Patent Application
20090197319
The invention relates to a Dengue (DEN) vector and in particular to a recombinant virus that is prepared by replacing DEN structural genes with non-DEN transgenes, such as, proteins and antigens, for the recombinant production thereof, and the production and application.
There is a constant need for new and useful cloning vectors for experimental use and for the development of scale up resources for the production of biological molecules in quantity. The flaviviruses are attractive candidates. Flaviviruses replicate cytoplasmically and therefore minimize interaction with the host genome, the genome can carry a large insert, the flaviviruses are easy to culture and maintain, expression is robust, the viruses do not inactivate the host and the genome is now being understood.
Thus, a flavivirus cloning vector can find utility in the laboratory for isolating genes, for maintaining and amplifying a gene, for gene delivery and so on.
Cancer continues to be a substantial threat to human health. Some cancers have been linked to a variety of viruses, most notably human papillomavirus (HPV), which can be responsible for cervical cancer. The prevalence of HPV infection of the female genital tract has a positive correlation with the pathological progression of the diseased cervix presenting in the normal progression of the disease beginning with chronic cervicitis→pseudocondyloma→verruca lesions→condyloma acuminate→cervical intraepithelial neoplasia→cervical cancers.
More than 75 percent of cervical cancers were found to be associated with HPV type 16 and/or HPV type 18 infection. Other types of HPV have been found to be associated with various forms of cancer in various populations. Those types include HPV, 30, 31, 39, 40, 45 and 59.
The current treatments for cervical cancer are surgery, radiotherapy, chemotherapy, and occasionally, such treatments are combined with therapies known as natural medicine, naturopathic medicine, herbal medicine, Chinese medicine and so on, using products or derivatives of natural products, such as plants, molds, fungi and the like. Unfortunately, the effect is not satisfactory, and the five-year survival rate is about 65 percent. The prognosis following the recurrence of disease is extremely poor, with only five percent survival over two years. The expense for cervical cancer screening and therapy in the USA is approximately 570 million dollars per year. Therefore, early detection is essential.
There are several new methods directed to treating cervical cancer including, use of inactivated vaccines, the earliest tumor vaccines with good safety, cannot induce effective cellular immune responses because the antigen generally cannot be expressed by a host cell; DNA virus vector vaccines, which to avoid oncogenicity, generally contains only parts of the oncogene, which in turn, reduces the immunogenicity of the antigen; Borysiewicz L K et al. (Lancet. 1996, Jun. 1:347(9014):1523-7) reported an HPV vaccine with vaccinia virus as vector; however, most people have antibody to vaccinia so the widespread use of such a vector is limited; WO01/53467 provides recombinant yellow fever virus (YFV) vectors, however, the recombinant YFV retained essentially the entire YFV genome without deletion of some relevant cis genes, and thus is replicable and infectious; and WO99/28487 provides an expression and delivery method of exogenous sequences by the flavivirus, kunjin virus (KUN); however, the titer of VLP was unsatisfactory and the booster immunization will be less effective because there is only one species of KUN and thus, repeated administration can lead to vector immunity.
The KUN genome is stable in plasmids which can be cloned and recombined with traditional methods. However, that strategy does not apply to other flaviviruses. For example, Dengue virus (DEN) sequences have lower stability in plasmids than KUN, probably because KUN belongs to the Japanese encephalitis virus group whereas DEN belongs to the different Dengue virus group. The two groups have only 45% homology at the level of the genome. So it is hard to construct DEN replicons and to obtain DEN VLPs directly following the teachings of WO99/28487.
WO02/072803 teaches a method for construction of DEN subgenomic replicons and use thereof for a DEN vaccine. Nevertheless, it is not possible to replicate those teachings.
In summary, there is an urgent requirement for novel cloning vectors with high capacity; and for host cells that are long-lived, can carry large inserts and are easy to propagate. Such vectors also are not oncogenic, and can be administered repeatedly.
The invention aims to provide novel products applicable to laboratory and industrial production of recombinant gene products which have good safety, high titer, good booster effects, and are versatile in what exogenous genes can be carried. That is achieved in the use of DEN replicons and virus-like particles.
In a first aspect the invention provides recombinant DEN replicons comprising deletion of one or more nucleotides including but not limited to, for example, the following nucleotide sequences:
In preferred embodiments, the 3′ end of any exogenous nucleotide sequence contains 3′ release elements upstream of the coding region of the NS1 signal peptide, which can be the last about 24 amino acids of the envelope protein. Other signal peptides can be used as well. These release elements can be a nucleotide sequence obtained from the autoprotease of foot-and-mouth disease virus (FMDV) (SEQ ID NO:3), such as the about 20 amino acids thereof known in the art as 2A.
