Patent Application
20060088937
The present invention generally relates to the field of gene expression and in particular to Flavivirus gene expression and delivery systems and to virus like particles produced from such systems.
The present invention generally relates to the field of gene expression and in particular to Flavivirus gene expression and delivery systems and to virus like particles produced from such systems.
Improved methodologies for maximising recombinant gene expression are an on-going effort in the art. Of particular interest is the development of methodologies that maximise recombinant expression of mammalian genes in safe vectors suitable for producing commercially useful quantities of biologically active proteins.
Currently, there are numerous expression systems available for the expression of genes. While procaryotic and yeast expression systems are extremely efficient and easy to use, these systems suffer from a number of disadvantages, including an inability to glycosylate proteins, inefficient cleavage of “pre” or “prepro” sequences from proteins (eg., inefficient post translational modification), and a general inability to secrete proteins.
Another expression system widely available is the baculovirus expression system. This system is arguably one of the most efficient in protein production, but is limited only to use in insect cell lines. Unfortunately, insect cell lines glycosylate proteins differently from mammalian cell lines thus this system has not proven useful for the production of many mammalian proteins. Another disadvantage of this system is that it relies on the use of homologous recombination for the construction of recombinant virus stocks. Thus, this system often proves very laborious when large numbers of genetic variants have to be analysed.
In view of these problems the art has sought eucaryotic host systems, typically mammalian host cell systems, for mammalian protein production. One feature of such systems is that the protein produced has a structure most like that of the natural protein species and purification often is easier since the protein can be secreted into the culture medium in a biologically active form.
One of the most efficient mammalian cell expression systems is based on Vaccinia virus. The main problem with this system, however, is that it uses recombinant viruses that express the heterologous gene upon infection. Thus there is no control over the virus once it has been release.
Recently researchers have started to explore the use of positive strand RNA viruses such as Semliki Forest Virus (SFV), Sindbis (SIN) virus, and poliovirus, as vectors for expression of heterologous genes in vitro and in vivo. The success of these expression systems has been mainly based on each virus' ability to produce high titer stocks of “pseudo” infectious particles containing recombinant replicon RNA packaged by structural proteins. In commercially available Semliki Forest virus (SFV) and Sindbis virus expression systems this is achieved by co-transfection of replicon RNA with defective helper RNA(s) expressing structural genes, but lacking the packaging signal. Replicon RNA expression provides enzymes for RNA replication and transcription of both RNA's, whereas helper RNA supports the production of structural proteins for packaging of replicon RNA via expression of its subgenomic region. The main problem with these expression systems is that the viruses used in the expression system are cytopathic and often compete out the host protein synthesis. Another major disadvantage of these systems includes possible contamination with infectious particles containing packaged full-length genomic RNA (in other words, infectious virus) due to the high probability of recombination between replicon and helper RNAs.
The present invention seeks to provide an improved expression and delivery system that at least ameliorates some of the problems associated with prior art systems.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers including method steps.
The present invention provides a gene expression system comprising:
Any replicon (self-replicating expression vector) derived from any flavivirus RNA may be used in the present invention. The replicon should however encode a sufficient amount of a flavivirus 5′ UTR and at least a portion of the 5′ flavivirus coding region for core protein, each of which is required for RNA replication. Both the 5′ UTR and the 5′ core protein coding region of a flavivirus genome contains regulatory elements that are required for flavivirus RNA replication. It will be appreciated that the flavivirus 5′ UTR and the 5′ core protein coding region may contain mutations or deletions in these regions and still be able to replicate. Preferably, the replicon should contain 5′ UTR and at least about between 60 and 80 nucleotides from the 5′ coding region for flavivirus core protein. The relative number of nucleotides from the 5′ core protein coding region that will be required in the replicon for RNA replication will largely depend on the type of flavivirus used in the vector. For example when the replicon is derived from Kunjin virus it must contain at least 60 nucleotides of the 5′ core protein coding region.
In one particular embodiment of the invention there is provided a gene expression system comprising:
According to the present invention, the replicon of flavivirus origin is adapted to receive at least a nucleotide sequence. Insertion of such a nucleotide sequence, into the replicon may be achieved at any point in the replicon that does not effect processing of flavivirus proteins. For example, heterologous genes may be inserted into the 3′ UTR of the flavivirus replicon, within a structural gene or within the locality of deleted structural genes. Preferably, heterologous genes are inserted into structural genes or in place of deleted structural genes since such insertions generally produce higher levels of expression and generally do not affect replication efficiency of the replicon. If, however, the nucleotide sequence(s) are inserted into the 3′ UTR they may be preceded by an internal ribosomal entry site (IRES) sequence. In an embodiment of the invention, the 3′ UTR is used only for insertion of IRES-Neo (neomycin transferase) or IRES-pac (puromycin N-acetyl transferase) sequences. Such insertions allow the generation of stable cell lines persistently expressing foreign genes via antibiotic (eg Geneticin or puromycin) selection.
In another preferred embodiment of the invention there is provided a gene expression system comprising:
When the nucleotide sequence is inserted into the replicon it should be introduced into the vector in a manner which avoids a frame shift in the open reading frame of the vector coding sequence. This may be achieved by either adapting the foreign nucleotide sequence or the vector to ensure the reading frame of the vector coding sequence is maintained. In an alternative arrangement foreign nucleotide sequence can be inserted without preserving open reading frame of the vector if it is followed by a termination codon and an internal ribosomal entry site (IRES) sequence to ensure initiation of translation of the vector's nonstructural proteins.
A replicon which encodes flavivirus structural and non-structural proteins may be either RNA or DNA based provided it is capable of self-replication and encodes flavivirus structural and non-structural protein coding information. Where the replicon is an RNA sequence the flavivirus genome is first reverse transcribed into complementary DNA sequence and cloned into appropriate plasmid vector containing prokaryotic (bacteriophage) DNA-dependent RNA polymerase promotor. The nucleotide sequence is then inserted into the resulting plasmid containing replicon complementary DNA sequence and the genomic sequence is then transcribed back into RNA prior to delivery to a host cell. Where the vector is DNA based the flavivirus genome is first reverse transcribed into complementary DNA sequence and cloned into appropriate plasmid vector containing eucaryotic expression promoter. A nucleotide sequence can then be inserted into the resulting plasmid containing replicon complementary DNA sequence, which is then introduced into a host cell as plasmid DNA.
While the replicon will in most circumstances be prepared from a single strain of flavivirus it should be appreciated that in some circumstances nucleotide sequences from more than one flavivirus strain may be brought together in a single vector. Preferably the replicon is derived from the genomic sequence of a single flavivirus species. Most preferably the replicon is derived from a single flavivirus species (such as Kunjin virus (KUN)) and includes the entire or a substantial portion of the genome of that strain, the genome being modified in at least one of its structural proteins to accept a nucleotide sequence such that the insertion of the nucleotide sequence into the structural protein nucleotide sequence disrupts coding for part or all of the structural protein.
Nucleotide sequences that may be inserted into the replicon include, for example, parts of flavivirus or non-flavivirus cDNA gene sequences. Nucleotide sequence(s) that are inserted into the replicon must, however, disrupt the expression of at least a structural protein thus preventing viral genome packaging. Desirably the inserted nucleotide sequence is a non-flavivirus nucleotide sequence (hereinafter referred to as a “heterologous nucleotide sequence”). The heterologous nucleotide sequence is not limited only to a sequence that encodes an amino acid sequence, but may also include sequences appropriate for promoting replication and or expression of a sequence that encodes an amino acid sequence.
