DNA sequence and a polyadenylation stop sequence, wherein (1 ) the HCMV /E-1 promoter is adapted to drive the expression of the DNA sequence, and (2) both the DNA sequence and the polyadenylation stop sequence are heterologous to the virus in which the cassette is inserted.
  • IE immediate-early
  • the cassette is inserted into the IE gene region in such a manner that it inactivates at least a gene that encodes for an IE protein.
  • insertion of the cassette into the viral genome leads either to activation of an identifiable phenotype or its deletion from the viral genome therein providing a means for selecting for the transformed virus.
  • the heterologous DNA sequence(s) employed in the cassette may encode part or all of any natural or recombinant protein that is heterologous to the CMV species from which the recombinant virus is formed.
  • the heterologous DNA sequence encodes one or more of the following: zona pellucida (ZP) protein(s); malarial surface antigen; beta-galactosidase; major antigenic viral antigen(s) (eg Haemagglutinin (HA) from influenza virus); eukaryotic polypeptide(s); enzyme inhibitor(s); hormone(s); lymphokine(s); cytokine(s); chemokine(s); plasminogen activator(s); or natural, modified or chimeric immunoglobulin or a fragment thereof.
  • the heterologous DNA sequence encodes either one or more of the ZP proteins or a viral antigen like HA.
  • the present invention also provides a prophylactic or therapeutic substance that comprises a recombinant virus vector produced according to the present invention in a pharmaceutically acceptable vehicle.
  • the pharmaceutically acceptable vehicle may also comprise one or more excipients, adjuvants, stabiliser or other like substances.
  • a pharmaceutical of this type should be prepared and used according to standard techniques known in the art.
  • the present invention also provides a a method for manufacturing a prophylactic or therapeutic substance, comprising the steps of: a) growing the recombinant CMV as herein described in a suitable host cell system; and b) mixing the resulting virus in an effective immunising amount with a pharmaceutically acceptable excipient.
  • the present invention employs the use of CMV as a vector for the delivery of polypeptides and therapeutic agents.
  • CMV is well suited for this role because it is ubiquitous in nature; it is a DNA virus; non-mutagenic; non-integrative; stable; it has benign effects in healthy individuals; and species specific CMV are commonly found in most animal populations.
  • Immunoglobulin responses to the expressed antigens are predominantly of a lgG2a isotype, predictive of a Type 1 immune response.
  • inoculation of animals with CMV generates a long lasting IgA response in the salivary gland.
  • the present inventors have found that a recombinant CMV produced in accordance with the present invention can boost an existing immune response to the expressed antigen to give long lasting IgA responses in the salivary gland. Responses can be detected in the absence of significant detectable virus replication in immunologically competent animals.
  • a recombinant cytomegalovirus comprising: a cytomegalovirus genome including within the immediate-early (IE) gene region of the virus genome, a genetic cassette containing at least a promoter adjacent to at least a DNA sequence and a polyadenylation stop sequence, wherein (1 ) the promoter is adapted to drive the expression of the DNA sequence, and (2) the promoter, DNA sequence and the polyadenylation stop sequence are heterologous to the virus in which the cassette is inserted.
  • IE immediate-early
  • rMCMV recombinant virus
  • rMCMV-ovalbumin OVA
  • rMCMV-HA replicate in the salivary glands of IFN-alpha/gamma receptor knock-out mice, to levels comparable to those attained by parental (RM427+ or K181 -WT) MCMV in immunocompentent animals.
  • rMCMV-OVA also replicates in the salivary gland of IFN-alpha R ko mice. These mice are defective in their immune responses. This suggests that rMCMV is able to replicate in salivary glands once the host immune response is defective. More specifically, it appears to be an early/innate immune response that is responsible for controlling virus replication.
  • rMCMV-OVA and rMCMV-HA both generate an immune reponse to OVA and HA antigens, respectively, in the immunocompetent BALB/c mice.
  • the predominant antibody response is of the lgG2a isotype, indicating a Th1-type response. This indicates that low levels of rMCMV replication are sufficient to generate an antibody response to the expressed foreign antigen.
  • Salivary gland rMVMC-OVA derived from the IFN-alpha/gamma R ko mice when passaged through IFN-alpha R ko mice or BALB/c weanling mice, in an attempt to generate SGV show no virus.
  • the cassette is inserted into the IE gene region in such a manner that it inactivates at least a gene which encodes for an IE protein.
  • the recombinant cytomegalovirus into which the cassette is inserted contains an identifiable phenotype such as lac Z or other antibiotic resistance markers.
  • insertion of the cassette into the viral genome leads either to activation of the identifiable phenotype or its deletion from the viral genome therein providing a means for selecting for the transformed virus.
  • the cassette employed in the present invention may be inserted into the CMV genome at any position that does not disrupt virus growth or replication.
  • the cassette is inserted into the CMV genome within or between the IE genes in the selected CMV.
  • the cassette is inserted within one or more of the IE genes in the viral genome the insertion should not disrupt virus growth or replication.
  • the cassette is inserted between the IE genes in the viral genome, again the insertion should not disrupt virus growth or replication. In mice, this may be achieved by inserting the cassette either between genes /E-1 and /E-2 or between genes /E-2 and /E-3. In humans, this may be achieved by inserting the cassette between genes /E-1 and /E-2 or after the /E-2 gene.
  • promoter includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation in eukaryotic cells, with or without a CCAAT box sequence and additional regulatory elements (ie. upstream activating sequences, enhancers and silencers). Promoters may also be lacking a TATA box motif, however comprise one or more "initiator elements” or, as in the case of yeast-derived promoter sequences, comprise one or more "upstream activator sequences" or "UAS" elements. For expression in prokaryotic cells such as bacteria, the promoter should at least contain the -35 box and -10 box sequences.