In another preferred embodiment, DEN is obtained from any one of the following: DEN type I, DEN type II, DEN type III or DEN type IV.
In another preferred embodiment, the coding region of the NS1 signal peptide is composed of the about last 72 nucleotides of the 5′ terminus of the E gene. Alternatively, a suitable signal peptide encoding sequence can be subcloned upstream in an operable situs of the NS1 coding sequence.
In another preferred embodiment, the 5′ untranslated region is followed by a sequence of about 60 nucleotides that are complementary and can base pair with the 3′ end of the replicon to enable circularization of the replicon for replication. In some embodiments, the about 60 nucleotides comprise the sequence encoding the first about 20 amino acids of the capsid protein.
In another preferred embodiment, the exogenous sequence encodes a human papilloma virus (HPV) antigen, an immunoregulator or both.
In yet another aspect, the present invention also provides virus-like particles (VLP) comprising a DEN recombinant replicon and deletion of one or more DEN structural proteins. Preferably, coding sequences of the capsid, membrane and envelope proteins are deleted.
In a further aspect, the present invention also provides cells for a packaging system for producing DEN recombinant replicons VLPs. These packaging cells are selected from:
and above cells express DEN structural proteins for complementing replicon packaging and the structural gene expression does not affect the growth of the host cells.
In another aspect, the present invention provides a method for production of a virus-like particle (VLP) as herein described comprising the steps of:
In another preferred embodiment, said packaging cells contain a DEN structural protein expression vector, selected from a DEN vector with a mutated NS3 or a Tet-regulated vector.
In another embodiment, the replicon is used to produce the transgene expression product. That expression product can be used in diagnostic assay, such as positive controls. The expression product can be used as an immunogen for developing, for example, antibody thereto. Alternatively, the expression product can be used as a positive control in a diagnostic assay or as a binding agent for antibody in an immunoassay. The expression product can be configured for secretion into the culture medium, for expression on the VLP or for expression on a host cell.
Additional features and advantages are described herein, and will be apparent from, the following Detailed Description and the figures.
The present invention relates to materials and methods for making a DEN-VLP preparation, as exemplified in the construction of a VLP carrying expressed HPV sequences with good safety, high titer and good booster effects.
“Nt” as used herein means a nucleotide.
“Release element” refers to nucleotide sequences at the 3′ end of a structural polypeptide coding sequence or at the 5′ end of the NS1 signal peptide coding region, used to enhance release of the polypeptide by a signal peptide protease without unrelated sequences after protein translation. A suitable release element is the about 60 nucleotide sequence of the Foot-And-Mouth virus hydrolytic enzyme, known as 2A, as provided in SEQ ID NO:3. Any nucleotide sequence that has the protease attracting properties of 2A can be used in a vector of interest. Usually there is only one “release element” per vector, but there may be plural “release elements” in a vector.
The terms “immunological activity” or “immunogenicity” refers to the ability to induce a specific humoral and/or cellular immunity of a mammal by natural, recombinant or synthetic immunogens, such as peptides.
The terms “antigen polypeptide” or “antigen peptide” refers to the amino acid sequence eliciting an immune response of a mammal whether single or combined with other helper molecules (for example, Human histocompatibility antigen (HLA) I or II).
The term “immune response” refers to a cellular and/or humoral immune response, for example, sufficient to inhibit or prevent, for example, infection or disease caused by microbes or to generate reagents that can be used in diagnostic assays, such as specific cytotoxic T cells, antibody and specific B cells.
The terms “object,” “individual” or “patient” refers to any object which needs diagnosis or therapy, especially mammals, such as human. Other objects include other mammals, such as, cow, dog, cat, cavy, rabbit, rat, mouse, horse etc.
A vector of interest will have one or more of the following characteristics to form a recombinant replicon vector that is disabled in the context of not containing an intact genome:
The DEN virus that can used to make a vector in the present invention has no special limitation and can be any subtype of DEN virus. Four subtypes of DEN genome that can be used, for example, have the following ATCC accession numbers:
DEN virus has the property of being bound by dendritic cells which can enhance immunogenicity effects. Furthermore, DEN has an ADE property (antibody dependent enhancement of infection) which refers to the observation that after a first immunization of DEN, antibody dependent infection is enhanced when the host is immunized with a different DEN subtype. So DEN is effective for repetitive, booster immunizations. The ADE phenomena can be used for exogenous protein expression systems, which not only avoids neutralization of vector caused by repetitive immunization, but also increases the efficiency of infection of vectors into cells and exogenous protein delivery efficiency. Because DEN has four subtypes, any one can be used for an exogenous expression vector, through further inoculation with different DEN recombinant replicons, that is, of a different subtype, tends to intensify immunization efficiency.