Insertion of a heterologous nucleotide sequence into the replicon may occur at any point in a flavivirus structural protein(s) or in any region of the nucleotide sequence where such a protein would normally be expressed in the native flavivirus sequence had the protein not been deleted. In one embodiment of the invention the heterologous nucleotide sequence is inserted into at least one of the structural genes deactivating that gene. In another embodiment at least a structural gene is deleted from the vector and the deletion site is adapted to serve as the insertion site for heterologous genetic sequences. Most preferably, the nucleotide sequence is inserted into the locality from where at least a structural gene was deleted.
By positioning heterologous nucleotide sequences within the locality of one or more sites in the replicon that might otherwise code for structural genes in a native flavivirus, the replicon is unable to produce structural proteins for viral packaging.
To induce viral packaging the invention employs a second vector that is engineered to prevent recombination with the replicon. Preferably, the second vector is heterologous in origin to the origin of the replicon. Any non-flavivirus vector that is engineered to prevent recombination with the replicon may be employed in the expression system to deliver the flavivirus structural protein that is deactivated in the replicon. For example, if a KUN replicon is used as the self-replicating expression vector, then the second vector may be derived from a virus other than a flavivirus. For example, the second vector could be derived from an alphavirus such as SFV or SIN, or from DNA virus such as adenovirus, fowlpox virus, or vaccinia virus. Those of ordinary skill in the field will know other vectors that may be employed in this role. In a highly preferred form of the invention the replicon is derived from KUN while the second vector is derived from SFV to take account of the impossible recombination between KUN RNA and SFV RNA.
In an alternative embodiment of the invention the second vector may be a plasmid DNA expression vector. For example, highly efficient packaging may be achieved by inserting structural genes into CMV based DNA expression cassettes which are inserted into baculovirus expression vectors which provide very efficient delivery of the cassettes into mammalian cells (see for example Shoji et al, (1997) J. Gen. Virol., 78: 2657-2664 and pBacMam-1 vector described on the Novagen homepage). In another example the second vector may be an inducible plasmid DNA expression vector (for example tetracycline inducible vector (Clontech)) allowing selection of packaging cell lines expressing KUN structural proteins in response to addition or removal of tetracycline in the incubation medium.
The present invention also provides a method for producing a stable cell line capable of persistently producing replicon RNA's, comprising the steps of:
Conditions that permit cell growth and replication will be known to those of ordinary skill in the field. In particular the conditions will vary depending on the type of cell that is used in the method. To prepare such cell lines, the described vectors are preferably constructed in selectable form by inserting an IRES-Neo or IRES-pac cassette into the 3′ UTR.
In another embodiment, the invention provides a method for producing a flavivirus like particles containing a replicon as herein described comprising the steps of:
Preferably the replicon containing virus like particles prepared by this method are purified from cellular and viral proteins and nucleic acids that may cause an adverse immunological or physiological reaction when introduced into an animal. Methods for purifying such viral particles are known in the art. Most preferably the replicon containing virus like particles are 50%, 60%, 70%, 80%, 90%, 95% or 99% free of all contaminating material including cellular and viral proteins, lipids and nucleic acids.
In further embodiment, the invention provides a flavivirus like particles containing a flavivirus replicon that is adapted to receive at least a nucleotide sequence without disrupting its replication capabilities. Desirably the virus like particles are purified from cellular and viral nucleic acids and amino acid sequences that may cause an adverse immunological or physiological reaction when introduced into an animal. Such particles may be used as a therapeutic agent. A person of ordinary skill in the field will appreciate that the described virus particles can be used to deliver to a subject any nucleotide sequence that is inserted into the replicon. For example the replicon within the virus like particles may be employed to deliver to a cell a nucleotide sequence encoding one or more amino acid sequences which are capable of inducing, for example, a protective immune response to a subject.
In further embodiment, the invention provides a DNA based replicon of flavivirus origin that is adapted to receive at least a nucleotide sequence without disrupting its replication capabilities. The DNA based replicon may be introduced into a cell as a naked vector (i.e. flavivirus structural proteins do not surround it) or alternatively used for preparation of virus like particles containing encapsidated replicon RNA in accordance with the described method. Whether the DNA based replicon is prepared as a naked vector or in virus like particles it should be purified from cellular and viral nucleic acids and amino acid sequences that may cause an adverse immunological or physiological reaction in an animal prior to introduction into that animal. Such particles may be used as a therapeutic agent. A person of ordinary skill in the field will appreciate that the described virus particles can be used to deliver to a subject any nucleotide sequence that is inserted into the replicon. In a particularly preferred form of the invention the replicon is prepared in DNA form and is used for preparation of virus like particles containing encapsidated replicon RNA for delivery into a cell via infection.
Although the present invention describes a means for producing proteins, the term “protein” should be understood to include within its scope parts of proteins such as peptide and polypeptide sequences.
In use, the replicon is introduced into a host cell where gene expression and hence protein production take place. Because the vector is capable of self-replication, multiple copies of the replicon will also be generated. This leads to an exponential increase in the number of replicons in the host cell as well as an exponential increase in the amount of protein that is produced.
Upon introduction of the second vector, containing the structural genes necessary to produce virus particles, structural proteins are produced. These proteins encapsulate the replicon therein forming a “pseudo” recombinant virus that is only capable of producing heterologous protein inside another cell. The pseudo-virus can not however replicate to produce new viral particles because the genes necessary for the production of the structural proteins are not provided in the replicon. Pseudo-virus stock will only be produced when co-transfection of the replicon and the vector bearing the structural genes occurs.
Some advantages associated with the use of the present invention include:
The replication of flaviviruses is quite different from other viruses. For example, flaviviruses differ from alphaviruses (such as SFV and SIN) by their genome structure (structural genes situated at the 5′ end of the genome) and by the absence of synthesis of subgenomic RNA. Furthermore, there are no data to date on packaging of flavivirus RNA. Substantial progress in the development of mammalian cell expression systems has been made in the last decade, and many aspects of these systems' features are well characterised. A detailed review of the state of the art of the production of foreign proteins in mammalian cells, including useful cell lines, protein expression-promoting sequences, marker genes, and gene amplification methods, is disclosed in Bendig, M., (1988) Genetic Engineering 7: 91-127.
It will be appreciated that any replicon derived from any flavivirus RNA, which is lacking at least a structural gene and which is adapted to receive at least a nucleotide sequence may be employed in the present invention. Preferably the replicon used in the invention should be adapted to include part or all of the following: at least, about the first 150 nucleotides of a flavivirus genome; at least about the last 60 nucleotides of E protein; substantially all of the nonstructural region; and part or all of the 3′ UTR. Replication of a flavivirus genome is dependent on the genes in the nonstructural region of the genome being present during transcription and translation. Preferably any modification made to the nonstructural region should not interfere with the functional activity of the genes within the nonstructural region of the genome. In a highly preferred form of the invention, the replicon is derived from KUN and includes the first 157 nucleotides of the KUN genome, the last 66 nucleotides of E protein, the entire nonstructural region, and all of the 3′ UTR.
