WO1999015692A2 - Dengue virus antigens and treatment of dengue fever - Google Patents
Dengue virus antigens and treatment of dengue fever Download PDFInfo
- Publication number
- WO1999015692A2 WO1999015692A2 PCT/EP1998/006009 EP9806009W WO9915692A2 WO 1999015692 A2 WO1999015692 A2 WO 1999015692A2 EP 9806009 W EP9806009 W EP 9806009W WO 9915692 A2 WO9915692 A2 WO 9915692A2
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- Prior art keywords
- dengue
- virus
- enhancement
- amino acid
- dengue virus
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
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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
-
- 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
- C12N2710/24011—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
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
- C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to monoclonal antibodies and antigen binding fragments specific for and recognising antigenic epitopes, including synthetic peptides, capable of eliciting antibodies which protect against infection by dengue viruses as well as the said antigenic epitopes and their use for vaccine purposes.
- the present invention also relates to recombinant vaccinia viruses and for example such which are derived from the Modified vaccinia virus Ankara (MVA) encoding for and capable of producing dengue virus antigens or antigenic epitopes recognised by said monoclonal antibodies, and the use of such recombinant viruses as vaccine.
- the invention also relates to pharmaceutical compositions comprising the above monoclonal antibodies, pharmaceutical compositions comprising dengue virus antigens, and pharmaceutical compositions comprising expression vectors 'synthesising dengue virus antigen.
- Dengue viruses are divided into four antigenically related serotypes, called dengue virus serotypes 1, 2, 3, and 4. Complete or partial nucleotide sequences of the dengue 1, 2, 3, and 4 type virus have been published (Chamber, T.J., Hahn, C.S., Galler, R. And Rice, CM. 1990. Annu. Rev. Microbiol., 44, 649, Zhao et al., 1986, Virology 155, 77-88).
- Dengue virus with its four serotypes Den-1 to Den-4, is the most important member of the Flavivirus genus with respect to infections of humans producing diseases that range from flu-like symptoms to severe or fatal illness, dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue outbreaks continue to be a major public health problem in densely populated areas of the tropical and subtropical regions, where mosquito vectors are abundant. Therefore, there is a substantial need for the development of prophylactic vaccines. Ideally, a single vaccine would confer protection against several pathogens and would induce both cellular and humoral arms of immune response.
- DHF dengue haemorrhagic fever
- DSS dengue shock syndrome
- the flavivirus genome consists of a single-stranded positive sense RNA molecule.
- the single encoded open reading frame (ORF) is translated into a polypeptide which is cleaved co- and post-translationally into at least 11 proteins.
- the order of proteins encoded in the ORF is 5'-C-preM(M)-E-NSl- NS2A-NS2B-NS3-NS4A-NS4B-NS5-3' (Venugopal, K., and Gould, E.A., 1994, Vaccine, Vol. 12, No. 11).
- the preM protein (18-19 kDa) is a precursor of the structural protein M, which is formed by cleavage and removal of the N-terminal (pre) segment, by a process presumed to be linked to maturation of the envelope glycoprotein and the development of virus infectivity.
- the E glycoprotein (53-54 kDa) is an outer structural protein of the dengue virus. It exhibits a number of biological activities including receptor binding and membrane fusion, and is the target for neutralising antibodies and T-helper cells.
- the E protein is a typical membrane glycoprotein with a C-terminal that spans the membrane.
- the non-structural protein NSl (39-41 kDa) is also modified by glycosylation. It is derived from the ORF by N-terminal signalase cleavage and C-terminal cleavage involving a protease. NSl may be involved in the assembly and release of virions. It is found on the cell surface and in the culture medium of infected cells.
- NSl protein evokes a strong antibody response which protects the host against challenge with flaviviruses, presumably through a complement mediated pathway, although recently it has been suggested that other mechanisms such as antibody-dependent cell cytotoxicity may also be responsible (Venugopal, K., & Gould, E.A., 1994, Vaccine, Vol. 12, No. 11).
- a successful vaccine against the prototype yellow fever virus (YFV) has long been in use, as has been the less celebrated vaccines against tick borne encephalitis virus (TBEV) and Japanese encephalitis virus (JEV). These successes are unlikely to be easily reproduced for the dengue viruses which are the causative agents for a two important disease syndromes - classical dengue fever (DF) and dengue haemorrhagic fever/ dengue shock syndrome (DHF/DSS). This is because DHF/DSS has been shown to be associated with secondary immune responses to heterologous dengue virus serotypes which may be due to antibody dependent enhancement (ADE) of virus replication in host monocytes via an Fc receptor mediated pathway.
- ADE antibody dependent enhancement
- the ADE phenomenon has been shown to be due to subneutralising concentrations of antibodies to the envelope glycoprotein (E).
- E envelope glycoprotein
- Fc receptors or C3 complement receptors can mediate attachment and uptake in the mechanism termed antibody-dependent enhancement (ADE).
- Modified vaccinia virus Ankara (MVA) is a host range restricted and highly attenuated vaccinia virus strain, which is unable to multiply in human and most other mammalian cell lines tested. But since viral gene expression is unimpaired in non-permissive cells recombinant MVA viruses may be used as exceptionally safe and efficient expression vectors.