Thus, the expression product of a DEN vector can be a useful immunogen for generating host antibody, for example, to obtain antiserum to the expressed transgene, because any host vector immunity that might occur can be overcome by delivering the transgene using a vector made from a different DEN subtype.
Also, the expression product of a DEN vector can be a useful absorbent of antibody directed to the expressed transgene. Accordingly, an expression product of a DEN vector can be used as a capture reagent in a solid phase assay for detecting antibody to the expressed transgene, the expression product of a DEN vector substituting for the capture antibody often used in such immunoassays. The expression product of a DEN vector is affixed to a solid phase, for example, a membrane, a surface, such as a plastic surface, a bead and the like, in a variety of formats, such as, in the case of a membrane, a strip, a sheet and the like. A sample suspected of containing antibodies to the transgene expressed by the DEN vector is exposed to the affixed DEN vector, the solid phase is washed as needed and as known in the art, and then exposed to a suitable reporter molecule that will reveal antibody bound to the solid phase. For example, the reporter can be an antibody, a receptor and the like, with specificity for antibody. The reporter is a molecule that is detectable and thus contains a label, such as a radioisotope, a fluorescent compound, a structure visible to the naked eye or with minimal magnification, such as a colored bead, such as a liposome containing a dye, a metal sol and so on.
The expression product of a DEN vector can be secreted and collected in the culture medium as known in the art. Alternatively, the expression product can be obtained from the host cells by lysis of the cells and purification, practicing methods known in the art. The artisan can also configure the vector and sequences contained therein for expression of the transgene product at the surface of the VLP. In that case, the VLP itself will substitute for the transgene expression product. Also, the expression product can be expressed at the surface of a host cell containing said DEN VLP. In that case, the host cell can substitute for the transgene expression product.
Exogenous genes used in the present invention have no special limitation. It can be any exogenous coding sequence, such as an aptamer, siRNA, polypeptide, ribozyme and the like, any therapeutic gene, such as a gene encoding an antibody to VEGF, an interferon, a cytokine and so on, a tumor antigen gene, a virus antigen gene, a microbe antigen gene, an immunoregulator gene and so on. The representative antigens include (but are not limited to): human HPV antigen (such as the E6 or E7 protein of the 16 and 18 subtypes), HIV antigen, HBV antigen, HCV antigen, EBV antigen, HTLV-1 antigen, MAGE, a Salmonella antigen, BCG, a Streptococcus antigen, an H. influenza antigen, an S. pneumoniae antigen, BAGE, CAGE etc. The length of an exogenous gene has no special limitation, usually the foreign coding sequence is from about 100 bp to about 2000 bp, optimally from about 150 bp to about 1200 bp. Any size of transgene can be used so long as the DEN genome can be packaged into a particle. Furthermore, a release element, such as 2A, can be added at the 3′ end of an exogenous gene to ensure a high level of expression of the expressed sequence. If the intercalated exogenous gene is a tumor antigen gene or a virus antigen gene, said VLP can be used for prevention and therapy of tumor and virus diseases.
Taking human papilloma virus (HPV) as an example, the present invention provides diagnostic, preventative and therapeutic compositions against diseases caused by HPV infection. Therein said composition contains a VLP packaged using a DEN recombinant replicon using necessary complementing DEN structural proteins. Therein said exogenous nucleic acid sequence of the DEN recombinant replicon encodes an antigen of one or many HPV subtypes. The vector can contain also a sequence encoding an immunoregulator, or the immunoregulator sequence may be carried by a second vector. Therein said HPV antigen protein can be E6, E7 or an E6/E7 fusion or composite molecule oncoprotein of, for example, HPV type 16 or HPV type 18, or can be an HPV major capsid protein L1 or minor capsid protein L2. Therein said immunoregulator can be a gene sequence encoding a polypeptide with an immunoregulating activity obtained from, for example, IL2, IL12, IL18, GM-CSF etc. or a functional portion thereof.