Optimal flavivirus replicon design for transfection into eukaryotic cells might also include sequences inserted into the replicon such as: sequences to promote expression of the heterologous gene of interest, including appropriate transcription initiation, termination, and enhancer sequences; as well as sequences that enhance translation efficiency, such as the Kozak consensus sequence; internal ribosomal entry site (IRES) of picornaviruses; an alphavirus subgenomic 26S promoter to enhance expression of inserted genes if cotransfection with alphavirus replicon RNA is used.
Flavivirus replicon RNA can be produced in in vitro transcription reaction with DNA-dependent RNA polymerase from corresponding plasmid cDNA constructs incorporating a prokaryotic (bacteriophage) promoter upstream of KUN genome sequence. Such replicon constructs are referred to as RNA-based replicon vectors. Resulting in vitro transcribed RNA can be delivered into the cell cytoplasm by RNA transfection followed by its self-amplification and translation resulting in expression of heterologous genes.
Alternatively, flavivirus replicon RNA can be produced in cells (in vivo) by the cellular transcription machinery after transfection of corresponding plasmid cDNA constructs incorporating a eucaryotic experession promoter upstream and transcription termination signal downstream of the KUN replicon sequence. These replicon constructs are referred to as DNA-based replicon vectors. Production of replicon RNA from these DNA-based vectors occurs in the nucleus of transfected cells by RNA polymerase II, followed by the transport of RNA into the cytoplasm where its amplification and translation takes place.
Finally, flavivirus replicon RNA produced in cells as a results of its self-amplification either after RNA transfection (RNA-based vector) or after plasmid DNA transfection (DNA-based vectors) can be packaged into the secreted virus-like particles by providing KUN structural proteins from a second vector. VLPs can then be used to deliver the encapsidated replicon RNA into cells by infection.
In one example of the invention the DNA-based replicon vector is derived from KUN virus and contains a eucaryotic promoter sequence (such as CMV or hybrid CMV enhancer-chicken β-actin promoter [CAG]) upstream of the KUN 5′ UTR and a hepatitis delta virus ribozyme sequence followed by an SV40, bovine growth hormone, or rabbit β-globin transcription terminator sequences downstream of the KUN 3′ UTR. Transfection of the resulting plasmid DNA in cells will ensure production of a KUN replicon RNA transcript with the authentic 5′-end by cellular RNA polymerase II and with the authentic 3′-end cleaved by hepatitis delta virus ribozyme, which is preferred for its efficient replication.
It will be appreciated that the nucleotide sequence inserted into the replicon may encode part or all of any natural or recombinant protein except for the structural protein sequence into which or in place of which the nucleotide sequence is inserted. For example, the nucleotide sequence may encode a single polypeptide sequence or a plurality of sequences linked together in such a way that each of the sequences retains their identity when expressed as an amino acid sequence. Where the nucleotide sequence encodes a plurality of peptides, the peptides should be linked together in such a way that each retains its identity when expressed. Such polypeptides may be produced as a fusion protein or engineered in such a manner to result in separate polypeptide or peptide sequences.
Where the vector is used to deliver nucleotide sequences to a host cell to enable host cell expression of immunogenic polypeptides, the nucleotide sequence may encode one or more immunogenic polypeptides in association with a range of epitopes which contribute to T-cell activity. In such circumstances the heterologous nucleotide sequence preferably encodes epitopes capable of eliciting either a T helper cell response or a cytotoxic T-cell (CTL) response or both.
The replicon described herein may also be engineered to express multiple nucleotide sequences allowing co-expression of several proteins such as a plurality of antigens together with cytokines or other immunomodulators to enhance the generation of an immune response. Such a replicon might be particularly useful for example in the production of various proteins at the same time or in gene therapy applications.
By way of example only the nucleotide sequence may encode the cDNA sequence of one or more of the following: malarial surface antigens; beta-galactosidase; any major antigenic viral antigen eg Haemagglutinin from influenza virus or a human immunodeficiency virus (HIV) protein such as HIV gp 120 and HIV gag protein or part thereof; any eukaryotic polypeptide such as, for example, a mammalian polypeptide such as an enzyme, e.g. chymosin or gastric lipase; an enzyme inhibitor, e.g. tissue inhibitor of metalloproteinase (TIMP); a hormone, e.g. growth hormone; a lymphokine, e.g. an interferon; a cytokine, e.g an interleukin (eg IL-2, IL-4, IL-6 etc); a chemokine eg macrophage inflammatory protein-2; a plasminogen activator, e.g. tissue plasminogen activator (tPA) or 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.
The nucleotide sequence may also code for one or more amino acid sequences that serve to enhance the effect of the protein being expressed. For example, ubiquitination of viral proteins expressed from DNA vectors results in enhancement of cytotoxic T-lymphocyte induction and antiviral protection after immunization. Thus, in a preferred embodiment of the invention the replicon 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.
In frame fusion of proteins other than flavivirus replicon encoded proteins to the C-terminus of ubiquitin also results in the efficient cleavage of such fusion protein after the last C-terminal residue of ubiquitin thus releasing free protein of interest. Preferably a ubiquitin sequence is inserted into the replicon vector. By way of example only the ubiquitin sequence is preferably inserted either prior to the 5′ end of the heterologous genetic sequence or at the 3′ end of the heterologous genetic sequence.
The second vector that contains the flavivirus structural gene(s) should be engineered to prevent recombination with the self-replicating expression vector. One means for achieving this end is to prepare the second vector from genetic material that is heterologous in origin to the origin of the self-replicating expression vector. For example, the second vector might be prepared from SFV when the replicon is prepared from KUN virus.
To optimise expression of the flavivirus structural genes, the second vector might include such sequences as: sequences to promote expression of the genes of interest, including appropriate transcription initiation, termination, and enhancer sequences; as well as sequences that enhance translation efficiency, such as the Kozak consensus sequence. Preferably, the second vector contains separate regulatory elements associated with each of the different structural genes expressed by the vector. Most preferably, the flavivirus C gene and the prME genes are placed under the control of separate regulatory elements in the vector.
The processing of flavivirus structural proteins during virus replication in cells is complex and requires a number of post-translational cleavages by host and viral proteases. Numerous in vitro and in vivo studies on processing of the C-prM region have established two cleavage events: cleavage at a dibasic cleavage site by viral NS2B-NS3 protease generating the carboxy terminus of mature virion C protein, which appears to be a prerequisite for the efficient cleavage at the NH2 terminus of prM by cellular signalase. While viral proteases are expressed by the replicon during expression of the genes forming the nonstructural region of a flavivirus, it will be appreciated that the second vector may also be adapted to include genes encoding viral NS2B-NS3 protease.
Further C-prM-E genes can be expressed as a single cassette only if C and prM genes separated by a self-cleaved peptide like for example 2A autoprotease of foot-and-mouth disease virus in order to ensure proper processing of C-pM region in the absence of KUN virus encoded NS2B-NS3 protease.
The present invention also provides stable cell lines capable of persistently producing replicon RNAs. To prepare such cell lines, the described vectors are preferably constructed in selectable form by inserting an IRES-Neo or IRES-pac cassette into the 3′ UTR.