- the modified vaccinia virus Ankara has been generated by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A., Hochstein-Mintzel, V. and Stickl, H: ⁇ 1975] Infection 3, 6-14; Swiss Patent No. 568, 392).
- the MVA virus was deposited in compliance with the requirements of the Budapest Treaty at CNCM (Institut Pasteur, Collection Nationale de Cultures de Microorganisms, 25, rue du Dondel Roux, 75724 Paris Cedex 15) on Dec. 15, 1987 under Depositary No. 1-721.
- the MVA virus has been analyzed to determine alterations in the genome relative to the wild type CVA strain.
- MVA replication in human cells was found to be blocked late in infection preventing the assembly to mature infectious virio s. Nevertheless, MVA was able to express viral and recombinant genes at high levels even in non-permissive cells and was proposed to serve as an efficient and exceptionally safe gene expression vector (Sutter, G. and Moss, B. [1992] Proc. Natl. Acad. Sci. USA 89, 10847-10851).
- an effective prevention and therapy of DF, DHF and DSS has been made possible by the identification of dengue virus neutralising monoclonal antibodies which does not cause immune enhancement or is involved in antibody dependent enhancement, and by using one such monoclonal antibody, an antigenic discontinuous epitope in the dengue virus envelope protein which elicits neutralising antibodies without being involved in immune enhancement or antibody dependent enhancement has been identified. Further using this monoclonal antibody to select peptides from a random peptide library, peptides which may be used to elicit dengue neutralising antibodies and which do not cause immune enhancement or is envolved in antibody enhancement have been identified. Further, according to the present invention, an antigen expression system for the in vivo or ex vivo production of a therapeutic agent against DF, DHF or DSS has been prepared.
- a still further object of the present invention is to provide recombinant vaccinia virus based expression systems expressing dengue virus antigens or antigenic epitopes to provide an efficient and safe dengue virus vaccine for animals and humans.
- Still one other object of the present invention is to provide heterologous and/ or synthetic peptide sequences recognised by non-immune enhancing dengue virus neutralising monoclonal antibodies and DNA sequences (genes) which code for the same.
- the present invention thus, inter alia, comprises the following, alone or in combination:
- Dengue antigens or antigenic epitopes as well as heterologous and/ or synthetic peptide sequences recognised by non-immune enhancing dengue virus neutralising monoclonal antibodies and DNA sequences (genes) which code for the same;
- proteins, polypeptides, peptides and synthetic peptides including the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any other amino acid sequence or functional analogues or other equivalents thereof which will elicit anti-dengue antibodies not able to effect immune enhancement or antibody dependent enhancement;
- a monoclonal antibody that specifically binds to and identifies dengue specific antigenic epitopes and especially dengue specific antigenic epitopes which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
- a monoclonal antibody as above which is a monoclonal antibody of subclass IgG recognising proteins, polypeptides or peptides including the amino acid sequences LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or functional analogues or other equivalents thereof and which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
- a monoclonal antibody as above which is a monoclonal antibody of subclass IgGl recognising proteins, polypeptides or peptides including the amino acid sequences LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or functional analogues or other equivalents thereof and which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
- a monoclonal antibody as above which is a mouse monoclonal antibody of subclass IgGl recognising proteins, polypeptides or peptides including the amino acid sequences LPWYNHS,APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or functional analogues or other equivalents thereof and which will elicit neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
- a vaccine comprising as a first component a recombinant MVA carrying and capable of expressing T7 RNA polymerase and as further components one or more recombinant DNA vectors each carrying at least one dengue virus antigen or synthetic antigen not being involved in immune enhancement or antibody dependent enhancement and being recognised by a dengue virus neutralising antibody when the said antigen or synthetic antigen is under transcriptional control of a T7 RNA polymerase promoter;
- a method for the treatment or prevention of a dengue virus infection comprising inoculating a living animal body, including a human, in need thereof with the first and further components of a vaccine, as above, either simultaneously or with a timelag but using the same inoculation site;
- a recombinant MVA containing and capable of expressing one or more DNA sequences encoding dengue virus antigens not able to effect immune enhancement or antibody dependent enhancement; a recombinant MVA as above, containing and capable of expressing DNA sequences encoding antigens from any of the four dengue virus serotypes (type 1, 2, 3 and 4) not able to effect immune enhancement or antibody dependent enhancement;
- a recombinant MVA as above containing and capable of expressing DNA sequences encoding peptides, polypeptides or proteins being ' recognised by monoclonal antibodies specific to the peptides, polypeptides and/ or proteins including the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL, any amino acid sequence or a functional or other equivalent thereof which will elicit dengue neutralising antibodies not able to effect immune enhancement or antibody dependent enhancement;
- a recombinant MVA as above, wherein the DNA sequences encoding antigen is under transcriptional control of a vaccinia specific promoter selected from the group of vaccinia virus early/ late promoter P7.5, vaccinia virus synthetic promoter or any other equivalent promoter;
- a method for the treatment or prevention of dengue virus infection comprising administering to a living animal body, including a human, in need thereof a therapeutically effective amount of a recombinant MVA as anyone above, or a vaccine as above.
- Monoclonal antibodies generated to be specifically directed against Dengue virus polypeptides are essential to identify and to target Dengue virus-specific epitopes which enables the rational design of novel prophylactic, therapeutic, and diagnostic tools for the control of human Dengue virus infection.
