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  • 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
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/002—Protozoa antigens
  • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
• • A—HUMAN NECESSITIES
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  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
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• • 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
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
• • 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
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  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
  • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/24011—Poxviridae
  • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
  • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
  • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the present invention relates to therapeutic and prophylatic vaccines, novel antigens for use in such vaccine(s), methods for their preparation and their use in human medicine.
• the present invention relates to antigens from Herpes Simplex (HSV) capable of stimulating a cytotoxic T lymphocyte response.
• HSV Herpes Simplex
• HSV causes lifelong infection and recurrent disease in man.
• HSV-1 and HSV-2 are two closely related serotypes of HSV, these are known as HSV-1 and HSV-2 respectively.
• HSV-1 and HSV-2 are two closely related serotypes of HSV, these are known as HSV-1 and HSV-2 respectively.
• primary infections after replication at a skin or mucosal site, the virus moves to the dorsal root ganglia and usually enters a latent phase. Reactivations then occur after appropriate stimuli, resulting in vesicles and ulcers at the mucocutaneous sites innervated by the ganglia. While neutralizing antibodies are shown to protect against primary infection and disease, their presence has no effect on the course or frequency of recurrent herpetic disease.
• T cell mediated immune responses particularly of the delayed type hypersensitivity (DTH) or cytolytic (CTL) effector types have also been shown to protect against primary disease in mouse animal models.
• DTH delayed type hypersensitivity
• CTL cytolytic
• individuals with compromised T cell functions may undergo severe and sometimes life-threatening herpetic disease.
• the major surface glycoproteins of Herpes Simplex Virus, gD and gC have been suggested for use in vaccines (EP 139417 Genentech). These primarily stimulate a neutralising antibody response.
• any virus coded polypeptide not just those that are integral membrane proteins like the glycoproteins can be a potential target of T cell mediated responses.
• HSV genome codes for several non structural proteins and internal virion proteins, in addition to external glycoproteins, this results in a large number of potential CTL targets and it is not known which protein would be the most relevant.
• HSV infection is characterized by minimal presence of free virus. During latency and reactivation virus is mainly intracellular. Accordingly, recurrent disease is not prevented even by high levels of neutralizing antibodies and virus control depends on cell ⁇ r stated immunity. In order to obtain protection by vaccination, it seems therefore c able to induce not just an antibody response, but also CTL. An effective vaccine should prime CTL capable of acting as early as possible as soon as signs of reactivation of latent virus appear.
• SUBSTITUTE SHEET simplex structural components such as glycoproteins gD, gB (Zarling et al. 1986), but the relevance of these CTL for virus clearance is not known. Moreover, such CTL were HLA class II restricted, and although expression of class II molecules is induced in keratinocytes during HSV replication, it may occur too late to prevent the appearance of lesions.
• HSV replicative cycle After primary infection and during reactivation from a latent state in neuronal ganglia, HSV is mostly intracellular, with minimal exposure to neutralizing antibodies. However, the beginning of viral protein synthesis inside a cell that harbours viral genome will generate viral protein fragments that will be presented by MHC molecules on the surface of the cell, making it a target for CTL of the appropriate specificity.
• the replication cycle of HSV lasts about 18-20 hours and involves an ordered expression of ⁇ or immediate early (IE) ⁇ or early (E) and ⁇ or late (L) gene products.
• IE immediate early
• E early
• L late
• CTL should detect the very first viral proteins that appear inside the cell after infection and reactivation.
• PBMC peripheral blood mononuclear cells
• the frequency of HS V-2 specific CTL ranged between 1/10000 and 1/36000.
• vaccinia virus recombinant ICP27 W the gene product was expressed in EBV transformed lymphoblastoid target cells for cytotoxicity assays.
• the recombinant infected target cells were recognized by a fraction of HS V-2 specific CTL induced by in vitro restimulation with HSV-2 infected lymphoblasts.
• This IE protein constitutes therefore a candidate component for HSV vaccines aimed at inducing CTL mediated immunity.