The composition provided by said invention has the advantage of being relatively non-immunogenic to the host, and thus can be administered repeatedly with effect. Such an anti-human papilloma virus composition is a useful immunogen, and provides prevention and therapy for diseases caused by papilloma virus, such as chronic cervicitis, pseudocondyloma, verrucous lesions, condyloma acuminata, cervical intraepithelial neoplasia, cervical cancer etc., is useful in diagnostic assays and so on.
Construction of said replicon can be processed as follows:
After obtaining the replicon, it can be introduced into packaging cells to produce VLP. A normal method is after the DEN replicon is introduced into cells, then the helper virus expressing a necessary structural protein is introduced into those cells. Another method is construction of expression packaging cells with an inducible or constitutive plasmid carrying the DEN structural gene as a vector and then introduction of the replicon (or VLP) into these cells to produce VLP. For example, constitutive packaging cells can be made that do not express a functional NS3 gene. The NS3 gene possesses a special packaging signal sequence and thus, a DEN genome with an NS3 deletion can't be packaged into a VLP. So if the packaging cell doesn't have a functional DEN NS3 gene, it can't be packaged into VLP themselves. VLP produced by this packaging cell only contains a DEN recombinant replicon, and thus, there is no need to screen amongst the replicons for those that are recombinant.
Specifically, such methods include:
The present invention also provides various compositions comprising such a recombinant DEN replicon and/or a VLP, including a pharmaceutical composition.
Various compositions comprising such a recombinant DEN can include different buffers according to practical purposes and substances suitable to other purposes required of a pharmaceutical delivery form, as known in the art. These compositions generally contain pharmaceutically available carriers, diluents and excipients as known in the pharmaceutics arts, “Remington: Pharmacology and Pharmacological Practice” 19th ed. (1995) Mack Publishing Co.
Pharmaceutical compositions can be made into a variety of forms, such as for injection, solid forms, such as grains, tablets, pills, suppositories and capsules, other liquid forms, such as a suspension, a spray etc. For example, some of the known carriers include water for injection, plant and animal oil and fats, and so on, as known in the art. The stabilizer, wetting agent, emulsifier, salt suitable for controlling osmolarity, various buffers suitable for maintenance of appropriate pH, surfactants, permeants to enhance transdermal movement and so on can be used for auxiliary materials as known in the art.
The said recombinant DEN can be put into dispensing means as known in the art. Thus, for example, if administered by injection or intravenously, the VLP can be suspended in a suitable medium or can be presented in a desiccated form, for example, by cryopreservation for reconstitution prior to use. For such desiccation, the preparation likely will include suitable excipients to facilitate the process, such as a cryoprotectant, such as glycerol, as known in the art.
Moreover, the said compositions can also contain other components such as an adjuvant, a stabilizer, a pH regulator, a preservative etc. These components are familiar to technicians of this field. Adjuvants include but are not limited to an aluminum adjuvant, a saponin adjuvant, a Ribi adjuvant (Ribi ImmunoChem Research Inc., Hamilton, Mont.), a Montanide ISA adjuvant (Seppic, Paris, France), a Hunter's TiterMax adjuvant (CytRx Corp., Norcross, Ga.), a Gerbu adjuvant (Gerbu Biotechnik GmbH, Gaiberg, Germany) and so on. In addition, other components for regulating and modifying the immunological response can be used.
The said recombinant DEN replicon and/or VLP can be administered to an individual through known methods. The said composition is often administered through a normal administering pathway or modeling pathogen infection pathway for a biologic. Pharmaceutically available vehicles can be used when administrating oral compositions such as flavorants, colorants, enteric coatings etc.
Normal and pharmaceutically available administration pathways include intranasal, intramuscular, intratracheal, subcutaneous, intracutaneous, endovaginal, intrapulmonary, intravenous, nasal, oral or other extraintestinal pathways. Combined administration can be made if needed or it can be regulated according to the transgene, the disease, the disease condition and so on. The vaccine composition can be administered as a single dose or in multiple doses, and also contain a booster dose to elicit and/or to maintain immunity.
“Effective dose” is given in an amount such that the availability of DEN recombinant replicon and/or VLP is of an amount so as to elicit an immune response and effectively prevent a host against a virus infection, tumor or other source of pathology etc. Usually, after infecting host cells, every dose of vaccine is enough to produce from about 1 to about 1000 ug, or about 1 to about 200 ug, or about 10 to about 100 ug of transgene. The vaccine effective dose calculated with recombinant DEN nucleic acids as a basis usually includes administrating about 1 to about 1000 ug nucleic acids. Furthermore, the average range of vaccine effective dose is about 102 to about 109, about 103 to about 107, or about 104 to about 105 plaque forming units (PFU). The optimal dose of vaccine can be determined, for example, by antibody titer of experimental objects and standard investigation methods of other reactions, as known in the art. Whether a booster dose is needed can be detected by supervising immunity levels using immune assays as known in the art. After evaluating serum antibody titer, one or more booster doses can be administered. Administering adjuvant and/or immunological stimulant can enhance an immune response to the target transgene.