- the locations in the envelope glycoprotein of dengue virus of a series of protective epitopes are identified.
- neutralising antibodies are screened for immune enhancing properties in order to determine that even at sub-neutralising concentrations the antibodies do not cause antibody dependent enhancement.
- a specific mouse monoclonal antibody identified as MASl which specifically recognises peptides, polypeptides and proteins which contain a sequence functioning as a neutralising but non immune enJiancing epitope for the elicitation of protective immune responses against dengue virus infection is provided.
- the monoclonal antibody MASl can be prepared by anyone skilled in the art and as described generally by Kohler, G. & Milstein, C (1975, Nature, 256: 495-497) and Harlow, E. & Lane, D. (1988, Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratories).
- the monoclonal antibody MASl according to one preferred embodiment of the present invention, is a mouse monoclonal antibody of subclass IgGl which has the property of being able to neutralise dengue virus without causing antibody dependent enhancement in Fc receptor bearing cells at any dilution tested.
- An epitope defined by this monoclonal antibody MASl has the advantage of being an epitope which is neutralizable but not enhanceable and therefore represents an epitope which would be safe and efficacious as a vaccine preparation, being able to elicit protective antibodies which will not cause immune enhancement at any concentration.
- neutralizing monoclonal antibodies which are not immune enhancing are used in the screening of different bacteriophage display libraries, peptide libraries etc. to identify peptide sequences recognised by the said non-immune enhancing dengue virus neutralising monoclonal antibodies.
- the said specific monoclonal antibody MASl has been found to recognise peptides, polypeptides or proteins which contain a sequence which functions as a protective but not enhancing epitope for the elicitation of a protective immune response against dengue virus infection.
- sequences may be derived from the authentic dengue virus proteins or glycoproteins themselves or from synthetic peptides or other mimetic peptides or polypeptides, including but not limited to anti-idiotypic antibodies and their derivatives as well as DNA sequences encoding any of the above-mentioned molecules.
- the monoclonal antibody MASl has been found to recognise polypeptides including the amino acids sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL.
- the monoclonal antibody MASl is further characterised as being a mouse monoclonal antibody within the IgGl family recognising the amino acid sequence LPWYNHS and furthermore by the fact it recognises a discontinuous antigenic epitope within the E gene of dengue 2.
- DNA sequences which code for dengue antigens or antigenic epitopes as well as heterologous and/ or synthetic peptide sequences recognised by non-immune enhancing dengue virus neutralising monoclonal antibodies, and especially by the MASl, capable of eliciting immune responses to dengue virus are thus provided for the treatment of and prevention of dengue virus infection.
- sequences are optionally introduced, with the aid of DNA recombination techniques, into the genome of viral expression systems including for example the MVA vector.
- the newly produced recombinant MVA will be infectious, that is to say able to infect foreign cells and it will express the integrated DNA sequence.
- the recombinant viruses and the recombinant MVA according to the invention will be useful as extremely safe live vaccines for the treatment or prophylactics of dengue infection.
- the recombinant MVA vaccinia viruses can be prepared as set out hereinafter.
- a DNA-construct containing sequences flanking the left and the right side of a naturally occurring deletion, e.g. deletion II or III, within the MVA genome (Altenburger, W., Suter, C.P. and Altenburger J. (1989) Arch. Virol. 105, 15-27) was constructed.
- a DNA-construct which contains a DNA-sequence which codes for a dengue antigen or antigenic epitope flanked by MVA DNA sequences adjacent to a naturally occurring deletion, e.g. deletion II or III, within the MVA genome is introduced into cells infected with MVA, to allow homologous recombination.
- the DNA-construct to be inserted can be linear or circular. A circular DNA is preferred, especially a plasmid.
- the DNA sequence encoding antigenic epitopes is inserted between the sequences flanking the naturally occurring deletion.
- promoters are known to those skilled in the art, and include for example those of the vaccinia 11 kDa gene as are described in EP-A-198, 328, those of the 7.5 kDa gene (EP-A-110, 385), the vaccinia specific synthetic promoter sP (Sutter et al., 1994, Vaccine 12, 1032-1040) or those of the bacteriophage T7 Polymerase.
- the DNA-construct can be introduced into the MVA infected cells by transfection, for example by means of calcium phosphate precipitation (Graham et al., Virol. 52, 456-467 [1973]; Wigler et al., Cell 777-785 [1979] by means of electroporation (Neumann et al., EMBO J. 1, 841-845 [1982]), by micro-injection (Graessmann et al., Meth. Enzymology 101, 482-492 (1983)), by means of liposomes (Straubinger et al, Methods in Enzymology 101, 512-527 (1983)), by means of spheroplasts (Schaffner, Proc. Natl. Acad. Sci. USA 11, 2163-2167 (1980)) or by other methods known to those skilled in the art. Transfection by means of calcium phosphate precipitation is preferred.
- the present invention therefore also relates to recombinant MVA which contains at least one DNA sequence which codes for dengue virus antigen or antigenic epitopes and relates further to vaccines containing such viruses in a physiologically acceptable form.
- the recombinant MVA according to the invention contain and are capable of expressing one or more DNA sequences encoding dengue virus antigens or antigenic epitopes capable of eliciting a protective immune response against dengue viruses.