Host cell lines contemplated to be useful in the method of the invention include any eukaryotic cell lines that can be immortalised, 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. Currently 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 BHK (baby hamster kidney), VERO, C6-36, COS, CHO (Chinese hamster ovary), myeloma, HeLa, fibroblast, embryonic and various tissue cells, eg., kidney, liver, lung and the like and the like. Desirably a cell line is selected from one of the following: BHK21 (hamster), SK6 (swine), VERO (monkey), L292 (mouse), HeLa (human), HEK (human), 2fTGH cells, HepG2 (human). Useful cells 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.
With respect to the transfection process used in the practice of the invention, all means for introducing nucleic acids into a cell are contemplated including, without limitation, CaPO.sub.4 co-precipitation, electroporation, DEAE-dextran mediated uptake, protoplast fusion, microinjection and lipofusion. Moreover, the invention contemplates either simultaneous or sequential transfection of the host cell with vectors containing the RNA sequences. In one preferred embodiment, host cells are sequentially transfected with at least two unlinked vectors, one of which contains flavivirus replicon expressing heterologous gene, and the other of which contains the structural genes.
The present invention also provides virus like particles containing flavivirus replicons and a method for producing such particles. It will be appreciated by those skill in the art that virus like particles that contain flavivirus derived replicons can be used to deliver any nucleotide sequence to a cell. Further, the replicons may be of either DNA or RNA in structure. One particular use for such particles is to deliver nucleotide sequences coding for polypeptides that stimulate an immune response. Such particles may be employed as a therapeutic or in circumstances where the nucleotide sequence encodes peptides that are capable of eliciting a protective immune response they may be used as a vaccine. Another use for such particles is to introduce into a subject a nucleotide sequence coding for a protein that is either deficient or is being produced in insufficient amounts in a cell.
The replicon containing flavivirus like particles that contain nucleotide coding sequence for immunogenic polypeptide(s) as active ingredients may be 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 flavivirus replicon therapeutic(s) may also be mixed with excipients that are pharmaceutically acceptable and compatible with the replicon encapsulated viral particle. Suitable excipients are, for example, water, saline, dextrose glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the therapeutic may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvant which enhance the effectiveness of the therapeutic.
The replicon containing flavivirus like particles may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%.
Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like, These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of virus like particles, preferably 25-70%.
The flavivirus like particles may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such an, 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, histidino, procaine, and the like.
The flavivirus like particles may be administered in a manner compatible with the dosage formulation and in such amount as will be prophylactically and/or therapeutically effective. The dose of viral particles to be administered depends on the subject to be treated, the type of nucleotide sequence that is being administered and the type of expression efficiency of that sequence and in the case where the nucleotide sequence encodes immunogenic peptide/polypeptides the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be peculiar to each subject.
The flavivirus like particles may be given to a subject in a single delivery schedule, or preferably in a multiple delivery schedule. A multiple delivery schedule is one in which a primary course of delivery may be with 1 - 10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or re-enforce the effect sought and if needed, a subsequent dose(s) after several months. The delivery regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.
Further features of the present invention are more fully described in the following Figures and Examples. In the figures:
The other abbreviations are as in (A).
Further features of the present invention are more fully described in the following Examples. It is to be understood that the following Examples are included solely for the purposes of exemplifying the invention, and should not be understood in any way as a restriction on the broad description as set out above.
Cells.
BHK<21 cells were grown in Dulbecco's modification of minimal essential medium (Gibco BRL) supplemented with 10% foetal bovine serum at 37° C. in a CO2 incubator.
Construction of the Replicons and Vectors.
(i) C20Rep
All deletion constructs were prepared from the cDNA clones used in the construction of the plasmid pAKUN for generation of the infectious KUN RNA (Khromykh and Westaway, J.Virol., 1994, 68:4580-4588) by PCR-directed mutagenesis using appropriate primers and conventional cloning. dME cDNA and its derivatives were deleted from nucleotides 417 to 2404, which represent loss of the signal sequence at the carboxy terminus of C now reduced to 107 amino acids, deletion of prM and E, with the open reading frame resumed at codon 479 in E, preceding the signal sequence for NS1. C20 rep and C2rep cDNAs represent progressive in frame deletions in coding sequence of C leaving only 20 or 2 amino acids of C, respectively, with the open reading frame continued at codon 479 in E, as in dME.
(ii) FLSDX
All RT reactions were performed with Superscript II RNase H-reverse transcriptase (Gibco BRL) essentially as described by the manufacturer using 100-200 ng of purified KUN virion RNA, or 1 μg of total cell RNA and appropriate primers. PCR amplification after RT of a 6895 bp DNA fragment was performed with the Expand High Fidelity PCR kit (Boehringer Mannheim) using 1/25 volume of RT reaction and appropriate primers as follows. The PCR reaction mixture (50 μl) containing all necessary components except the enzyme mixture (3 parts of Taq polymerasse and 1 part of Pwo polymerase) was preheated at 95° C. for 5 min, then the enzyme mixture was added and the following cycles were performed: 10 cycles of 95° C. for 15 sec and 72° C. for 6 min, followed by 6 cycles of 95° C. for 15 sec and 72° C. for 6 min with an automatic increase of extension time at 72° C. for 20 sec in each following cycle. All PCR reactions with Pfu DNA polymerasse (Stratagene) were performed essentially as described by the manufacturer using 1/25- 1/10 volumes of RT reactions and appropriate primers.
All plasmids shown in
Initially the SacII1481-DraIII8376 (6895 bp) fragment in pAKUN clone (
(iii) C20DXrep
KUN replicon cDNA construct C20DXrep was constructed from described above C20rep by replacing an SphI2467 XhoI11021 fragment representing the sequence coding for the entire nonstructural region and the 3′ UTR with the corresponding fragment from a stable full-length KUN cDNA clone FLSDX. Transfection of BHK cells with 5-10 μg of C20DXrep RNA resulted in detection of 80% replicon-positive cells compare to only ˜10% positive after transfection with the same amount of C20rep RNA.
(iv) SFV-C.
An SFV replicon construct expressing KUN core (C) gene was obtained by cloning of the Bg/II-BamHI fragment, representing the sequence of the last 7 nucleotides of the KUN 5′ UTR and the sequence coding for the first 107 of the 123 amino acids of KUN C protein, from the plasmid pCINeoC107 (Khromykh, A. A. and E. G. Westaway. Arch. Virol., 1996, 141:685-699) into the BamHI site of the SFV replicon expression vector pSFV1 (Gibco BRL;
(v) SFV-prME
KUN prME sequence was PCR amplified from another highly efficient full-length KUN cDNA clone FLBSDX modified from FLSDX (which will be described elsewhere), using appropriate primers with incorporated Bg/II sites. The amplified fragment was digested with Bg/II and cloned into the BamHI site of the SFV replicon expression vector pSFV1 to obtain the SFV-prME construct (
(vi) SFV-prME-C
SFV replicon construct expressing both KUN prME and KUN C genes was obtained by cloning a MscI-SpeI fragment from the SFV-C plasmid containing the SFV 26S subgenomic promoter, KUN C sequence and SFV 3′ UTR into the SFV-prME vector digested with SmaI and SpeI (
RNA Transcription and Transfection.