The vector of interest can be configured into a kit for diagnostic use, relating to the transgene contained therein. The kit can contain the expressed transgene, the VLP expressing the transgene or a cell expressing the transgene affixed on a surface usable for the assay. The kit also can contain a suitable reporter system for detecting the specific analyte of interest. The reporter can be, for example, an antibody, a receptor, a ligand and the like that has a specific binding function. The reporter will contain a detectable marker, such as a bead, a metal sol, a nanoparticle, a Quantum Dot, a fluorescent molecule and so on. A preferred detectable marker is one that can be detected with a minimum of intervention. Thus, one that is visible to the naked eye is desirable. The kit will include instructions suitable for making use of the replicon of interest.
Compared with known technology, the present invention has the following advantages:
1. enhancement of immunological effectiveness;
2. enhancement of vaccine safety;
3. little treatment distress; and
4. low cost:
The invention now is exemplified in the following representative examples. Clearly, these examples only clarify and do not limit this invention. The experimental methods taught herein can be performed practicing such known methods, such as those taught in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York, Cold Spring Harbor Laboratory Press, 1989) or recommended by manufacturers.
1) Full-length DEN cDNA clone pRS/FLD2) produced in step 1 (c) of Example 1 was linearized with BamHI.
Summary: Using the tetracycline-regulated gene expression system, BHK-21 cells were transformed to produce a DEN structural protein regulated expression cell line. The Tet-Off Gene Expression systems were purchased from Clontech Inc. This plasmid expresses a regulatory protein named “rtTA” which regulates gene expression and plasmid transcription. This gene expression system contains pTet-Off regulatory vectors, pTRE2 response vectors and pTK-Hyg selection vectors.
To examine the immunity of an HPV expressing VLP, the HPV VLP was used to determine any effect on tumor cells using C57BL/6 mice.
C57BL/6 mice were inoculated with JHU-1 HPV cells as the tumor model, JHU-1 HPV cells contained E6 and E7 oncogenes of HPV. Eight-week-old mice were inoculated in the flank with 500 μl PBS buffer containing 105 JHU-1 HPV cells. JHU-1 HPV cells can completely induce solid tumors. Seven days after inoculation of JHU-1 HPV cells, tumors could be felt. After fourteen days, the tumors could reach over 6 mm in diameter.
1. To examine immunity of HPV VLP:
2. Results:
In group 3, after inoculation of C57BL/6 mice with 105 JHU-HPV for fourteen days, tumors were induced, as expected. The tumor bumps sometimes were larger than 6 mm in diameter. Then those mice were inoculated ip with 107 HPV-VLP. After two days it can be seen that most tumors began to shrink. After first inoculation, after seven days, the mice were inoculated with 107 HPV-VLP again. After two inoculations for one week, tumors in almost half the mice disappeared. In other mice, the trend was for diminution of tumor size. In group 2, after inoculation of C57BL/6 mice with 105 JHU-HPV for thirty-five days, tumors of 75% of the mice were larger than 25 mm.
The effects of the HPV VLP on E6-E7-specific CD8+ T cells were examined by cytoplasm staining. The HPV VLP induced CD8+ T cells to secrete IFNγ and TNFα.
A recombinant DEN II replicon was constructed as in Examples 1, 2 and 3 as described, but the difference was that the sequence of the 5′ end of the C gene and the length of the NS1 signal peptide were different. VLP were produced as in Example 4 described above.
A recombinant DEN replicon was constructed as in Examples 1, 2 and 3 above, but the difference is that DEN I replaced DEN II of Examples 1, 2 and 3. Then VLP were produced by method 4 of Example 4. Four mice of Example 5 with tumors were inoculated as group 3 of Example 5 described above.
All publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.
It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention, which is defined by the following claims.
It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| CN03115272.4 | Jan 2003 | CN | national |
| CN03115273.2 | Jan 2003 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11194342 | Aug 2005 | US |
| Child | 11971343 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2004/000088 | Jan 2004 | US |
| Child | 11194342 | US |