- the dengue virus antigen includes the amino acid sequence LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL or any functional analogue or other equivalents thereof.
- the invention therefore in a preferred embodiment relates to a viral expression vector, and preferably a pox virus based expression system and especially a MVA based expression system coding for peptides, polypeptides or proteins including the amino acid sequences LPWYNHS, APWYTHP, TPWYL, LPWYPSP, TPWYTHL including for example the following synthetic expression sequence containing one start codon and two stop codons for translation: 5'- CAG CAG CCC GGG ATG CTT CCG TGG TAT AAT CAT TCT GGT CCT -3' and 5'-CAG CAG CCC GGG CTA TTA GCC CTC ATA GTT AGC GTA ACG -3'
- the invention also relates to vaccines comprising recombinant MVA according to the invention containing and capable of expressing one or more DNA sequences encoding dengue virus antigens.
- a vaccine according to the invention comprises a recombinant MVA encoding dengue virus type 1 antigen; recombinant MVA encoding dengue virus type 2 antigen; recombinant MVA encoding dengue virus type 3 antigen; recombinant MVA encoding dengue virus type 4 antigen; and/ or includes the amino acid sequence LPWYNHS APWYTHP, TPWYL, LPWYPSP, TPWYTHL or any functional analogue or other equivalents thereof.
- the invention further relates to vaccines comprising recombinant MVA according to the invention containing and capable of expressing one or more DNA sequences encoding peptides or polypeptides comprising antigenic epitopes recognised by monoclonal antibody MASl which elicit protective immune responses against dengue virus infection.
- the vaccine according to the invention comprises a peptide, polypeptide or protein which contains an epitope recognised by the said monoclonal antibody MASl.
- the epitope can exist within an amino acid sequence derived from random peptide libraries and identified by the said monoclonal antibody MASl, or the epitope can exist within an anti-idiotypic antibody generated against said monoclonal antibody MASl, or can exist within polypeptides and proteins encoded by the envelope genes of dengue viruses and includes but is not limited to nucleic acid sequences which encode for the epitope described by monoclonal antibody MASl.
- a DNA vectors carrying DNA sequences encoding dengue antigens under transcriptional control of a T7 RNA polymerase promoter were provided.
- recombinant MVA encoding T7 RNA polymerase (MVA-T7pol) is used in combination with DNA vectors carrying DNA sequences encoding dengue antigens under transcriptional control of a T7 RNA polymerase promoter.
- a coding sequence of a given dengue antigen capable of binding to a monoclonal antibody not being involve in immune enhancement is cloned under control of a T7 RNA polymerase promoter preferably in a plasmid vector and the resulting DNA construct is amplified and purified using standard laboratory procedures.
- the vector DNA is inoculated simultaneously or with appropriate timelags together with MVA- T7pol.
- An appropriate timelag allows a cell to take up the inoculated DNA vector, before MVA infection takes place.
- the mode of administration, the dose and number of administration can be optimised by one skilled in the art.
- the recombinant gene of interest is expressed transiently in cells containing both the vector DNA and MVA-T7pol and the corresponding antigen is presented to the host immune system stimulating an antigen-specific immune response.
- the recombinant MVA according to the invention can also carry a marker gene.
- Said marker genes are preferably selected from the group consisting of marker genes which codes for proteins such as ⁇ -galactosidase, neomycin, alcohol dehydrogenase, luciferase, puromycin, hypoxanthine phosphoribosyl transferase (HPRT), hygromycin, and green or blue fluorescent proteins.
- marker genes can either facilitate the purification of recombinant MVA or be used as a independent antigenic marker to control status of immune response after a antigen specific vaccination.
- the invention also relates to methods for the preparation of such recombinant MVA and vaccines, and to the use of these vaccines for the prophylactics or treatment of infections caused by dengue virus.
- the recombinant MVA according to the invention are converted into a physiologically acceptable form. This can be done based on the experience in the preparation of MVA vaccines used for vaccination against smallpox (as described by Stickl, H. et al. [1974] Dtsch. med. Wschr. 99, 2386-2392).
- PBS phosphate-buffered saline
- the lyophilisate can contain extenders (such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone) or other aids (such as antioxidants, stabilisers, etc.) suitable for parenteral administration.
- extenders such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone
- other aids such as antioxidants, stabilisers, etc.
- the lyophilisate can be dissolved in 0.1 to 0.5 ml of an aqueous solution, preferably physiological saline, and administered either parenterally, for example by intramuscular inoculation or locally.
- Vaccines or therapeutics according to the invention are preferably injected intramuscularly (Mayr, A. et al. [1978] Zbl. Bakt. Hyg., I. Abt. Orig. B 167, 375-390).
- the mode of administration, the dose and the number of administrations can be optimised by those skilled in the art in a known manner. It is expedient where appropriate to administer the vaccine several times over a lengthy period in order to obtain appropriate immune responses against the foreign antigen.
- a continuous mosquito cell line, C6/36 was grown to confluency in a 25 mm 2 tissue culture flask in Leibovitz 15 medium supplemented with 3% fetal calf serum (FCS), 10% tryptose phosphate broth, penicillin (100 units/ ml), streptomycin (100 mg/ml). The medium was then replaced with 4.5 ml of maintenance medium (Leibovitz 15 medium supplemented with 1% FCS, 10% tryptose phosphate broth, and antibiotics in the concentrations described above. Half a ml of Dengue infectious virus stock culture supernatant was added and the flask was rocked gently at room temperature overnight.