• the present invention is therefore, directed towards an immediate early HSV- 2-viral protein ICP27 that is recognised by cytolytic T lymphocyte (CTL) in humans.
• ICP 27 having substantially the sequence as shown in ID Sequence No.l (protein sequence).
• the term substantially means at least 85% homologous, preferably 90 to 95% homologous, more preferably greater than 95% homologous.
• the present invention provides a vaccine composition, for therapeutically or prophylactically treating HSV infections, comprising HSV-2, immediate early protein ICP27 or an immunologically active fragment thereof.
• the ICP27 protein may be expressed as a fusion protein or on a carrier such as a Hepatitis B surface antigen, or presented by a live bacterial carrier, such as listeria, shigella, BCG or Salmonella.
• the protein may be presented as in a live viral vector, such as vaccina, adenovirus or poliovirus.
• the protein may be incorporated into an HSV light particle, as described in British patent application No. 91147140.0 and 9109763.4.
• a preferred embodiment of the invention is a vaccinia recombinant which expresses an HSV-2 ICP27 protein or an immunologically active fragment thereof.
• HSV-2 ICP27 for use in medicine.
• ICP27 is an immediate early protein and its function in the virus is poorly understood, but it is known to be essential for viral replication and involved in virus genome transactivation [McCarthy, A.M., McMahan, L. Schaffer, P.A. (1989).
• Herpes simplex virus type 1 ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient J. Virol. 63:18-27.; Rice, S.A., Su, L, Knipe, D.M. (1989).
• Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities. J. Virol. 83:3899-3407].
• an immunological fragment of ICP27 is a portion of the protein which is capable of eliciting a functional immunological response.
• a further aspect of the invention provides a process for the preparation of the ICP 27 HSV-2 protein or an immunogenic derivative thereof, which process comprises expressing DNA encoding said protein or derivative thereof in a recombinant host cell and recovering the product, and thereafter, optionally, preparing a derivative thereof.
• a DNA molecule comprising such coding sequence eg as shown in ID Sequence No.2 or a fragment thereof forms a further aspect of the invention and can be synthesized by standard DNA synthesis techniques, such as by enzymatic ligation as described by D.M. Roberts el ai in Biochemistry 1985, 24, 5090-5098, by chemical synthesis, by in vitro enzymatic polymerization, or by a combination of these techniques.
• ID sequence 2 the coding sequence for the mature protein ends at base no.1536.
• Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50 ⁇ l or less.
• a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50 ⁇ l or less.
• Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01M MgCl2, 0.01M dithiothreitol, ImM spermidine, ImM ATP and O.lmg ml bovine serum albumin, at a temperature of 4°C to ambient, generally in a volume of 50 ⁇ l or less.
• the chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in 'Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual' (ed.
• the coding sequence can be derived from HSV-2 mRNA, using known techniques (e.g. reverse transcription of mRNA to generate a complementary cDNA strand), and commercially available cDNA kits.
• the invention is not limited to the specifically disclosed sequence, but includes all molecules coding for the protein or an immunogenic derivative thereof, as described above.
• DNA polymers which encodes mutants of the protein of the invention may be prepared by site-directed mutagenesis of the cDNA which codes for the protein by conventional methods such as those described by G. Winter ⁇ i al in Nature 1982, 299, 756-758 or by ZoUer and Smith 1982; Nucl. Acids Res., 10, 6487-6500, or deletion mutagenesis such as described by Chan and Smith in Nucl. Acids Res., 1984, 12, 2407-2419 or by G. Winter et al in Biochem. Soc. Trans., 1984, 12, 224-225.
• the process of the invention may be performed by conventional recombinant techniques such as described in Maniatis et. al., Molecular Cloning - A Laboratory Manual; Cold Spring Harbor, 1982-1989.
• the process may comprise the steps of: i) preparing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide
• SUBSTITUTE SHEET sequence that encodes said HSV-2 ICP 27 protein or an immunogenic derivative thereof; ii) transforming a host cell with said vector, iii) culturing said transformed host cell under conditions permitting expression of said DNA polymer to produce said protein; and iv) recovering said protein.