RNA transcripts were prepared from C20DXrep plasmid DNA linearized with XhoI, and from SFV plasmids linearised with SpeI using SP6 RNA polymerase. Electroporation of RNAs into BHK21 cells was performed. Briefly, 10-20 μg of in vitro transcribed RNAs were electroporated into 2×106 BHK21 cells in 400 μl in a 0.2-cm cuvette (Bio-Rad) using the Bio-Rad Gene Pulser apparatus.
Immunofluorescence Analysis.
Replication of KUN replicon RNA C20DXrep after initial electroporation, and after infection of BHK cells in packaging experiments, was monitored by immunofluorescence (IF) analysis with antibodies to KUN NS3 protein. Expression of KUN E protein after electroporation with SFV-prME and SFV-prME-C RNAs was detected by IF with a cocktail of mouse monoclonal antibodies to KUN E protein. These antibodies designated 3.91D, 10A1, and 3.67G were generously provided by Roy Hall, University of Queensland, Brisbane, Australia. All three antibodies were mixed in equal amounts and a 1/10 dilution of this mixture was used in IF analysis. Expression and nuclear localisation of KUN C protein after electroporation with SFV-C and SFV-prME-C RNAs was monitored by IF analysis with rabbit polyclonal antibodies to KUN C protein.
Metabolic Labeling and Radioimmunoprecipitation Analysis.
Metabolic labeling with 35S-methionine/cysteine of electroporated BHK cells was performed essentially as described in the SFV Gene Expression System Manual with some minor modifications. Briefly, cells at 18 h after the electroporation with SFV RNAs (with or without prior electroporation with KUN replicon RNA), were pulse labeled with 35S-methionine/cysteine for 4h, or for 1-2h followed by different periods of incubation (chase) in medium with an excess of unlabeled methionine/cysteine. Cell culture fluid was collected for analysis of secreted proteins by electrophoresis and radioimmunoprecipitation (RIP). Labeled cells were lysed in buffer containing 1% Nonidet P40 (NP40), 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 2 mM EDTA, the nuclei removed by low speed centrifugation and the lysate supernatant was used for parallel analysis with the culture fluid.
For RIP analysis, labeled cell culture fluids were first filtered through 0.45 μm filter (Sartorius AG, Gottingen, Germany) and digested with RNase A (20 μg per ml) for 30 min at 37° C. to ensure the removal of membrane particulate material and naked RNA. Filtered and RNase treated culture fluids, or untreated cell lysates, were then mixed with 1/20 volume of the pooled anti-E monoclonal antibodies (see above) or with rabbit anti-C antibodies, and incubated overnight at 4° C. with constant rotation in microcentrifuge tubes. Protein A-Sepharose beads were then added to a final concentration of about 1%, and incubation was continued for another 1h at 4° C. After three washes with RIPA buffer (50 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1% NP40; 0.5% deoxycholic acid sodium salt [DOC]; 0.1% sodium dodecyl sulfate [SDS]) and one wash with phosphate buffered saline (PBS), beads were resuspended in the SDS-gel sample buffer, boiled for 5 min and subjected to electrophoresis in an SDS-polyacrylamide gel. After electrophoresis gels were dried and exposed to X-ray film.
Northern Blot Hybridisation.
Five μg total RNA, isolated using Trizol reagent (Gibco BRL) from BHK21 cells infected with culture fluid collected from cells doubly transfected with C20DXrep RNA and SFV RNAs expressing KUN structural proteins, was electrophoresed for Northern blotting. The hybridisation probes were [32P]-labelled cDNA fragments representing the 3′-terminal 761 nucleotides of the KUN genome including the 3′ UTR region (see
Expression of KUN C Gene by the Recombinant SFV-C Replicon.
For the expression of KUN C gene in the pSFV1 vector the Bg/II-BamHI fragment from plasmid pCINeoC107 was subcloned into the BamHI site of pSFV1 (
Electroporation of SFV-C RNA into BHK21 cells resulted in expression of KUN C protein in almost 100% of cells as judged by IF with antibodies to KUN C protein (Fig.3A, panel 1). KUN C protein expressed in SFV-C RNA transfected cells was localised in the cytoplasm (
Expression of KUN prME Genes by the Recombinant SFV-prME Replicon.
The full-length prME sequence plus the preceding signal sequence in our SFV-prME construct (see
When SFV-prME RNA was electroporated into BHK21 cells, nearly 100% of cells were found to be positive in IF analysis with monoclonal antibodies to KUN E protein at 12-18h after electroporation (
An apparent increase in the intensity of labelling of E and possibly pr proteins in the culture fluid (
When samples from the pulse-chase labelling experiment with SFV-prME replicon were immunoprecipitated with KUN anti-E monoclonal antibodies, E and prM were coprecipitated from the transfected cell lysates (
Expression of all Three KUN Structural Proteins by the Recombinant SFV-PrME-C Replicon.
Although KUN replicon was packaged using transfection with two SFV RNAs expressing prME and C genes separately (see results in the next example), the efficiency of packaging was low. To increase the efficiency of packaging and to simplify the procedure a single SFV replicon construct was prepared expressing prME genes and C gene simultaneously. Because of the difficulties experienced with cloning of the entire C-prM-E region into the pSFV1 vector (see the first section of the Results) and also in order to avoid possible uncertainty regarding cleavage at the carboxy terminus of C in the absence of flavivirus protease NS2B-NS3, an SFV replicon expressing prME and C genes under the control of two separate 26S promoters was prepared (see SFV-prME-C in
IF analysis of SFV-prME-C electroporated BHK cells with anti-E and anti-C antibodies showed expression of both E and C proteins in nearly 100% of cells by 18h after transfection (results not shown). Both E and C proteins were expressed in the same cell (compare dual labelling by anti-C and anti-E antibodies in
Overall, the immunofluorescence and labelling patterns in cells transfected with SFV-prME-C RNA were very similar to those observed in cells transfected with two different RNAs expressing prME and C proteins separately (compare
For all infections with encapsidated particles, cell culture fluid was filtered through a 0.45 μm filter (Sartorius AG, Gottingen, Germany) and treated with RNase A (20μg per ml) for 0.5h at room temperature (followed by 1.5h incubation at 37° C. during infection of cells). To prepare partially purified particles, filtered and RNase A treated culture fluids from transfected cells were clarified by centrifugation at 16,000 g in the microcentrifuge for 15 min at 4° C., and the particles were pelleted from the resulting supernatant fluid by ultracentrifugation at 40,000 rpm for 2h at 4° C. in the AH650 rotor of the Sorvall OTD55B centrifuge. The pellets were resuspended in 50 μl PBS supplemented with RNAse A (20μg per ml), left to dissolve overnight at 4° C., and then used for infection of BHK21 cells or for RT-PCR analysis. To determine the titer of encapsidated particles, BHK21 cells on 1.3 cm2 coverslips were infected with 100-200 μl of serial 10-fold dilutions of cell culture fluid or of pelleted material for 1.5h at 37° C. The fluid was then replaced with 1 ml of DMEM medium supplemented with 2% FBS; cells were incubated for 24h at 37° C. in the CO2 incubator and then subjected to IF analysis with anti-NS3 antibodies as described above. The infectious titer of packaged particles was calculated using the following formula:
Titer (IU) per 2×106 of initially transfected cells=N×(SW:SIA)×10n×(V:VI),
where N is the average number of anti-NS3 positive cells in the image area, calculated from 5 image areas in different parts of the coverslip; SW is the surface of the well in a 24-well plate (200 mm2); SIA is the surface of the image area (1.25 mm2 using defined magnification parameters, calculated according to the manual for the Wild MPS46/52 photoautomat [Wild Leitz, Heerburg, Germany]); V is the total volume of the culture fluid (usually 3-5 ml per 60 mm dish) collected from the population of 2×106 initially electroporated BHK21 cells; VI is the volume used for infection of the cover slips (usually 100-200 μl); and 10n on is the dilution factor.