- the flask was then maintained in a stationary position in an incubator set at 28°C for 4 to 5 days until some signs of a cytopathic effect were seen.
- the medium was then changed to 5 ml of fresh maintenance medium and the cells were incubated at 28°C until syncytia formation occured.
- the supernatant was harvested for infectious virus.
- the supernatant was clarified by centrif ugation for 10 minutes at 4 °C at 5000 rpm in a Beckman Avanti J25 using a JS7.5 rotor.
- the clarified supernatant was the Dengue infectious virus stock and was used for seeding new cultures as well as for all Dengue virus plaque assays including neutralization assays and immune enhancement assays.
- the Dengue infectious virus stock was titrated in a plaque assay in the following manner.
- a confluent monolayer of PS Clone D cells were trypsinized and resuspended at 2 x 10 5 cells/ ml maintenance medium (composition as described in Example 1) and 0.5 ml was dispensed in each well of a 24 well tissue culture cluster plate.
- Ten fold serial dilutions of Dengue infectious virus stock were prepared in maintenance medium and 100 ⁇ l of these dilutions or maintenance medium was added in duplicate to each well containing PS Clone D cells in suspension. The plate was gently tapped to mix the contents of each well and the plate then was incubated at 37 °C for 4 hours until the cells had adhered to the plate.
- the cells were then overlaid with 0.6 ml of maintenance medium containing 1.5% carboxymethylcellulose, and incubated at 37 °C for 4 to 5 days until plaques were visible under an inverted microscope.
- the medium was then tipped out into a container containing 5% chlorox and the monolayers gently washed with phosphate buffered saline, pH 7.4.
- the monolayers were then stained for 15 minutes with naphthalene black and washed with water. The plates were allowed to airdry and the plaques counted.
- Hybridoma culture supernatant containing the monoclonal antibody to be tested was diluted in four fold serial dilutions in maintenance medium. 100 ⁇ l of undiluted hybridoma supernatant or dilutions were dispensed into 24 well tissue cluster plates in triplicate.
- Control supernatant from hybridomas secreting irrelevant monoclonal antibody (for example to Hepatitis B virus) was also dispensed in triplicate and these wells containing monoclonal antibody or control were then seeded with 100 ⁇ l of virus dilution prepared previously.
- the plate was tapped gently to mix the contents and incubated at 37 °C for one hour.
- a confluent monolayer of PS Clone D cells was resuspended at 3 x 10 5 cells/ ml maintenance medium as described above and dispensed at 0.3 ml per well containing the virus-antibody incubation mixtures or controls. Cells were also added to triplicate wells containing no incubation mixtures. The plate was incubated at 37 °C for 4 hours until the cells had adhered to the plastic and 0.5 ml of maintenance medium containing 1.5% carboxymethylcellulose was overlayed. The plate was incubated at 37 °C for 4 to 5 days until plaques were visible using an inverted microscope. The monolayers were then washed and stained with naphthlene black as described above and plaques counted. Neutralization of Dengue virus was deemed to have occured when the mean number of plaques in wells containing antibody was at least 50% less than in the control wells containing irrelevant antibody or no antibody.
- a dilution of Dengue virus infectious stock which would give approximately 30- 40 plaques per 100 ⁇ l based on the above titration was then selected and prepared by dilution into maintenance medium.
- Hybridoma culture supernatant containing the monoclonal antibody to be tested was diluted in ten fold serial dilutions in maintenance medium. Similar dilutions were prepared of irrelevant hybridoma supernatants as controls.
- Duplicate wells containing 100 ⁇ l of maintenance medium was used as the negative control.
- 100 ⁇ l of undiluted hybridoma supernatant or dilutions were dispensed into 5 ml round bottomed cell culture tubes.
- 100 ⁇ l of the Dengue virus dilution was added to each tube. The mixture was gently mixed and incubated at 37 °C for 1 hour.
- a confluent monolayer of P388D1 cells were resuspended at 5 x 10 s cells/ml into Leibovitz 15 maintenance medium and dispensed at 0.5 ml per tube.
- the tubes were incubated at 37 °C overnight.
- the cells were then washed by centrifugation for 10 minutes at 800 rpm at room temperature in a Beckman Avanti J25 using a JS7.5 rotor.
- the supernatant was discarded and 1 ml of fresh maintenance medium was added per tube.
- the tubes were centrifuged again as described and replenished with 1 ml of fresh maintenance medium.
- the cells were resuspended and incubated at 37 °C for 5 days.
- Immune enhancement or antibody dependent enhancement is deemed to have occured when the number of plaque forming units (pfu) per ml of culture was double or greater than that of the negative controls containing no antibody.
- a Balb/c mouse was immunised through the ip route with Dengue antigen prepared as follows. C6/36 mosquito cell monolayers wre inoculated with
- Dengue 2 virus as described in Example 1.