  • a promoter is usually, positioned upstream or 5', of the heterologous DNA sequence, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
  • promoter is also used to describe a synthetic or fusion molecule, or derivative that confers, activates or enhances expression of the heterologous DNA sequence in a cell.
  • Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the gene and/or to alter the spatial expression and/or temporal expression.
  • Placing a heterologous DNA sequence operably under the control of a promoter sequence means positioning the said sequence such that its expression is controlled by the promoter sequence.
  • the preferred positioning of a regulatory sequence element with respect to a heterologous DNA sequence to be placed under its control is defined by the positioning of the element in its natural setting, ie. the gene(s) from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
  • promoters suitable for use in regulating the expression of the heterologous DNA sequence include viral, fungal, yeast, insect, animal and plant derived promoters.
  • Preferred promoters are capable of conferring expression in a eukaryotic cell, especially mammalian cell.
  • the promoter may regulate the expression of a gene constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as environmental stress, or hormones amongst others.
  • promoter will depend upon the nature of the cell being transformed and the heterologous DNA sequence to be expressed. Such persons will be readily capable of determining functional combinations of minimum promoter sequences and operators for cell types in which the inventive method is performed.
  • the promoter is a viral promoter, mammalian promoter, a bacterial or bacteriophage promoter sequence.
  • the promoter is the CMV promoter sequence, more preferably the CMV-IE promoter or alternatively, the SV40 promoter and, in particular, the SV40 late promoter sequence.
  • the present invention consists in a recombinant cytomegalovirus comprising: a cytomegalovirus genome including within the immediate-early (IE) gene region of the virus genome, a genetic cassette containing at least a HCMV /E-1 promoter adjacent to at least a DNA sequence and a polyadenylation stop sequence, wherein (1) the HCMV /E-1 promoter is adapted to drive the expression of the DNA sequence, and (2) both the DNA sequence and the polyadenylation stop sequence are heterologous to the virus in which the cassette is inserted.
  • IE immediate-early
  • the cassette used in the present invention possesses its own promoter, it need not necessarily include other regulatory elements (eg enhancers) to promote gene expression.
  • the cassette is preferably inserted into the viral genome in such a manner ensuring that the endogenous viral regulatory elements influence expression of the heterologous DNA sequence in the cassette via the HCMV /E-1 promoter.
  • high constitutive levels of expression may be achieved by inserting the HCMV /E-1 promoter adjacent to the MCMV /E-1/ /E-2/ /E-3 enhancer.
  • the cassette includes all of the regulatory elements to enhance expression via the HCMV /E-1 promoter. Such elements will be well known to those of ordinary skill in this field.
  • the cassette also includes a heterologous polyadenylation stop sequence.
  • a heterologous polyadenylation stop sequence may be used in the present invention.
  • the polyadenylation stop sequence is derived from the SV40 polyadenylation stop sequence; human or bovine growth factor p[A], retroviral 3' LTR, together with any other long lived hormonal (cytokine for eg) polyadenylation stop sequence.
  • the heterologous DNA sequence encodes at least a polypeptide.
  • polypeptide as used herein includes within its scope parts of polypeptides such as peptides, oligopeptides as well as complete or substantially complete proteins.
  • heterologous DNA sequence(s) employed in the cassette may encode part or all of any natural or recombinant protein that is heterologous to the CMV species from which the recombinant virus is formed.
  • the heterologous DNA sequence might encode a polypeptide which consists of a plurality of antigenic and/or immunogenic peptides linked together in such a way that each of the peptides retains its immunological identity when expressed in a protein.
  • the heterologous DNA sequence may encode one or more of the following, by way of example: ZP proteins; malarial surface antigens; beta-galactosidase; major viral antigen eg HA from influenza virus or in the case of human immunodeficiency virus (HIV) the heterologous DNA sequence might encode for HIV gp 120 and or the DNA sequence for the HIV gag protein; eukaryotic polypeptide such as, for example, a mammalian polypeptide; enzyme(s), e.g. chymosin or gastric lipase; enzyme inhibitor(s), e.g. tissue inhibitor of metalloproteinase (TIMP); hormone(s), e.g. growth hormone; lymphokine(s), e.g.
  • cytokine(s) e.g interleukin-2, IL-4, IL-6 etc
  • chemokine(s) eg macrophage inflammatory protein-2
  • plasminogen activator(s) e.g. tissue plasminogen activator (tPA) or prourokinase
  • tPA tissue plasminogen activator
  • prourokinase or a natural, modified or chimeric immunoglobulin or a fragment thereof including chimeric immunoglobulins having dual activity such as antibody-enzyme or antibody-toxin chimeras.
  • heterologous DNA sequence encodes either one or more of the ZP proteins from the animal species for which the CMV virus is specific or a viral antigen such as HA from influenza virus.
  • Zona pellucida is an extracellular matrix that surrounds growing oocytes and ovulated eggs. Antibodies directed against these proteins have been shown to exert a contraceptive effect in some animals.
  • the heterologous DNA sequence may encode one or more of the murine ZP1 , ZP2 or ZP3 proteins. Desirable the DNA sequence encodes ZP3 where immunocontraception of mice is required.
  • heterologous DNA sequence may encode only a single polypeptide sequence in the cassette, it will be appreciated by those skilled in the art that a plurality of heterologous DNA sequences may be linked together and inserted into the cassette to produce a plurality of polypeptides.
  • Such polypeptides may be either produced as a fusion protein or engineered in such a manner to result in two separate polypeptide sequences. Where the polypeptides are fused, at least one of the polypeptide sequences is preferably capable of being membrane bound. In such circumstances the polypeptide is preferably linked to a polypeptide which is expressed on a cells surface.
  • the polypeptide may be a transferrin receptor (eg.
  • the human transferrin receptor or any other polypeptide sequence that comes with a natural membrane bound anchoring sequence.
  • haemagglutinin and ZP-3 come with their own anchoring sequences.