• transformation is used herein to mean the introduction of foreign DNA into a host cell by transformation, transfection or infection with an appropriate plasmid or viral vector using e.g. conventional techniques as described in Genetic Engineering; Eds. S.M. Kingsman and A.J. Kingsman; Blackwell Scientific
• the expression vector is novel and also forms part of the invention.
• the replicable expression vector may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment encode the desired product, such as the DNA polymer encoding the 16 kDa protein, or fragments thereof, under ligating conditions.
• the DNA polymer may be preformed or formed during the construction of the vector, as desired.
• vector The choice of vector will be determined in part by the host cell, which may be prokaryotic or eukaryotic. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses.
• the preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis et al cited above.
• the recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions. Suitable transforming conditions are conventional and are described in, for example, Maniatis si al cited above, or "DNA Cloning" Vol. II, D.M. Glover ed., IRL Press Ltd, 1985.
• a bacterial host such as E. coli may be treated with a solution of CaCl2 (Cohen et al, Proc. Nat Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnCl2, potassium acetate and glycerol, and then with 3-[N-mo ⁇ pholino]-propane-sulphonic acid, RbCl and glycerol.
• SUBSTITUTE SHEET culture may be transformed by calcium co-precipitation of the vector DNA onto the cells.
• the invention also extends to a host cell transformed with a replicable expression vector of the invention.
• the cell is supplied with nutrient and cultured at a temperature below 45°C.
• the product is recovered by conventional methods according to the host cell.
• the host cell is bacterial, such as E. coli it may be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate.
• the product may generally be isolated from the nutrient medium or from cell free extracts.
• Conventional protein isolation techniques include selective precipitation, absorption chromatography, and affinity chromatography including a monoclonal antibody affinity column.
• the expression may be carried out in insect cells using a suitable vector such as the Baculovirus.
• the protein is expressed in Lepidoptera cells to produce immunogenic polypeptides.
• baculovirus expression system For expression of the protein in Lepidoptera cells, use of a baculovirus expression system is preferred.
• an expression cassette comprising the protein coding sequence, operatively linked to a baculovirus promoter, typically is placed into a shuttle vector.
• a shuttle vector contains a sufficient amount of bacterial DNA to propagate the shuttle vector in E. coli or some other suitable prokaryotic host.
• Such shuttle vector also contains a sufficient amount of baculovirus DNA flanking the desired protein coding sequence s as to permit recombination between a wild-type baculovirus and the heterologous gene.
• the recombinant vector is then cotransfected into Lepidoptera cells with DNA from a wild-type baculovirus.
• baculoviruses arising from homologous recombination are then selected and plaque purified by standard techniques. See Summers et al., TA ⁇ S Bull (Texas Agricultural Experimental Station Bulletin) NR 1555, May, 1987. A process for expressing the CS protein in insect cells is described in detail in
• Production in insect cells can also be accomplished by infecting insect larvae.
• the protein can be produced in Heliothis virescens caterpillars by feeding the recombinant baculovirus of the invention along with traces of wild type baculovirus and then extracting the protein from the hemolymph after about two days. See, for example, Miller et al., PCT/WO88/02030.
• novel protein of the invention may also be expressed in yeast cells as described for the CS protein in EP-A-0278 941.
• SUBSTITUTE SHEET Vaccina constructs can be made by methods well known in the art, see for example European Patent Application EP-083-286 Health Research Inc., Inventors Paoletti and Panicali. The construction of such a vaccinia construct is presented in more detail in the examples. ICP27 has been shown by the present inventors, to be recognised by human
• HSV specific CTL induced by in vitro stimulation of PBMC (peripheral blood mononuclear cells) with HS V-2 infected cells.
• PBMC peripheral blood mononuclear cells
• HS V-2 infected cells By using infected cells, as stimulator cells in vitro, viral epitopes which are synthesized in the cytoplasm, are preferentially presented by class I molecules.