- the monolayer was washed once with phosphate buffered saline and lysed with 2 ml of isotonic buffer (1.5 mM magnesium chloride, 5 mM potassium chloride, 10 mM HEPES) containing 1% Nonidet P 40 after gentle rocking at room temperature for 10 minutes, the lysate was harvested and spun for 5 minutes in a microfuge. The supernatant was loaded into a single well comb of a 12% SDS PAGE gel (Biorad Mini Protean II) and run at 100 Volts until the dye front reached the end of the gel. Bands corresponding to Dengue Envelope glycoprotein and pre Membrane protein were excised from the gel, placed in a dialysis bag, electro-eluted in a minimal volume and used as the immunogen.
- the immunised mouse was boosted through the ip route after two weeks with 0.5 ml infectious dengue virus culture supernatant prepared as described in Example 1 and left to rest for a week before the spleen was removed and spleen cells harvested.
- Cell fusion was attained by the method as essentially described by Kohler & Milstein, Nature 256, 495-497 (1975) and Kohler et al., Eur.J.Immunol. 6, 292-295 (1976) and clones were screened for anti Dengue activity by a dot enzyme immunoassay (Cardosa, M.J. & Tio, P.H., 1991.
- Dot enzyme immunoassay Alternative diagnostic aid for dengue fever and dengue haemorrhagic fever, Bulletin of the World Health Organisation, 69, 741-745.
- a mouse immunoglobulin capture ELISA using 96 well ELISA plates coated with anti-mouse immunoglobulin incubated with tissue culture supernatants overnight at 4 °C After washing with phosphate buffered saline containing 0.05% Tween 20, the immunoglobulin bound was identified using dengue virus prepared as described in Example 1 followed by an HRP conjugated anti dengue convalescent human serum (Innis, B.L. Nisalak, A., Nimmannitya, S., et al., 1989. An enzyme-linked immunosorbent assay to characterise dengue infections where dengue and Japanese encephalitis co-circulate. Am J Trop Med Hyg 40:418- 27) Positive clones were expanded and recloned by limiting dilution twice.
- the hybridoma which yielded MAS 1 was grown to large quantities in 75 mm 2 tissue culture flasks and supernatants harvested every 2 or 3 days.
- the supernatant was clarified by centrifugation at 3000 rpm at 4 °C for 15 minutes in a Beckman Avanti J25 using a JS7.5 rotor, and the clarified supernatant was pushed through a 0.22 micron filter before purification through a HiTrap Protein G column (Pharmacia Biotech, Product Code No. 17-040-01) according to the manufacturer's instructions. Fractions containing protein were pooled and checked for specific activity in a dot enzyme immunoassay (Cardosa & Tio, 1991).
- the affinity purified monoclonal antibody MAS 1 was used in panning experiments to select peptide ligands from a phage display peptide library purchased from New England Biolabs (Catalog number 8100). The experiment was performed according to the instructions of the manufacturer using MAS 1 as the panning antibody bound to the plastic surface of an microtitre ELISA plate. Three pannings were performed and phage selected were purified and DNA sequences of the heptapeptide displayed in each clone was determined. The DNA sequence was translated and concensus peptides were determined using the software Lasergene. The following are the concensus sequences obtaine hy comparison of amino acid sequences.
- First line indicates the concensus sequence. Clones (characterised by a number) from all three pannings are listed in the first row.
- First line indicates the concensus sequence. Clones (characterised by a number) from the second and third pannings only are listed in the first row.
- First line indicates the concensus sequence. Clones (characterised by a number) from the third panning are listed in the first row.
- First line indicates the concensus sequence. Clones (characterised by a number) from the third panning are listed in the first row.
- First line indicates the concensus sequence. Clones (characterised by a number) from the second and third pannings are listed in the first row.
- the MVA virus is a highly attenuated vaccinia virus derived from the vaccinia virus strain Ankara (CVA) by long-term serial passages on primary chicken embryo fibroblast (CEF) cultures.
- CVA vaccinia virus strain Ankara
- CEF primary chicken embryo fibroblast
- CEF cells To prepare CEF cells, 11-days old embryos were isolated from incubated chicken eggs, the extremities are removed, and the embryos are minced and dissociated in a solution composed of 0.25% trypsin at 37 °C for 20 minutes. The resulting cell suspension was filtered and cells were sedimented by centrifugation at 2000 rpm in a Sorvall RC-3B centrifuge at room temperature for 5 minutes, resuspended in 10 volumes of medium A (MEM Eagle, for example obtainable from Life Technologies GmbH, Eggenstein, Germany), and sedimented again by centrifugation at 2000 rpm in a Sorvall RC-3B centrifuge at room temperature for 5 minutes.
- medium A MEM Eagle, for example obtainable from Life Technologies GmbH, Eggenstein, Germany
- the cell pellet was reconstituted in medium A containing 10% fetal calf serum (FCS), penicillin (100 units/ml), streptomycin (100 mg/ml) and 2 mM glutamine to obtain a cell suspension containing 500 000 cells/ml.
- CEF cells obtained in this way were spread on cell culture dishes. They were left to grow in medium A in a 6% CO2 atmosphere with 95% humidity at 37°C for 1-2 days, depending on the desired cell density, and were used for infection either directly or after one further cell passage.