  • Multiple expression of polypeptides may also be useful for example if the CMV cassette was engineered to express a plurality of antigens (ie. protective antigens) along with cytokines or other immunomodulators to enhance the generation of an immune response.
  • the cassette may be used to encode the immunogenic polypeptides in association with a range of epitopes which contribute to T-cell activity. In such circumstances the cassette preferably encodes epitopes capable of eliciting either a T helper cell response or a cytotoxic T-cell (CTL) response or both.
  • CTL cytotoxic T-cell
  • the heterologous DNA sequence may also encode one or more proteins which serve to enhance the effect of the protein being expressed.
  • proteins which serve to enhance the effect of the protein being expressed.
  • ubiquitination of viral proteins expressed from DNA vectors results in enhancement of cytotoxic T-lymphocyte induction and antiviral protection after immunisation.
  • the heterologous genetic sequence may encode ubiquitin in association with the protein to be expressed thus targeting the resulting fusion protein to proteosomes for efficient processing and uptake by the MHC class I complexes.
  • Interferon gamma causes increased surface expression of MHC class I proteins.
  • increasing the expression of class I heavy chains in CMV-infected cells is preferred for the efficient generation of polypeptide-/E-specific CTLs. Therefore in a preferred form of the invention those genes capable of down regulating the major histocompatibility complex in CMV are preferably deleted from the recombinant virus. This may be achieved using the method described in US Patent specification US 5720957 incorporated herein by reference [ii]. This patent specification teaches a method for identifying a gene sequence capable of down regulating the major histocompatibility complex and deleting the identified gene sequence from the cytomegalovirus genome.
  • the present invention also provides a prophylactic or therapeutic substance that comprises a recombinant virus vector produced according to the present invention in a pharmaceutically acceptable vehicle.
  • the pharmaceutically acceptable vehicle may also comprise one or more excipients, adjuvants, stabiliser or other like substances.
  • a pharmaceutical of this type should be prepared and used according to standard techniques known in the art.
  • the prophylactic or therapeutic substance comprises a pharmaceutically acceptable excipient and an effective immunising amount of a recombinant CMV prepared according to the present invention.
  • An effective immunising amount of the recombinant CMV may be determined according to techniques well known in the art.
  • the present invention also provides a method for manufacturing a prophylactic or therapeutic substance, comprising the steps of: a) growing the recombinant CMV as herein described in a suitable host cell system; and b) mixing the resulting virus in an effective immunising amount with a pharmaceutically acceptable excipient.
  • Host cell lines contemplated to be useful in the method of the invention include any eucaryotic cell lines that can be immortalised and which is capable of supporting CMV replication, ie. are viable for multiple passages, (eg., greater than 50 generations), without significant reduction in growth rate or protein production.
  • Useful cell line should also be easy to transfect, be capable of stably maintaining foreign RNA with an unarranged sequence, and have the necessary cellular components for efficient transcription, translation, post-translation modification, and secretion of the protein.
  • preferred cells are those having simple media component requirements, and which can be adapted for suspension culturing.
  • Most preferred are mammalian cell lines that can be adapted to growth in low serum or serum-free medium.
  • Representative host cell lines include human fibroblast (for human cells) or fibroblast cells derived the species for which the CMV vector is specific, immortal NIH3T3 cells, E6/E7 immortalised cells, astorocytoma cell lines such as U373-MG, stromal cells and macrophage like cell lines and the like.
  • Other useful cells and cell lines can be obtained from the American Type Culture Collection (ATCC), Rockville, Md. or from the European Collection of Animal Cell Cultures, Porton Down, Salisbury SP40JG, U.K.
  • all means for introducing viral vectors into a cell are contemplated including, without limitation, CaPO.sub.4 co-precipitation, electroporation, DEAE-dextran mediated uptake, protoplast fusion, microinjection and lipofusion.
  • the invention contemplates either simultaneous or sequential transfection of the host cell with vectors containing the RNA sequences to be integrated into the genome.
  • Recombinant CMV may be purified by any protein purification method known in the field. Purification may be achieved by techniques such as, for example, salt fractionation, chromatography on ion exchange resins, affinity chromatography, centrifugation, and the like. See, for example, Methods in Enzymology or Current Protocols in Protein Chemistry for a variety of methods for purifying proteins [iii][iv]. Preferably they are purified by a combination of sucrose and caesium chloride gradient centrifugation using methods which are well described in the literature.
  • therapeutics are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified, or the particles encapsulated in liposomes.
  • the virus particles are formulated into therapeutics with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • excipients which may be used in such a formulation include, water, saline, ethanol, dextrose glycerol, or the like and combinations thereof.
  • the virus-like particle formulation may also contain minor amounts of auxiliary substances such as adjuvants, wetting, pH buffering agents, or emulsifying agents that enhance the effectiveness of the vaccine.
  • Suitable adjuvants which may be include in such formulations for example, aluminium hydroxide, N-acetyl-muramy1-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor- muramyl -L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-(r-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) methylamine (MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS).
  • thr-MDP N-acetyl-muramy1-L-threonyl-D-isoglutamine
  • nor-MDP N-acetyl
  • Virus particles may also be formulated into therapeutics as neutral or salt forms.
  • Pharmaceutically acceptable salts include, for example, the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from in- organic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamins, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • Virus particle formulations may also be administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
  • the quantity of virus particles to be administered will generally depend on the regulatory elements included in the cassette and the half-life of the polyadenylation site.
  • the dosage will also depend on the subject to be treated, the capacity of the subject's immune system to respond, and the degree of protection desired. Precise amounts of active ingredient required to be administered will depend on the judgment of the practitioner and may be peculiar to each subject.
  • Formulations may be administered by the intradermal, subcutaneous or intramuscular routes, or by other routes including oral, aerosol, parenteral, intravenous, intraperitoneal, rectal or vaginal administration.
  • the virus-like particles may be administered parenterally, by injection, for example, either subcutaneously or intramuscularly. All the above formulations are commonly used in the pharmaceutical industry and are known to suitably qualified practitioners.