• class I molecules By using infected cells, as stimulator cells in vitro, viral epitopes which are synthesized in the cytoplasm, are preferentially presented by class I molecules.
• the spectrum of effector cells stimulated in vitro by this approach will include both class I and class II restricted T cells.
• HSV infection is characterised by the ability to establish latency and reactivate periodically. During latency and reactivation there is minimal exposure of free virus to antibodies as the virus is mainly maintained intracellularly.
• the vaccine may also preferably contain one or more other HSV proteins, other immediate early, early or late proteins capable of stimulating a CTL response in humans, such as gD or gC Vmw65, RR2, ICPO or ICP4.
• the vaccine may advantageously contain a truncated gD derivative from HSV-2 as described in EP 139417 B.
• the vaccine may contain HSV-1 proteins or cocktails of variants of the same proteins where they exist
• the vaccine may contain HSV-1 proteins or cocktails of variants of the same proteins where they exist
• the vaccine of the present invention will preferably be adjuvanted.
• Known adjuvants will include alum (aluminium hydroxide) mycobacterium derived antigens such as Freunds complete or incomplete adjuvants, and muramyldipeptide (MDP) and derivatives, saponin type adjuvants such as QS21 (US Patent No 5057540) and the like.
• a particularly preferred adjuvant preparation is 3-0-de-acylated monophosphoryl lipid A (3D-MPL) which is commercially available i ,om Ribi
• Immunochem may be prepared according to the method of GB 2220211, or QS21 commercially available from Cambridge Biotech.
• 3D-MPL and or QS21 will be present in the range lO ⁇ g -
• the vaccine containing 3D-MPL or QS21 will typically be presented on alum or in an oil in water emulsion.
• Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978.
• Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877.
• Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.
• each vaccine dose is selected as an amount which induces an immunoprotective response without significant adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Generally, it is expected that each dose will comprise 1-1000 ⁇ g of protein, preferably 2-100 ⁇ g, most preferably 4-40 ⁇ g. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects may receive one or several booster immunisation adequately spaced.
• compositions of the present invention may be used to treat, immunotherapeutically, patients suffering from HSV infections, in order to prevent or significantly decrease recurrent herpes disease, frequency, severity and duration of episodes.
• the rationale for immunotherapeutic use of the invention is that the frequency of HSV specific CTL, that exert an immune surveillance function against the virus, may physiologically decline with time after the last antigen-triggered expansion. Alternatively virus infection may not trigger a strong enough CTL response.
• a suitable protocol of therapeutic vaccination may be defined as a pharmacologically acceptable amount of vaccine preparation administered at regular
• the missing 3' terminal region was reconstituted by a synthetic oligonucleotide:
• the expression cassette was used to transform vaccinia virus following the M. MACKETT et al. protocol except that 20 ⁇ g of plasmid DNA was used by experiment ( Mackett M. eial- 1985. ibid).
• CV1 cells ATCC CCL70
• WR vaccinia virus strain were used for the transfection.
• Selection of recombinants was realized in rat 2 cells (ATCC CRL 1764) following Mackett et al. ⁇ -galactosidase activity was detected as described in Chakrabarti S. et al., Mol. Cell. Biol. 1985 5:3403-3409.
• Plasmid pMG27N is a derivative of the vector pASl, which has been used to synthesize large quantities of numerous foreign proteins (ref. 2-9).
• the pMG27N plasmid, as pASl, utilizes signals from 1 phage DNA to drive the transcription and translation of inserted foreign genes.
• the plasmid contains the 1 promoter PL; operator OL; two utilization sites (NutL and NutR) to relieve transcriptional polarity effects when N protein is provided; the ell ribosome binding site including the ell translation initiation codon incorporated in an Ndel restriction site (ref. 1).
• Plasmid pMGl has been constructed by inserting the 81 first amino acids of the NS1 coding region from influenza strain A/PR/8/34 cleaved from plasmid pASl EH/801 (ref. 4) by BamHI and Ncol into pMG27N digested by BamHI and Sacl.