- FCS fetal calf serum
- penicillin 100 units/ml
- streptomycin 100 mg/ml
- 2 mM glutamine 2 mM glutamine
- MVA viruses were used for infection as follows. CEF cells were cultured in 175 cm ⁇ cell culture bottles. At 90-100% confluence, the medium was removed and the cells were incubated for one hour with an MVA virus suspension (0.01 infectious units (IU) per cell, 0.02 ml/ cm 2 ) in medium A. Then more medium A was added (0.2 ml/ cm 2 ) and the bottles were incubated at 37°C for 2-3 days (until about 90% of the cells show cytopathogenic effect). Crude virus stocks were prepared by scraping cell monolayers into the medium and pelleting the cell material by centrifugation at 3000 rpm in a Sorvall RC-3B centrifuge at 4°C for 5 minutes. The crude virus preparation was stored at -20°C before further processing (e.g. virus purification).
- MVA virus suspension (0.01 infectious units (IU) per cell, 0.02 ml/ cm 2 ) in medium A. Then more medium A was added (0.2 ml/ cm 2 ) and the bottles
- MVA virus obtained from Prof. Anton Mayr was cloned by limiting dilution during three consecutive passages in CEF cultured on 96-well tissue culture plates.
- the MVA clone F6 was selected and amplified in CEF to obtain working stocks of virus that served as starting material for the generation of recombinant MVA viruses described in this patent application as well as for the generation of recombinant MVA viruses described previously (Sutter, G. and Moss, B. [1992] Proc. Natl. Acad. Sci. USA 89, 10847-10851; Sutter, G., Wyatt, L., Foley, P., Bennink, J. and Moss, B.
- the gradient was centrifuged in Beckmann SW41 rotor at 13,000 rpm for 50 minutes at 4°C After centrifugation, discrete bands containing virus particles were harvested by pipetting after aspirating volume above band.
- the obtained sucrose solution was diluted with three volumes PBS and the virus particles were sedimented again by centrifugation (Beckmann SW 27/28, 60 minutes at 13,500 rpm, 4° C).
- the pellet which now consisted mostly of pure virus particles, was resuspended in PBS and equilibrated to virus concentrations corresponding on average to l-5x l ⁇ " IU/ml.
- the purified virus stock solution was stored at -80°C and used either directly or diluted with PBS for subsequent experiments.
- the primers for the left 600 bp DNA • flank were 5 X CAG CAG GGT ACC CTC ATC GTA CAG GACGTT CTC-3' and 5'- CAG CAG CCC GGG TAT TCG ATG ATT ATT TTT AAC AAA ATA ACA-3' (sites for restriction enzymes Kpnl and Smal are underlined).
- the primers for the right 550 bp DNA flank were 5'-CAG CAG CTG CAG GAA TCA TCC ATT CCA CTG AAT AGC-3' and 5'-CAG CAG GCA TGC CGA CGA ACA AGG AAC TGT AGC AGA-3' (sites for restriction enzymes PstI and SphI are underlined).
- the E. coli lacZ gene under control of the vaccinia virus late promoter Pll (prepared by restriction digest from pill LZ, Sutter, G. and Moss, B. [1992] PNAS USA 89, 10847-10851) was inserted, using the BarnHI site, to generate the MVA insertion vector pUCII LZ [ Figure 1].
- a 289 bp fragment containing the vaccinia virus early-late promoter P7.5 together with a Smal site for cloning prepared by restriction digest with EcoRI and Xbal from the plasmid vector pSCll [Chakrabarti et al.
- coli LacZ open reading frame was amplified by PCR (primers were 5'-CAG CAG GTC GAC CCC GAC CGC CTT ACT GCC GCC-3' and 5'-GGG GGG CTG CAG ATG GTA GCG ACC GGC GCT CAG-3') and cloned into the Sail and PstI sites of pUC II LZ P7.5 to obtain the MVA vector pUC II LZdel P7.5.
- this vector plasmid can be used to insert DNA sequences encoding a foreign gene under transcriptional control of the vaccinia virus promoter P7.5 into the MVA genome.
- a 147 bp minigene encoding a polypeptide including the N-terminal amino acids LPWYNHS and containing one start codon and two stop codons for translation was prepared by PCR from cloned DNA of a phage M13 library using synthetic oligonucleotides 5'- CAG CAG CCC GGG ATG CTT CCG TGG TAT AAT CAT TCT GGT CCT -3' and 5'- CAG CAG CCC GGG CTA TTA GCC CTC ATA GTT AGC GTA ACG -3' and cloned into the Smal restriction of site of the MVA vector plasmid pUCII LZdel P7.5 to create the plasmid pUCII LZdel P7.5- DENminil carrying the minigene under transcriptional control of the vaccinia virus early/ late promoter P7.5.
- the minigene under control of the vaccinia virus early/late promoter P7.5 was inserted into deletion II within the MVA using homologous recombination.
- CEF cells infected with MVA at a multiplicity of 0.05 TCID50 per cell were transfected with DNA of plasmid pUC II LZdel P7.5-DENmini as described previously (Sutter, G, Wyatt, L., Foley, P., Bennink, J. and Moss, B. [1994] Vaccine 12, 1032-1040).
- coli LacZ marker gene were selected by consecutive rounds of plaque purification in CEF cells stained with 5- bromo-4-chloro-3-indolyl ⁇ -D-galactoside (300 mg/ml).