  • the virus particles may be delivered with diluents (water, saline etc) and/or delivery vehicles (tablets, capsules) which do not interfere with the activity of the particles.
  • Oral formulations may include for example excipients such as, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Such formulations may also take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders
  • Rectal or vaginal administration also requires specific formulation into acceptable forms that contain lubricants and or emulsifying agents.
  • such formulations usually include, traditional binders and carriers such as, polyalkylene glycols or triglycerides.
  • the therapeutic may be given in a single dose schedule, or preferably in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of delivery may be with for example 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at I-4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • the dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.
  • Figure 1 illustrates the plasmids and the cloning vectors used in the construction of recombinant MCMV expressing the genes mouse zona pellucida 3, chicken ovalbumin (OVA) linked to the human transferrin cytoplasmic tail, influenza virus haemagglutinin from strain PR8 containing the transmembrane binding region and the murine interieukin 6 gene.
  • OVA ovalbumin
  • the plasmids containing the forementioned genes were all constructed in a similar way as described in the following examples, the difference being in the restriction enzyme sites used.
  • Figure 2 illustrates plasmid pMV11 containing human CMV promoter and
  • Figure 3 illustrates the nucleotide sequence of the OVA/TfR construct cloned into pMV11.
  • Figure 4 illustrates the sequence of MCMV /E-2 region showing the location of Hpal restriction enzyme site into which the gene cassettes were inserted removing the TATA and AUG start region of /E2.
  • a unique restriction site possibly suitable for insertion of the same cassette is also highlighted Nru I.
  • Figure 5 illustrates plasmid pK181-H3L containing the 7.5 kb fragment of the MCMV K181 strain after digestion with the restriction enzyme Hindlll and cloned into the promega plasmid pGEM11Zf.
  • Figure 6A illustrates restriction maps and salient features of completed plasmids containing OVA/TfR genes for the cotransfection and generation of recombinant MCMV.
  • Figure 6B illustrates restriction maps and salient features of completed plasmids containing ZP-3RJ genes for the cotransfection and generation of recombinant MCMV.
  • Figure 7A(i) illustrates restriction maps and salient features of completed plasmids containing a HA (PR8) gene for the cotransfection and generation of recombinant MCMV.
  • Figure 7A(ii) illustrates restriction maps and salient features of completed plasmids containing a mlL-6 gene for the cotransfection and generation of recombinant MCMV.
  • Figure 7b illustrates the process by which the recombinant MCMV is created and the constituent viral and vector constructs.
  • Figure 8 illustrates the replication of K181 -OVA/TfR in the salivary gland of
  • FIG. 9 illustrates the replication of K181- OVA/TfR in the visceral organs of BALB/c mice.
  • Figures 10(A-C) illustrates the replication of the recombinant MCMVs in tissue culture as measured by plaque assay.
  • Figure 11 provide CTL assay results for BALB/c mice were inoculated with
  • Figure 13 provides a summary of the in vivo replication of recombinant MCMV containing different gene cassettes as described above in BALB/c mice ip innoculated with 2x10 4 pfu of tissue culture derived (MEF) recombinant virus.
  • MEF tissue culture derived
  • Figure 14 provides a summary table of the results obtained from ELISA assays conducted on serum obtained from BALB/c mice inoculated with 2x10 4 pfu tissue culture derived recombinant MCMV expressing membrane bound OVA, HA soluble and OVA with adjuvant or ultraviolet light inactivated (non-replicating) recombinant MCMV.
  • Figure 15 provides a summary ELISA result of antibody isotype (lgG1 and lgG2a) titres to ZP-3 from animals inoculated with 2x10 4 pfu of recombinant MCMV-ZP3 virus 100 days previously.
  • Figures 16(A-C) shows the stimulation of a mucosal immune response in the salivary gland induced by recombinant MCMV expressing membrane bound OVA as measured by the ELISPOT assay.
  • Figure 17 shows the stimulation of a mucosal immune response in the salivary gland induced by recombinant MCMV expressing membrane bound OVA as measured by the ELISPOT assay.
  • Figures 18(A-C) illustrates the replication of K181 -OVA/TfR in interferon- receptor gene knock-out mice.
  • Figures 19(A-C) illustrates the replication of K181-OVA/TfR in interferon- alpha receptor gene knock-out mice.
  • Figure 20 provides a summary table demonstrating the replication of MCMV-IL6 (non-membrane antigen expression) and restoration of replication in the CMV organ of tropism for the virus in the salivary gland.
  • Figure 21 provides summary data and a graph for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2x10 4 pfu virus in C57BL/6 mice.
  • Figure 22 provides summary data and a graph for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2x10 4 pfu virus in BALB/c, mice.
  • Figure 23 provides summary data for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2x10 4 pfu virus in outbred ARC/s mice.
  • Figure 24 provides summary data for immunocontraception delivered by recombinant MCMV-ZP3 in mice infected with 2x10 4 pfu virus in wild outbred mice.
  • Figure 25 provides summary data for immunocontraception in BALB/c mice infected with non-recombinant MCMV or MCMV-LacZ prior to infection with MCMV-ZP3.
  • Figure 26 provides a graph showing the titration of IL-6.
  • Figure 27 provides summary data for the protective effect of rMCMV-HA from lethal influenza virus infection 6 days after inoculation with rMCMV- HA.
  • Figure 28 provides summary data for the protective effect of rMCMV-HA from lethal influenza virus infection 21 days after inoculation with rMCMV- HA.
  • K181 MCMV was the wild-type virus used throughout this study.
  • the parental MCMV used for construction of the OVA recombinant MCMV was the RM427+ lacZ containing K181 (smith)MCMV obtained from Prof. Ed. Mocarski, Stanford USA.
  • This virus was constructed on the K181 (Smith) strain of MCMV and contains the lacZ gene, inserted into the Hpa I sites of the /E2 gene. Construction and characterisation of RM427 recombinant has been described previously in Manning et al., 1992 [vi].