• a synthetic DNA linker resulting from the ligation of two synthetic oligonucleotides (5 ' ATCCCGGGATAAAAACAACCAAGGTAATGGACA3' and 5 'GCCCTATTTTTGTTGGTTCCATTACCTGTT 3, was introduced between the Ncol and the Sacl sites.
• pMG81 is a derivative of pMGl. pMGl has been digested with Bgl ⁇ -Pstl to remove the ampicillin resistance gene.
• the kanamycin resistance gene from transposon Tn903 was isolated from plasmid pOTS207 (16) by a EcoRI-PstI digestion and ligated into pMGl along with a synthetic DNA linker resulting from the ligation of two synthetic oligonucleotides ( ⁇ AATTCGTACCTA 3'
• the AR58 bacterial lysogen used for the production of the NS1-ICP27 protein is a derivative of the standard NIH E. coli K12 strain N99 (F- su-galK2 lacZ- thr-). It contains a defective phage lambda lysogen (galE::TnlO, 1 Kil- cI857 HI) which is Kil- (i.e.
• the AR58 strain was generated by transduction of N99 with a PI phage stock previously grown on an SA500 (galE::TnlO, 1 Kil- cI857 HI) derivative.
• the introduction of the defective lambda lysogen into N99 was selected with tetracycline by virtue of the presence of a TnlO transposon coding for tetracycline resistance in the adjacent galE gene.
• N99 and SA500 are E. coli K12 strains derived from Dr. Martin Rosenberg's laboratory at the National Institute of Health.
• plasmids containing the PL promoter are introduced into an E. coli lysogenic host to stabilize the plasmid DNA (ref. 3). Cloning into lysogens also precludes the synthesis of proteins that may be toxic to the cells (10). For these purposes, defective lambda phage lysogens are employed so that no phage production ever occurs.
• the integrated lambda phage DNA in the host genome directs the synthesis of a cl repressor protein which binds to the Ol operator on the plasmid and prevents the binding of RNA polymerase to the PL promoter. The inserted gene is therefore transcriptionally silent and no synthesis of the recombinant protein can occur.
• a 1.7 kb fragment containing the ICP27 gene has been prepared by digesting plasmid pSCl 1 ICP27 with EcoR I, filling the protruding ends using the T4 DNA polymerase, and finally digesting the linear fragment with Nco I. Upon isolation on agarose gel and electroelution, this fragment has been ligated with plasmid pMG81 previously digested with Xba I, treated with the T4 DNA polymerase and then digested with Nco I. The ligation mixture has been transformed into Escherichia coli strain AR58. The transformants were selected onto solid medium containing kanamycin.
• NS1/ICP27 A strain AR58( ⁇ MG81/ICP27) has been incubated in 20 ml LB medium containing kanamycin, at 30°C up to an optical density (620 nm) of 0.6. The culture has then been incubated at 42°C for 3 hours.
• mice mice group were immunized with 10 ⁇ pfu of ICP27.W in the footpad and challenged 2 weeks later with wild type HSV-1 (17 + ) or HSV-2 (MS). The occurrence of zosteriform lesions and death was then recorded.
• mice were immunized with 10? pfu of ICP27.VV in the footpad. Draining popliteal lymph nodes were removed 5 days later for CTL assays, without in vitro restimulation, in order to evaluate the primary response.
• Herpes simplex virus The HG52 strain of herpes simplex virus type
• ICP27 vaccinia recombinant was produced as herein described.
• Medium PBMC cultures were grown in RPMI 1640 (Gibco, Ghent, Belgium) supplemented with 10% (v/v) heat inactivated foetal calf serum (FCS)(Flow laboratories, Irwine, Scotland), 2 x 10" 3 M L-glutamine, 100 IU/mL penicillin, 100 ⁇ g mL streptomycin, 5 x 10" ⁇ M mercaptoethanol, 1% MEM non-essential amino acids (Gibco), 1 x 10" 3 M sodium pyruvate MEM (Gibco). Cells.