Packaging of the KUN Replicon RNA into Transmissible “Infectious” Particles by the KUN Structural Proteins Expressed from the Recombinant SFV Replicons.
Because the KUN replicon construct C20rep was able to successfully transfect only 10-20% of cells a KUN replicon of greater transfection efficiency was used for attempted packaging in doubly transfected cells (i.e. KUN replicon, and recombinant SFV replicons expressing KUN structural proteins). This significantly improved the efficiency of transfection in BHK21 cells to about 80% using the replicon construct C20DXrep, which was used in all packaging experiments. As noted above, all cell culture fluids from packaging experiments were filtered to remove large membrane fragments and treated with RNase A to remove naked RNA.
Initial cotransfection experiments showed that simultaneous transfection of C20DXrep RNA and SFV RNAs expressing KUN structural proteins did not result in the detection of infectious particles. Therefore a delay period of 12h or longer between electroporations was used in subsequent experiments to allow KUN replicon RNA to accumulate before electroporation of SFV RNAs. IF and Northern blot analyses of BHK cells infected with the tissue culture fluid collected at 27h after the first electroporation with C20DXrep RNA, and at 26h after the second electroporation with recombinant SFV RNAs, showed higher efficiency of packaging when the single SFV-prME-C RNA was used compared to that obtained with two SFV RNAs, SFV-prME and SFV-C (compare panels 1 and 2 in
To optimize the conditions for efficient packaging of C20DXrep RNA in cotransfection experiments with SFV-prME-C RNA, variable time points between electroporations (
In a separate study BHK cells were electroporated with SFV-prME-C RNA at 30h after electroporation with C20DXrep RNA and seeded into one 60 mm culture dish. Single aliquot's of the culture fluid (lml of total 3ml) were then collected at 24h, 30h, and 42h after the second electroporation. The volume of the remaining culture fluid after removal of each aliquot was adjusted to the original volume by adding fresh media, and cells were re-incubated. Collected aliquots were then used to infect BHK cells and total cell RNA recovered from these infected cells at 24h was then analysed for relative amounts of amplified KUN replicon RNA using IF analysis with anti-NS3 antibodies and Northern blot hybridization with a labelled KUN-specific cDNA probe. The gradual increase in amplified KUN replicon RNA from 24h to 42h after the second electroporation with SFV-prME-C RNA detected by Northern blot analysis (
In a separate experiments we compared efficiency of packaging using SFV replicon RNA SFV-prME-C105 expressing prME genes and a C gene coding precisely for the first 105 amino acids of KUN core protein (representing an exact copy of mature KUN core protein found in infectious virions) with that of SFV-prME-C RNA expressing prME genes and a C gene coding for the first 107 amino acids of KUN core protein plus an extra 4 amino acids derived from the vector (see
Characterisation of the Infectious Particles.
To prove that infectious particles secreted into the culture fluid of cells transfected with C20DXrep and SFV-prME-C105 RNAs were in fact virus-like particles incorporating KUN structural proteins, a virus neutralisation test was performed. An hour incubation of this tissue culture fluid at 37° C. with a 1/10 dilution of the cocktail of monoclonal antibodies to KUN E protein with known neutralising activity resulted in almost complete loss of infectivity (panel 1 in
To show that the infectious particles can be concentrated by pelleting, a clarified culture fluid of cotransfected cells was subjected to ultracentrifugation. When pellet and supernatant after ultracentrifugation were used to infect BHK cells which were later (at 24h) analysed by IF with anti-NS3 antibodies, virtually all the infectious particles were present in the pelleted material (compare panels 1 and 2 in
To identify the proteins and to detect the presence of KUN replicon RNA in the recombinant infectious particles, they were immunoprecipitated in the absence of detergents from the culture fluid of cotransfected and radiolabeled cells using anti-E antibodies. Half of the immunoprecipitated sample was used for separation in the SDS-polyacrylamide gel, and the other half was used to extract RNA by proteinase K digestion. Radioautography of the polyacrylamide gel showed the presence of E, prM, and C proteins in the immunoprecipitates of culture fluid collected from cells either sequentially transfected with C20DXrep and SFV-prME-C RNAs or infected with KUN virus (
RNA extracted from the immunoprecipitates was reverse transcribed and PCR amplified using KUN-specific primers. A DNA fragment of predicted size (˜700 bp, NS2A region) was observed in the RT-PCR reactions of RNAs extracted from the immunoprecipitates of the culture fluid collected from cells either transfected sequentially as in
Further characterization of the packaged particles containing replicon RNA was performed by sedimentation analysis. In parallel with KUN virions (both concentrated by ultracentrifugation) they were sedimented through 5-25% sucrose density gradients. 0.5 ml fractions were collected, diluted and assayed for infectivity by IF assay using anti-NS3 antibodies at 1 8h for KUN virions or at 24h for replicon particles (see legend to
Cells.
BHK21 cells were grown in Dulbecco's modification of minimal essential medium (DMEM, Gibco BRL) supplemented with 10% of fetal bovine serum (FBS). Vero cells were grown in M199 medium (Gibco BRL) supplemented with 5% FBS.
Construction of the Plasmids.
(I) C20DXrepNeo.
The dicistronic replicon construct C20DXrepNeo used for generation of replicon-expressing BHK cells (repBHK) was prepared from C20DXrep by cloning an Ires-Neo cassette into the 3′ UTR 25 nucleotides downstream of the polyprotein termination codon. An XmaI-XhoI fragment from ΔME/76Neo plasmid (Khromykh and Westaway, J.Virol.1997, 71:1497-1505) representing nucleotides 10260 -10422 of KUN sequence, followed by the IRES-Neo cassette and the last 522 nucleotides of KUN sequence was used to substitute XmaI10260-XhoI11021 fragment in C20DXrep construct. Note, that IRES-Neo cassette was initially derived from the mammalian expression vector pIresNeo1 (a derivative of pCIN1, provided by S. Rees (Rees et al., BioTechniques, 1996, 20:102-110)). The nucleotide sequence at the C-terminus of IRES element in this IRES-Neo cassette was modified by authors in order to decrease the level of Neo expression thus forcing selection of cells expressing only high levels of inserted genes when using this (pIresNeo1) vector and high concentrations of antibiotic G418.
(i) C20DX2Arep and C20DX2ArepNeo.
To ensure cytosolic cleavage of heterologous genes expressed from the KUN replicon vectors, the C2ODxrep, C20DXrepNeo constructs were modified by inserting sequence coding for 2A autoprotease of the food-and-mouth disease virus (FMDV-2A) between the first twenty amino acids of KUN C and the last twenty two amino acids of KUN E proteins in each plasmid preserving the KUN polyprotein open reading frame. (C20DX2Arep,
Two unique sites for cloning of foreign genes were also incorporated into these vectors: (1.) a Spel site between the first 20 amino acids of C protein and the 2A sequence, and (2.) a EagI site between the 2A sequence and the rest of the KUN replicon sequence. Cloning into SpeI site ensures the correct cleavage of C20-FG-2A fusion protein from the rest of the KUN polyprotein sequence. Cloning into the EagI site permits correct N-terminus cleavage, but it will have its C-terminus fused to the 22aa of E protein.