  • This virus contains the lacZ gene under transcriptional control of the human CMV immediate-early promoter-enhancer (positions -219 to -19 relative to the start site of transcription).
  • RM427+ has been rescued for the sggl gene. The presence of sgg1 + has been confirmed by PCR. All virus used in this study was propagated on MEF tissue culture in minimal essential medium supplemented with 10% Newborn Calf Serum.
  • mice 5-6 week old female BALB/c mice were obtained from Animal Resources Centre, Murdoch, Western Australia, Australia. Mice were kept under SPF conditions.
  • Plasmid Plasmid cloning was by standard methods [vii]. All restriction enzymes were obtained from commercial suppliers and used according to manufacturers specifications, using the supplied buffers. A recombination plasmid designed to replace the lacZ gene in the Hindlll L fragment of RM427+ with an OVA gene was constructed as described.
  • a pGEMI 1Zf(+) plasmid carrying the MCMV K181 Hindlll L fragment in its Hindlll site was obtained from Scalzo et al Department of Microbiology, University of Western Australia, Western Australia, Australia (see figure 1 ). This plasmid was designated pK181-H3L (see figure 5).
  • the TfROVA sequence was excised from pBlueRIP-TfROVA with Hindlll and blunt-ended by a fill-in reaction using Klenow DNA polymerase. The resulting fragment was blunt-end ligated into the Smal site of pMV11 (see figure 2 & 3), located between the human CMV IE1 promoter and a poly-adenylation stop sequence.
  • the Hindlll fragment, containing the regulatory elements and TfROVA was again blunt-ended and inserted into the two Hpal sites of the Hindlll L fragment on pK181-H3L, thereby removing the TATA box and transcriptional start site of /E2 gene [vi] (see figure 4).
  • This plasmid was designated pK181- H3L/MV11 /OVA TfR (see figure 6A).
  • Plasmids containing murine ZP-3 (see figure 6B) Plasmid pUC19/ZP-3 containing the murine ZP-3 cDNA was obtained from Dr Ron Jackson VBCRC, Canberra , ACT, Australia. The cDNA coding for the ZP-3 was excised using the restriction endonucleases Bam HI and Eco Rl obtained from commercial suppliers. The cDNA was then ligated into the Bam Hl/Eco Rl site of the pMV11 vector and the DNA used to transform competent E. coli bacteria. After transformation, putative clones were screened and the clone was verified by standard molecular biological quality control methods.
  • the expression cassette containing the cDNA coding for ZP-3 was excised from pMV 11 using Hindlll, the fragment blunt-ended using DNA polymerase (Klenow) and blunt-end ligated into the pK181-H3L plasmid.
  • the pKI8l-H3L plasmid had first been digested to completion with the restriction enzyme Hpal the larger fragment purified by standard techniques of ethanol precipation, agarose gel electrophoresis and gel purification. Once again the ligation products were transformed into competent E. coli and putative clones verified by standard molecular biological techniques.
  • the resultant plasmid DNA (pK 181-H3L containing ZP-3) was extracted, purified and used in the co-transfection reaction.
  • Plasmid pZP-3/1 containing the ZP-3 cDNA was obtained by extracting mRNA from ovaries of BALB/c mice, random priming and DNA synthesis, the cDNA amplified using primers containing restriction sites and specific for the ZP-3 by
  • the resultant DNA was cloned into pUC 19 and the DNA sequenced for confirmation.
  • the cDNA coding for the ZP-3 was excised using the restriction endonucleases Bam Hl/Eco Rl obtained from commercial suppliers.
  • the cDNA was then ligated into the Bam HI/ Eco Rl site of the pMV 11 vector and the DNA used to transform competent E. coli bacteria. After transformation, putative clones were screened and the clone was verified by standard molecular biological quality control methods.
  • the expression cassette containing the cDNA coding for ZP-3 was excised from pMV 11 using Hindlll, the fragment blunt-ended using DNA polymerase (Klenow) and blunt-end ligated into the pK181-H3L, plasmid.
  • the pK181-H3L plasmid had first been digested to completion with the restriction enzyme Hpal the larger fragment purified by standard techniques of ethanol precipation, agarose gel electrophoresis and gel purification. Once again the ligation products were transformed into competent E. coli and putative clones verified by standard molecular biological techniques.
  • the resultant plasmid DNA (pKI8l-H3L containing ZP-3) was extracted, purified and used in the co-transfection reaction.
  • Plasmid containing Influenza virus (PB8) haemagglutinin (see figure 7A(i)) Plasmid pKG4 containing the Influenza virus (PR8) haemagglutinin cDNA was obtained from Dr. Bernadette Scott, University of Western Australia, Western Australia, Australia. The cDNA coding for the HA was excised using the restriction endonucleases Bam HI and Bgl II obtained from commercial suppliers. The cDNA was then ligated into the Bam HI site of the pMV 11 vector and the DNA used to transform competent E. coli bacteria. After transformation, putative clones were screened and the clone was verified by standard molecular biological quality control methods.
  • the expression cassette containing the cDNA coding for HA was excised from pMV 11 using Hindlll, the fragment blunt-ended using DNA polymerase (Klenow) and blunt-end ligated into the pK181-H3L plasmid.
  • the pKI8l-H3L plasmid had first been digested to completion with the restriction enzyme Hpal, the larger fragment purified by standard techniques of ethanol precipitin, agarose gel electrophoresis and gel purification. Once again the ligation products were transformed into competent E. coli and putative clones verified by standard molecular biological techniques.
  • the resultant plasmid DNA (pK181-H3L containing HA) was extracted, purified and used in the co-transfection reaction.