• PBMC Peripheral blood mononuclear cells
• lymphoblastoid cell lines LCL
• EBV Epstei ⁇ -Barr virus
• Mass cultures PBMC were thawed and stimulated in 24- well plate cultures containing 2 x 10° responder cells and 5 x 10 ⁇ stimulator cells, in the presence of 1 U/mL human recombinant IL-2 (rIL-2, Boehringer Mannheim) and 5% (v/v) supernatant from PHA activated lymphoblasts. Cultures were fed every 3-4 days with medium supplemented with 5 U/mL rIL-2 and 5% PHA-blast supernatant. Cultures were re-stimulated on day 10 and tested on day 20 for cytolytic activity.
• rIL-2 human recombinant IL-2
• PBMC peripheral blood mononuclear cells
• the number of responder cells per well ranged between 10 3 and 4 x 10 4 and 24 to 32 wells were set up for each input cell concentration.
• Autologous stimulator cells (5 x 1 Orwell) were added to all wells. Control wells without responder cells were included.
• Cultures received 1 U/ml rIL-2 and 5% (v/v) PHA-blast supernatant at the onset and were fed with 5U/ml rIL-2 and 5% (v/v) PHA-blast supernatant every 4-6 days.
• Target cells were then washed twice, incubated on ice for 30 min, washed once and 2 x 10 3 cells per well were distributed into the wells containing responder cells and control wells containing medium or Triton X- 1003% in water (spontaneous release and maximum release, respectively). Effector and target cell mixtures were incubated for 4 hours at 37°C in a total of 200 ⁇ L, then 100 ⁇ L of supernatant were harvested and released ⁇ Cr counted. Results were expressed as % specific lysis according to the formula:
• HSV-2 specific CTL Frequency of HSV-2 specific CTL.
• varying numbers of patient PBMC were cultured in the presence of a constant number of autologous stimulator cells (HSV-2 infected PHA- blasts prepared as described in materials and methods). Each well was tested for lysis of autologous LCL infected with HSV-2 or psCl 1. W as negative control. Out of 14 patients tested in this way, 3 008, 114 and K01) had high frequencies of effectors that lysed psCl 1. VV infected target cells, and were therefore not considered.
• Viral antigens recognized by HSV-2 specific CTL Viral antigens recognized by HSV-2 specific CTL.
• limiting dilution cultures of patient PBMC stimulated with HSV-2 infected stimulators were split 4-ways and 3 aliquots from each well tested on autologous LCL infected with HSV-2, psCl 1 vaccina virus. VV or ICP27.W. (Table 4)
• ICP27 specific CTL Frequencies of 1/22000 and 1/50000.
• ICP27 seems to be the main HS V-2 antigen recognized by CTL.
• Intermediate frequencies of CTL specific for ICP27 antigen occurred in patients like 107.
• Patient 109 had very low frequencies of CTL specific for ICP27.
• Patient PBMC were stimulated in mass cultures with HSV-2 infected stimulator cells and tested for cytolytic activity on uninfected or HSV-2 infected autologous or heterologous LCL targets.
• This patient is the asymptomatic sexual partner of a patient with recurrent lesions.
• PBMC peripheral blood mononuclear cells
• PBMC from patients with good responses to HSV-2 were evaluated for recognition of ICP27.
• the frequencies were calculated after exclusion of wells with lytic activity on control target cells infected with psCl l.W.
• ICP27 HSV-2 is recognized by human HSV specific CTL induced by in vitro stimulation of PBMC with HSV-2 infected cells.
• ICP27 is a 63 Kdalton polypeptide coded by one of the five alpha genes that are expressed first upon infection, and reaches peak synthesis at 2-4 hours. 1CP27 has regulatory functions and is probably essential for expression of late genes.
• frequencies of HSV-2 specific CTL ranging between 1/10000 and 1/36000
• two patients had frequencies of 1CP27 specific CTL of 1/22000 and 1/5000. These frequencies are calculated after exclusion of all cultures scoring positive on control target cells, and constitute therefore minimal estimates.

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