(iii) C20DX/CAT/2Arep, and C20DX/CAT/2ArepNeo.
The FMDV-2A-CAT sequence was PCR amplified from the plasmid pT3CAT2A/NAmodII (Percy et al., J.Virol. 1994, 68:4486-4492), by using the same as for FMDV-2A amplification reverse primer and a forward primer with incorporated MluI restriction site, and cloned into the AscI site of the C20DXrep and C20DXrepNeo plasmids to obtain C20DX/CAT/2Arep, and C20DX/CAT/2ArepNeo constructs, respectively (
(iv) C20DXIRESrep and C20DX/CAT/IRESrep.
C20DXIRESrep was constructed by cloning EMCV IRES sequence PCR amplified from ΔME/76Neo plasmid (Khromykh and Westaway, J.Virol., 1997, 71:1497-1505) using the appropriate primers with incorporated AscI (forward primer) and MluI (reverse primer) restriction sites into the AscI site of the C20DXrep plasmid. C20DX/CAT/IRESrep construct was prepared by cloning CAT gene PCR amplified from the plasmid pT3CAT2A/NAmodII (Percy et al., J.Virol. 1994, 68:4486-4492) using primers with incorporated MluI restriction sites into the AscI site of C20DXIRESrep plasmid (
(v) C20DX/GFP/2Arep, C20DX/GFP/2ArepNeo, C20DX/hcvCORE160/2Arep, C20DX/hcvCORE191/2Arep, C20DX/hcvNS3/2Arep, C20DX/VSV-G/2Arep, and C20DX/β-GAL/2Arep.
All these constructs (
RNA Transcription and Electroporation.
Recombinant KUN replicon RNA transcripts were prepared using SP6 RNA polymerase from the corresponding recombinant KUN replicon plasmid DNAs linearized with XhoI or from the SFV-prME-C105 μlasmid linearized with SpeI. Electroporation of RNAs into BHK21 cells was performed according to the method described in Example 1.
Immunofluorescence Analysis.
Immunofluorescence (IF) analysis of electroporated or infected cells was performed as described using antibodies specific to expressed proteins or KUN anti-NS3 antibodies. Rabbit polyclonal anti-CAT antibodies were purchased from 5 Prime→3 Prime (Boulder, Colo., USA), rabbit polyclonal anti-VSV-G antibodies were obtained from Michael Bruns (Heinrich-Pette-Institut, Hamburg, Germany), human anti-HCV polyclonal serum was provided by Eric Gowans (Sir Albert Sakzewski Virus Research Centre, Brisbane, Australia). Preparation and characterization of KUN anti-NS3 antibodies were described previously (Westaway et al., J.Virol., 1997, 71:6650-6661).
In Situ β-Galactosidase Staining and β-Galactosidase Assay.
X-gal staining of BHK21 cells either electroporated with C20DX/β-GAL/2Arep RNA or infected with VLP containing encapsidated C20DX/β-GAL/2Arep RNA and determination of β-galactosidase activity in the cell lysates was performed using commercial β-GAL Enzyme Assay System Kit (Promega, Madison, Wis., USA) essentially as described by the manufacture.
CAT Assay.
CAT activity in lysates of BHK21 cells either electroporated with TRCAT and C20DX/CAT/2Arep RNAs, or after infection with VLPs containing encapsidated C20DX/CAT/2Arep RNA, or in stable cell line expressing C20DX/CAT/2ArepNeo RNA was determined using TLS or LSC assays as described previously (Khromykh and Westaway, J.Virol., 1997, 71:1497-1505).
Preparation of Encapsidated Particles and Determination of Their Titer.
Preparation of the recombinant VLPs expressing CAT, GFP, and VSV-G proteins and determination of their titers was performed as described in Example 1.
Optimal Time of Expression of Heterologous Products
In order to estimate the level and the optimal time of expression of heterologous products using this system, as well as to evaluate possible effects of the size of inserted sequences on the replication and packaging efficiency of resulting recombinant KUN replicon RNAs, KUN replicons expressing CAT (218 amino acids), GFP (237 amino acids), and β-Gal (1017 aa) genes were prepared in C20DX2Arep vector (
Expression of HCV Proteins.
To express HCV proteins using the KUN replicon system, Core and NS3 genes of an Australian isolate of HCV (Trowbridge and Gowans, Arch.Virol., 1998, 143:501-511) were expressed using the replicon vector C20DX2Arep. A truncated form of HCV NS3 gene (coding for amino acids 183 to 617), containing most of the HCV NS3 cytotoxic T cell epitopes was cloned into C20DX2Arep vector. Transfection of the recombinant C20DX/hcvNS3/2Arep RNA into BHK21 cells resulted in detection of expression of HCV NS3 gene in ˜20-30% of transfected cells (panel 2 in
Kinetics of Expression, Processing and Glycosylation of Heterologous Proteins using KUN Replicon Vectors.
The kinetics of expression of two different size reporter genes CAT (218 amino acids) and β-Gal (1017 amino acids) after electroporation of corresponding recombinant replicon RNAs into BHK21 cells were compared by appropriate reporter gene assays. Similar to previous results with detection of replicating KUN replicon RNA, a delay of about 10-16h in detectable expression of both reporter genes was observed (
To examine whether proper proteolytic cleavage mediated by FMDV-2A protease occurred during translation of recombinant KUN replicon RNAs in electroporated cells, the sizes of the radiolabelled protein products expressed from C20DX/CAT/2Arep RNA were examined using radioimmunoprecipitation (RIP) analysis with anti-CAT antibodies. Strong radiolabelled band of ˜30 kDa, corresponding to a predicted size of C20/CAT/2A fusion protein (257 amino acids) was observed (lane 1,
Expression and proper processing of heterologous genes from the dicistronic KUN replicon vector C20DXIRESrep was demonstrated by detection of ˜27.5 kDa radiolabelled band corresponding to a predicted size of C20CAT protein (240 amino acids) in the anti-CAT immunoprecipitate from the lysate of BHK21 cells transfected with C20DX/CAT/IRESrep RNA (lane 2,
Packaging of Recombinant KUN Replicon RNAs into Pseudoinfectious Virus-like Particles.
Although relatively high level of expression of heterologous genes was achieved in BHK21 cells after electroporation of recombinant KUN replicon RNAs, it is well known that the efficiencies of transfection of different cell lines varies tremendously. Therefore it was desirable to prepare a stocks of virus-like particles (VLP) containing encapsidated recombinant replicon RNAs in order to broaden the spectrum of cells which could be used for expression. According to the present invention a heterologous packaging system has been developed allowing production of VLPs containing KUN replicon RNA encapsidated by the KUN structural proteins using consecutive transfections with KUN replicon RNA and SFV replicon RNA SFV-prME-C105 expressing KUN structural genes. The highest titer of VLPs was achieved when the second electroporation with SFV-prME-C 105 RNA was performed at the time of the maximum replication of KUN replicon RNA (delay of ˜24-27h). Therefore in packaging experiments with recombinant KUN replicon RNAs, second electroporation with SFV-prME-C105 RNA was performed at the estimated time of maximum replication of recombinant KUN replicon RNAs (for time intervals see legend to
Essentially all recombinant replicon RNAs were packaged into VLPs (
Establishment of Stable Cell Lines Expressing CAT and GFP Genes Using C20DX2ArepNeo Vector.