  • Piasmid containing murine IL-6 (see figure 7A(ii)) Plasmid pCD-mlL6 containing the IL-6 cDNA was obtained from Dr Alistair Ramsay, Australian National University, Canberra, Australia. The cDNA coding for the IL-6 was excised using the restriction endonucleases Nla IV obtained from commercial suppliers. The cDNA was then ligated into the Sma I site of the pMV 11 vector and the DNA used to transform competent E coli bacteria. After transformation, putative clones were screened and the clone was verified by standard molecular biological quality control methods.
  • the expression cassette containing the cDNA coding for IL-6 was excised from pMV 11 using Hindlll, the fragment blunt-ended using DNA polymerase (Klenow) and blunt-end ligated into the pK181-H3Lplasmid.
  • the pK181-H3L plasmid had first been digested to completion with the restriction enzyme Hpal, the larger fragment purified by standard techniques of ethanol precipation, agarose gel electrophoresis and gel purification. Once again the ligation products were transformed into competent E. coli and putative clones verified by standard molecular biological techniques.
  • the resultant plasmid DNA (pK181 -H3L containing IL-6) was extracted, purified and used in the co-transfection reaction.
  • Infectious MCMV DNA was prepared from RM427+ propagated in tissue culture and extracted from both supernatant and infected cells by protein kinase digestion, followed by phenol chloroform extraction and ethanol precipitation.
  • Recombinant MCMV was generated by cotransfecting 10-50 ⁇ g of K181-RM427+ DNA and 2 ⁇ g of Hindlll linearised transfer plasmid, into MEF cells at 60-70% confluency.
  • Cells were transfected by Calcium phosphate co-precipitation (Cellphect, Pharmacia) was allowed to proceed for 7hrs and followed with osmotic shock.
  • the Transfected MEF monolayers were incubated for 4 days prior to sonication and the supernatant replated onto confluent fresh MEFs monolayers. The monolayer was overlaid with carboxymethylcellulose (CMC) during viral plaque development.
  • CMC carboxymethylcellulose
  • MEFs were fixed in 0.5% gluteraldehyde, permeabilized, X-gal added in a Fe 2+ /Fe 3+ containing solution and plaques were screened for blue colour.
  • a high titre tissue-culture-propagated viral stock was subsequently prepared of the OVA recombinant MCMV.
  • the OVA-recombinant MCMV (rMCMV-TfROVA) was generated by cotransfection of RM427+ DNA with pH3L/MV11 /TfROVA; the recombinant MCMV deleted for part of the IE2 gene (rMCMV-D/E2) was generated by cotransfection of RM427+ DNA with pK181-H3LD/E2; the /acZ-containing revertant (rMCMV-TfROVA-REV) was generated by cotransfection of rMCMV-TfROVA DNA with pON427. This is schematically represented in Figure 7B.
  • Expected sizes were 4.1 kb for lacZ containing virus, 2.6kb for the recombinant MCMV clone containing the TfROVA construct and a difference of 79bp for the D/E2 construct when compared to wild type MCMV.
  • Primer used to confirm intact sggl gene were as follows; forward primer H3JSG1 F: ACAAGAGTCTGTCCGACCAC and reverse primer H3JSG2R: GCGGTACGTATACTGCCGTTA which amplify a 843bp fragment within the H3J fragment of MCMV spanning the potential 323bp deletion of RM427.
  • the Geneamp kit Perkin Elmer was used as per manufacturers instructions. The template was amplified for 30 cycles: 94°C, 30sec; 52°C, 30sec; 72°C, 45sec in a 2400 PCR machine and products resolved by 0.9% AGE.
  • MEF cells were infected at a multiplicity of infection of 5 with rMCMV-TfROVA, RM427+, rMCMV-TfROVA-REV, rMCMV- D/E2, K181 -HA, K181-HA_REV and K181 MCMVs. After the initial 1 hr incubation, the cell monolayers were washed with citric acid buffer of pH 3.0 containing 40mM citric acid, 10 mM KCI and 135 mM NaCI, to remove reversibly bound virus particles.
  • virus was harvested from the monolayer and the supernatant, sonicated, and virus titres quantitated by plaque assay on MEF cells.
  • the limit of detection was 10 pfu/ml for each monolayer assayed. The results are illustrated in Figures 10(a), (b) and (c).
  • mice were inoculated i.p. with OVA-adjuvant emulsion, and given two consecutive boosters two weeks apart. Blood was collected by exsanguination (heart puncture) and allowed to clot at room temperature for 1 hour prior to centrifugation for 1 min. Sera were stored at -20°C until use.
  • hyperimmune sera against ZP-3 Similar experiments were performed for the generation of hyperimmune sera against ZP-3, and HA.
  • the hyperimmune sera for HA was raised from mice infected with influenza PR8 with the hyperimmune sera for ZP-3 was raised against the purified B-cell epitope protein in ZP-3. Detection of expression of OVA by recombinant MCMV.
  • the expression of OVA by recombinant virus was measured by immunostaining viral plaques infected with the recombinant virus using hyperimmune serum raised in mice against the purified OVA protein.
  • the enzyme-linked immunosorbent assay (ELISA) used was a modification of the method described in [ix].
  • MCMV antigen prepared by infecting MEF monolayers with MCMV until near 100% CPE observed, clarifying the culture fluid by centrifugation at 4500g/20min/4°C and then ultracentrifugation at 30000g/2hrs/4°C [i].
  • Viral pellets were resuspended in MOBS and the antigen titrated for optimum dilution.
  • Antigen was coated onto the plates in carbonate/bi-carbonate buffer pH 9.6, overnight at 4°C.
  • the secondary antibodies used were biotinylated goat anti-mouse anti-lgM, lgG1 or lgG2a (Southern Biotechnology, Birmingham, USA). Streptavidin alkaline phosphatase (Pierce, USA) was added in an additional step.
  • the substrate solution contained 5 mg p-Nitrophenyl Phosphate tablets (Sigma) in 5 ml diethanolamine buffer pH 9.8.