- recombinant MVA viruses containing the E sequence and having deleted the LacZ marker gene were isolated by three additional consecutive rounds of plaque purification screening for non-staining viral foci in CEF cells in the presence of 5-bromo-4-chloro-3-indolyl ⁇ -D-galactoside (300 mg/ml). Subsequently, recombinant viruses were amplified by infection of CEF monolayers.
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AU10249/99A AU754386B2 (en) | 1997-09-23 | 1998-09-21 | Dengue virus antigens and treatment of dengue fever |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004067567A2 (en) * | 2003-01-31 | 2004-08-12 | Novartis Ag | Anti-dengue virus antibodies, compositions, methods and uses |
WO2007059715A2 (en) | 2005-11-22 | 2007-05-31 | Centro De Ingenieria Genetica Y Biotecnologia | Methods and proteins for the prophylactic and/or therapeutic treatment of the four serotypes of dengue virus and other flaviviruses |
AU2002356690B2 (en) * | 2001-12-04 | 2008-07-24 | Bavarian Nordic A/S | Flavivirus NS1 subunit vaccine |
WO2017060650A1 (en) | 2015-10-08 | 2017-04-13 | Jean-Marc Limacher | Anti-tumoral composition |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992003545A1 (en) * | 1990-08-15 | 1992-03-05 | Virogenetics Corporation | Flavivirus recombinant poxvirus vaccine |
WO1996040933A1 (en) * | 1995-06-07 | 1996-12-19 | The Government Of The United States Of America, Represented By The Secretary Department Of Health And Human Services | Infectious dengue 2 virus pdk-53 as quadravalent vaccine |
WO1998013500A2 (en) * | 1996-09-24 | 1998-04-02 | Bavarian Nordic Research Institute A/S | Recombinant mva virus expressing dengue virus antigens, and the use thereof in vaccines |
-
1998
- 1998-09-21 WO PCT/EP1998/006009 patent/WO1999015692A2/en not_active Application Discontinuation
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992003545A1 (en) * | 1990-08-15 | 1992-03-05 | Virogenetics Corporation | Flavivirus recombinant poxvirus vaccine |
WO1996040933A1 (en) * | 1995-06-07 | 1996-12-19 | The Government Of The United States Of America, Represented By The Secretary Department Of Health And Human Services | Infectious dengue 2 virus pdk-53 as quadravalent vaccine |
WO1998013500A2 (en) * | 1996-09-24 | 1998-04-02 | Bavarian Nordic Research Institute A/S | Recombinant mva virus expressing dengue virus antigens, and the use thereof in vaccines |
Non-Patent Citations (5)
Title |
---|
A. FALCONAR ET AL.: "Precise location of sequential dengue virus subcomplex and complex B cell epitopes on the nonstructural-1 glycoprotein." ARCHIVES OF VIROLOGY, vol. 137, no. 3-4, 1994, pages 315-326, XP002101238 Vienna, Austria * |
G. SUTTER ET AL.: "A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus." VACCINE, vol. 12, no. 11, August 1994, pages 1032-1040, XP002101241 Oxford, GB * |
L. WYATT ET AL.: "Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells." VIROLOGY, vol. 210, no. 1, 20 June 1995, pages 202-205, XP002101240 Orlando, FL, USA * |
M. CARDOSA: "Dengue vaccine design: issues and challenges." BRITISH MEDICAL BULLETIN, vol. 54, no. 2, 1998, pages 395-405, XP002101242 Edinburgh, GB * |
M. PUPO-ANTUNEZ ET AL.: "Monoclonal antibodies raised to the Dengue-2 virus (Cuban A15 strain) which recognize viral structural proteins." HYBRIDOMA, vol. 16, no. 4, August 1997, pages 347-353, XP002101239 New York, NY, USA * |
Cited By (9)
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AU2002356690B2 (en) * | 2001-12-04 | 2008-07-24 | Bavarian Nordic A/S | Flavivirus NS1 subunit vaccine |
US7759116B2 (en) | 2001-12-04 | 2010-07-20 | Bavarian Nordic A/S | MVA virus vector expressing dengue NS1 protein |
EP2345665A2 (en) | 2001-12-04 | 2011-07-20 | Bavarian Nordic A/S | Flavivirus NS1 subunit vaccine |
US8815501B2 (en) | 2001-12-04 | 2014-08-26 | Bavarian Nordic A/S | Flavivirus NS1 subunit vaccine |
WO2004067567A2 (en) * | 2003-01-31 | 2004-08-12 | Novartis Ag | Anti-dengue virus antibodies, compositions, methods and uses |
WO2004067567A3 (en) * | 2003-01-31 | 2004-11-25 | Novartis Ag | Anti-dengue virus antibodies, compositions, methods and uses |
JP2007524350A (en) * | 2003-01-31 | 2007-08-30 | ノバルティス アクチエンゲゼルシャフト | Anti-dengue virus antibodies, compositions, methods and uses |
WO2007059715A2 (en) | 2005-11-22 | 2007-05-31 | Centro De Ingenieria Genetica Y Biotecnologia | Methods and proteins for the prophylactic and/or therapeutic treatment of the four serotypes of dengue virus and other flaviviruses |
WO2017060650A1 (en) | 2015-10-08 | 2017-04-13 | Jean-Marc Limacher | Anti-tumoral composition |
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EP1021545A2 (en) | 2000-07-26 |
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