To demonstrate the utility of this dicistronic KUN-Neo replicon vector for the establishment of stable cell lines expressing heterologous genes two constructs, C20DX/CAT/2ArepNeo and C20DX/GFP/2ArepNeo were prepared by cloning CAT and GFP sequences into the SpeI site of the C20DX2ArepNeo vector (
The above examples show that noncytopathic flavivirus KUN replicon vectors can be used for transient or stable expression of heterologous genes in mammalian cells. They also show that recombinant KUN replicon RNAs expressing heterologous genes can be encapsidated into pseudoinfectious virus-like particles by subsequent transfection with SFV replicon RNA expressing KUN structural genes. These virus-like particles can be used for delivery of the recombinant self-replicating RNAs into a wide range of cells or animals leading to a long-term production of heterologous proteins. Importantly, because of the heterologous nature of the developed packaging system, no recombination between KUN and SFV RNAs producing an infectious virus can occur.
While the amounts of produced heterologous proteins using KUN replicon vectors were lower than those reported in using alphavirus replicon vectors, replication of KUN replicons in contrast to alphavirus replicons did not produce any cytopathic effect in cells. This noncytopathic nature and persistence of replication of KUN replicons allowed the development of a vector for generation of stable cell lines continuously expressing heterologous genes by inserting IRES-Neo cassette into the 3′ UTR of C20DX2Arep replicon. Using such a selectable vector (C20DX2ArepNeo), a stable BHK cell lines continuously expressing GFP and CAT genes were rapidly established by selection of transfected cells with antibiotic G418. The expression of these genes in the established cells lines maintained at the same level for at least 17 passages.
Mouse ubiquitin gene was PCR amplified from the plasmid pRB269 (Baker et al., J Biol Chem 269:25381-25386) using appropriate primers with incorporated unique cloning sites (see
To produce KUN replicon transcripts with authentic 3′-termini we incorporated hepatitis delta virus (HDV) antigenomic ribozyme sequence (Perrotta and Been, 1991, Nature (London) 350:434-436) followed by the simian virus 40 (SV40) polyadenylation signal (HDVribo/SV40polyA) immediately downstream of the last nucleotide of KUN replicon sequence (
Resulting PCR product was digested with XmaI (5′ end) and XhoI (3′ end) and cloned into XmaI/XhoI digested C20DXUb2Arep DNA, producing C20DXUb2A_HDVrep vector (
Electroporation of ˜5-10 μg RNA transcribed from this construct resulted in its efficient replication in ˜100% BHK21 cells compared to ˜60% positive cells obtained after transfection with the same amounts of parental C20DXUb2Arep RNA (
To allow in vivo transcription of the KUN replicon RNA by cellular RNA polymerase II after transfection of the corresponding plasmid DNA we modified existing KUN replicon vector C20DXUb2A_HDVrep by inserting cytomegalovirus immediate-early (CMV-IE) enhancer/promoter region immediately upstream of the KUN replicon sequence. The fragment containing CMV-IE promoter sequence followed by 5′ end of the KUN replicon sequence was produced in fusion PCR reaction (Karreman, 1998, BioTechniques 24:736-742) using Pfu DNA polymerase, appropriate primers and pCI (Promega) and C20DXUb2Arep plasmid DNAs as templates. Primers were: CMV_F (CMV IE promoter, forward)—5′-GCG CTT AAG ACA TTG ATT ATT GAC TAG TTA -3′; CMV5′ UTR (junction of CMV promoter and 5′ UTR of the KUN sequence)—5′ -CGT TTA GTG AAC CGA GTA GTT CGC CTG TGT GA -3′; FMDV2AR (end of FMDV-2A autoprotease sequence, reverse)—5′-GTG ACG CGT CGG CCG GGC CCT GGG TTG GA -3′ . Resulting PCR product was digested with EagI (3′ end) and cloned into NruI (blunt)/EagI digested C20DXUb2A_HDVrep plasmid, producing pKUNRepl vector (
Transfection of the plasmid DNA pKUNRep1 into BHK21 cells using FuGENE 6 transfection reagent (Boehringer Mannheim) resulted in successful detection of expression of the KUN NS3 protein (indicator of the replicating KUN replicon RNA) at 42 h post transfection (
Two female BALB/c mice were immunized intra-nasally with ˜106 IU per mouse of the recombinant KUN VLPs expressing GFP (for details of the VLP preparation and determination of their titre see Example 3). Mice were anaesthetized with ketamine/xylazine (100 ul per 20 g of mouse weight) via intra-peritoneal route prior to immunization. At days 2, and 4 after immunization mice were euthanased with CO2, their lungs were collected, rinsed in PBS and fixed in 4% paraformaldehyde for 2-4 hours at 4° C. Lungs were also collected from the control nonimmunized mouse using the same procedure. All the specimens were paraffin embedded and microtome sectioned at ˜5 micron, mounted on a microscope slide and analyzed under ultraviolet light using FITC filter. Strong GFP fluorescence was observed in epithelial cells lining the airways passages of the lung sections prepared from mice immunized with recombinant KUN VLPs but not in the lung section prepared from the control mouse (
In order to evaluate immunogenic properties of KUN replicon VLPs, three BALB/C mice were immunized intra-dermally (in the base of a tail) with ˜5×105 IU of VLPs containing packaged C20DX/GFP/2Arep RNA (see Example 3). Two weeks after immunization their serum was analyzed on the presence of anti-GFP antibodies by ELISA with purified GFP protein. The results of 50% end point titrations (ELISA t50) for each mouse were: mouse #1- 1/200, mouse #2- 1/130, mouse #3- 1/100. These results clearly demonstrate that specific humoral immune response to the heterologous protein encoded by the KUN replicon vector can be developed as early as at 2 weeks after only a single immunization with the recombinant KUN VLPs. It is anticipated that the antibody response will be greatly enhanced after the second immunization.
It should be understood that the foregoing description of the invention including the principles, preferred embodiments and Examples cited above are illustrative of the invention and should not be regarded as being restrictive on its scope. Variations and modifications may be made to the invention by others without departing from the spirit of that which is described as the invention and it is expressly intended that all such variations and changes which fall within this ambit are embraced thereby is intended merely to be illustrative thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| PP0627 | Nov 1997 | AU | national |
| PP6096 | Sep 1998 | AU | national |
The present application is a continuation of U.S. Ser. No. 09/580,476, filed May 26, 2000, which is a continuation of International Application No. PCT/AU98/00993 (published as International Publication No. WO 99/28487), filed 30 Nov. 1998 and designating the United States, which in turn claims priority from Australian Application Nos. PP 0627, filed 28 Nov. 1997 and PP 6096, filed 23 Sep. 1998, the teachings of all of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09580476 | May 2000 | US |
| Child | 11098283 | Apr 2005 | US |
| Parent | PCT/AU98/00993 | Nov 1998 | US |
| Child | 09580476 | May 2000 | US |