  • the reactions were read after 20 min at 405 nm using a BioRad Model 3550 Microplate reader (BioRad, Hercules, California, USA). All incubation steps were performed for 1 hour at 37°C. Absorbance values (y-axis) were plotted against the log of the dilution factors (x-axis). Serum titre is given as the preceding value on the x -axis of the intersect of the linear region of the curve with NMS + 3 standard deviations and expressed as reciprocal dilutions.
  • ELISAs for the detection of anti-OVA or anti- ⁇ -galactosidase antibodies were performed as described above, using plates coated with either 1.0 ⁇ g/ml OVA (Grade VI; Sigma) or 0.5 ⁇ g/ml ⁇ -galactosidase, respectively.
  • MEF monolayers on 8-well glass microscope slides (LAB-TEK, Nalge Nunc International) were infected for 4h with rMCMV- TfR/OVA or K181-WT, washed in MOBS/1 %FCS and fixed with methanol/acetone (50%/50%) for 2 mins. Fixed cells were washed in MOBS/1 %FCS, blocked in 10% normal mouse serum/MOBS/1 %FCS for 30 min at room temperature and again washed in MOBS/1 %FCS.
  • Detection of expression of expression of ZP-3 by recombinant MCMV The expression of ZP-3 was measured by immunostaining viral plaques infected with the recombinant virus using hyperimmune serum raised in mice against the purified B-cell epitope.
  • Detection of expression of expression of HA by recombinant MCMV The expression of Influenza virus HA was measured by immunostaining viral plaques infected with the recombinant virus using hyperimmune serum raised in mice infected with influenza virus PR8
  • Detection of expression of expression of IL-6 by recombinant MCMV Expression of the IL-6 was measured in a bioassay whereby indicator cells dependent on the presence of IL-6 were fed serial dilutions of cell culture supernatants that had been infected with the IL-6 containing recombinant MCMV. This is illustrated in Figure 26.
  • mice In vivo growth of recombinant MCMV.
  • Three week-old female BALB/c mice were inoculated intraperitoneally (ip) with 2 x 10 4 pfu of tissue-culture-propagated (TCV) MCMV.
  • TCV tissue-culture-propagated
  • mice per time point were sacrificed and salivary glands, spleens and livers harvested.
  • IFN ⁇ / ⁇ and ⁇ receptor knockout mice 129Sv(Ev)
  • control mice were inoculated with 2x10 4 pfu of TCV MCMV.
  • K181 -OVA/TfR In BALB/c mice inoculated with 2x10 4 pfu TCV, replicating K181 -OVA/TfR was not detected in the salivary gland, which is the organ of persistence for MCMV (see figure 8). Virus was only detected using the following PCR experiment. However, K181 -OVA/TfR did replicate in visceral organs at levels comparable to parental RM427+MCMV during early infection times following i.p. inoculation with a higher dose (5x10 4 pfu TCV) (see figure 9). K181 -OVA/TfR was still not detected by plaque assay in the salivary glands of BALB/c mice given 10 6 pfu TCV ip. 5x10 4 pfu ip for in vivo replication in the liver and spleen, organs collected at days 3 and 5 post infection.
  • mice were inoculated with 2x10 4 pfu K181 -OVA/TfR or K181 -wild type (wt). Spleens were harvested post infection restimulated in vitro and used in a CTL assay against K181-wt infected mouse embryo fibroblast (MEF) targets. The data from these experiments are shown in figures 11 and 12.
  • Figure 13 provides a summary of the in vivo replication of recombinant MCMV containing different gene cassettes as described above in BALB/c mice ip innoculated with 2x10 4 pfu of tissue culture derived (MEF) recombinant virus.
  • MEF tissue culture derived
  • ELISA For the detection of anti-MCMV antibodies, plates coated with MCMV antigen prepared by infection of MEF monolayers and at 90% CPE, the culture fluid clarified by centrifugation at 3000RPM and then ultracentrifugation at 40000RPM, 3hrs. Viral pellets re-suspended in PBS and used to coat plates. Primary antibody 1hr, wash, 2 antibody conjugate either IgM/lgG or anti-lgG1 or lgG2a used. Serum titre given as intersect of linear region of curve with NMS+3SD and expressed as reciprocal dilutions [i].
  • Figure 14 provides a summary table of the results obtained from ELISA assays conducted on serum obtained from BALB/c mice inoculated with 2x10 4 pfu tissue culture derived recombinant MCMV expressing membrane bound OVA, HA and soluble OVA with adjuvant or ultraviolet light inactivated (non-replicating) recombinant MCMV.
  • the recombinant MCMV OVA or HA stimulates an immune response to the heterologous antigen of a predominantly lgG2a isotype when compared to administration of the antigen by other means and indicates an ability to switch the antibody response from what otherwise occurs. Serum boosting of the lgG2a response occurs after a second (boost) with MCMV expressing HA.
  • Figure 15 provides a summary ELISA result of antibody isotype (lgG1 and lgG2a) titres to ZP-3 from animals inoculated with 2x10 4 pfu of recombinant MCMV-ZP3 virus 100 days previously.
  • Figures 16 and 17 show the stimulation of a mucosal immune response in the salivary gland induced by recombinant MCMV expressing membrane bound OVA as measured by the ELISPOT assay.
  • Antibody secreting cells from the cervical lymph nodes of BALB/c mice were infected with 2x10 4 rMCMV-OVA ip. IgA secreting cells were detected by this method and not by inoculation with OVA/adjuvant.
  • FIG 18(A), (B) and (C) illustrate the replication of K181 -OVA/TfR in interferon- receptor gene knock-out mice.
  • K181-OVA TfR replication in IFN-gamma R gene- knock out (gko) mice is not significantly different to that obtained in parental 129/Ev/Sv mice, however, it appears that IFN-alpha does have a role in controlling viral replication in the spleen. Loss of both IFN-alpha and IFN-gamma function significantly increases virus replication in the visceral organs and demonstrates that K181 -OVA/TfR is not defective in salivary gland replication. Mice were inoculated with 2x10 4 pfu TCV ip.
  • Figures 19 (A), (B) and (C) illustrate the replication of K181 -OVA/TfR in interferon- alpha receptor gene knock-out mice.
  • a dose dependent effect is observed in 129/Ev/Sv control mice, inoculated with 2x10 5 pfu TCV, where K181 -OVA/TfR replication is detected at higher levels in the visceral organs and also in the salivary gland.
  • IFN-alpha plays a role in controlling K181 -OVA/TfR replication in these organs.
  • the parental RM427+ replication in IFN-alpha R gko mice was increase by the same order of magnitude.
  • Figure 20 provides a summary table demonstrating the replication of MCMV-IL6 (non-membrane antigen expression) and restoration of replication in the CMV organ of tropism for the virus in the salivary gland.
  • mice at 6-8 weeks of age were inoculated ip. 2x10 4 recombinant virus containing the gene encoding ZP-3.
  • Males were added 21 days post infection in the following ratios: Uninfected, 1 male + 3 females, 3 groups. (9 females)
  • Figure 21 provides summary data and a graph for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2x10 4 pfu virus in C57BL/6 mice.
  • Figure 22 provides summary data and a graph for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2x10 4 pfu virus in BALB/c, mice.
  • Figure 23 provides summary data for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2x10 4 pfu virus in outbred ARC/s mice.
  • Figure 24 provides summary data for immunocontraception delivered by recombinant MCMV ZP3 in mice infected with 2x10 4 pfu virus in wild outbred mice.
  • mice innoculated with wild parent MCMV and boosted twice with MCMV-ZP3 is shown in figure 25. This figure illustrates the immunocontraceptive effect in wild outbred mice.
  • mice were vaccinated with MCMV and subsequently challenged with a lethal dose of live influenza virus either 6 days or 21 days post infection.
  • the results of these experiments highlight the protective effect of the recombinant McMV-HA (see figures 27 & 28).
  • a desired gene of interest or antigenic sequence is cloned into a transfer vector such that it is flanked by the bacterial transposable element sequences, Tn7L and Tn7R.
  • a bacterial plasmid (pCMH151 ) has been constructed by standard cloning techniques that contains a bacterial origin of replication, a kanamycin resistance (Km R ) marker gene, and a lacZ gene that contains the attachment sequence (att:Tn7) for the bacterial transposable element, Tn7.
  • This plasmid has subsequently been digested with the appropriate restriction enzyme (Notl) and inserted into a second plasmid (pK181 H3L-No /) that is resistant to ampicillan (Amp R ) and has been engineered to contain a Notl restriction site within the /E2 gene of the Hindlll L fragment (previously a Hpal site).
  • the resulting clone (pK181 H3L/pCMH151 ) contains the elements of pCMH151 flanked by MCMV sequences that are homologous to the /E1 and /E2 regions of the MCMV genome.
  • This transfer vector can be transformed into bacterial cells that contain the MCMV genome that has been cloned as a bacterial artificial chromosome (BAC).
  • BAC bacterial artificial chromosome
  • the desired antigenic sequence in the transfer vector can be transposed into a specific attachment site (att:Tn7) that has been engineered into the MCMV BAC plasmid.
  • Positive MCMV BAC plasmids containing the inserted antigenic sequence is indentified by disruption of a lacZ gene sequence associated with the att:Tn7 site.
  • the MCMV BAC plasmids can then be purified and transfected onto eukaryotic cells to recover infectious viral progeny containing the foreign antigenic sequence.
  • a model for the in vitro transposition procedure was developed.
  • Bacterial cells (DH10B) were transformed with the plasmid pK181 H3L/pCMH151. Positive transformants were identified by their brilliant blue colour on L-agar plates containing X-gal, kanamycin and ampicillin.
  • a positive transformed colony was grown in culture and subsequently these cells were transformed with the plasmid pMON7124, a helper plasmid containing the genes for the bacterial transposition proteins and a tetracyline (Tc R ) resistance marker.
  • Tc R tetracyline
  • a transfer vector, pFASTBAC, containing a gentamycin resistance marker (Gm R ) flanked by the transposable element sequences Tn7R and Tn7L was transformed into the host bacteria containing pK181 H3L/pCMH151 and pMON7124. It was observed that several white colonies grew on L-agar plates containing Km, Gm, Amp and Tc, indicating that the transposable element from pFASTBAC had inserted into pK181 H3L/pCMH151 , disrupting the lacZ gene. No colonies were observed when the host bacteria alone were plated onto L-agar plates containing Km, Gm, Amp and Tc.
  • Gm R gentamycin resistance marker
  • CMV-1 A genetic locus that controls murine cytomegalovirus replication in the spleen.

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Abstract

L'invention concerne un cytomégalovirus de recombinaison composé d'un génome de cytomégalovirus contenu à l'intérieur de la région du gène précoce immédiat (IE) du génome du virus, une cassette génétique comprenant au moins un promoteur contigu à au moins une séquence d'ADN et une séquence d'arrêt de polyadénylation, (1) ce promoteur étant conçu pour commander l'expression de la séquence d'ADN et (2) le promoteur, la séquence d'ADN et la séquence d'arrêt de polyadénylation étant hétérologues par rapport au virus dans lequel est insérée la cassette.
EP99924573A 1998-05-22 1999-05-24 Systeme d'expression de cytomegalovirus de recombinaison ciblant la region du gene precoce immediat Withdrawn EP1080217A1 (fr)

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AUPP9584A AUPP958499A0 (en) 1999-04-01 1999-04-01 Recombinant cytomegalovirus expression system
PCT/AU1999/000395 WO1999061636A1 (fr) 1998-05-22 1999-05-24 Systeme d'expression de cytomegalovirus de recombinaison ciblant la region du gene precoce immediat

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