Patent Application
20090149405
The invention relates to dengue virus (DEN) and in particular to a recombinant virus that is prepared by replacing DEN structural protein genes with non-dengue transgenes, such as, therapeutic antigens, and its production and application.
Cancer threatens human health seriously. Some cancers have been linked to a variety of viruses, most notably human papillomavirus (HPV), which is 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. HPV infection has been shown to be extremely common affecting 10-40 percent of women in China and 40-60 percent of women in Europe and the Western Countries.
Cervical cancer is the second most common cancer after breast cancer among women worldwide. According to WHO, there are 1.7 million cervical cancer patients diagnosed worldwide, and approximately 500,000 new cases are diagnosed per year. There are as many as 200,000 deaths per year. There are nearly 140,000 cervical cancer patients in China, and 40,000 new cases are diagnosed every year. Moreover, more than 75 percent of cervical cancers were found to be associated with HPV 16 and/or HPV 18 infection.
The current treatments for cervical cancer are surgery, radiotherapy, chemotherapy, and occasionally such treatments are combined with therapies known as Chinese medicine, herb medicine, and so on. Unfortunately, the effect is not satisfactory, and the five-year survival rate is 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.
The three traditional methods for cervical cancer therapy, surgery, chemotherapy and radiotherapy, have many limitations, such as high disease recurrence rate, serious side effects and tolerance. Because of its theoretically high specificity and low side effects, biotherapy, especially immunotherapy and gene therapy, is being viewed as a new strategy for tumor therapy. The sciences of immunotherapy and gene therapy are now being applied in the treatment and cure of many different diseases and the promise of biotherapy is being realized.
In the area of cancer treatment vaccines, a number of different strategies are being pursued.
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:
A kind of live vaccine with a DNA genome as the vector, can effectively replicate and express an oncogene in vivo and can continually stimulate the cellular immune system to produce cytotoxic T cells (CTLs) against tumors. But to avoid oncogenicity, only parts of the oncogene are commonly employed as the antigen. Thus the immunogenicity of the antigen is reduced. Another problem of this type of vaccine is safety.
Borysiewicz L K et al (Lancet. 1996 June 1:347(9014):1523-7.) reported an HPV vaccine with vaccinia virus as vector. But, most people have antibody to vaccinia so its application is limited. Moreover, boosters of the vaccine are useless because of the existing or developed immune response to the vector.
WO0153467 provides recombinant yellow fever virus (YFV), which comprise exogenous nucleotide sequences. Infection of a host cell with a recombinant YFV provides for expression of the exogenous nucleic acid in a host cell and production of an antigenic polypeptide encoded by the exogenous nucleic acid. Such recombinant YFV are useful in eliciting an immune response to the exogenous polypeptide. But the recombinant YFV, retaining the whole YFV genome without deletion of some relevant cis genes, is replicable and thus, infectious. So the recombinant YFV as a vaccine is not safe, although its preparation does not need a packaging system.
WO9928487 provides an expression and delivery method of exogenous sequences by the flavivirus, kunjin virus (KUN). KUN replicons are constructed by the deletion of part of the KUN structural genes and the insertion of an exogenous sequence (i.e., non-KUN) into the genome, followed by packaging as KUN virus-like particles (VLP) in host cells, along with infection by other virus carrying the complementing coding region of the deleted KUN structural protein gene(s). However, the titer of VLP was unsatisfactory. Also, the booster immunization will be less effective. 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, 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 WO9928487.
WO02072803 teaches a method for construction of DEN subgenomic replicons and use thereof for a DEN vaccine. Nevertheless, it is difficult to apply the invention because there is some insufficient information in the application.
Antigen Presenting Cell (APC) Vaccines:
The preparation and use of APC vaccines is, for example, based on the approach whereby dendritic cells (DC) from a patient are cultured and amplified, transformed to express a tumor-associated antigen (TAA) or tumor-specific antigen (TSA), and the treated DC cells then become an APC vaccine for the patient. The APCs will activate T cells of the patient to kill tumor cell when the vaccine is injected into patient. However, such an APC vaccine is patient-specific so these vaccines can't be administered en masse to a wide population. Moreover, this therapy is time-consuming, labor-consuming, is associated with a high-cost because it is difficult to culture DCs, it is costly to amplify the transformed cells, it can be difficult to activate and obtain maturation of DCs for proper and effective antigen presentation and there are quality control issues because of the difficult vaccine preparation process and the individual nature of the product.
In summary, there is an urgent requirement for novel vaccines with high capacity, host cells are long-lived, can carry large inserts and are easy to propagate. Such vaccines also are not oncogenic and can be administered repeatedly.
The invention aims to provide novel products applicable to industrial production, which have good safety, high titer, good booster effects, especially the vaccines against tumor and virus.
In a first aspect the invention provides recombinant DEN with a deletion of preM and comprising of follow nucleotide sequences at least:
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 5′ release elements. These release elements can be a nucleotide sequence obtained from the autoprotease of foot-and-mouth disease virus (FMDV) (SEQ ID NO: 3), or nucleotide sequence encoding the substrate of signal peptide protease (SEQ ID NO: 44) or both.
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 3′ 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 exogenous sequence encodes an HPV antigen, immunoregulators or both.
In a second aspect, the present invention also provides VLPs comprising a DEN recombinant replicon and DEN structural proteins.
In a third aspect, the present invention also provides pharmaceutical compositions containing a DEN recombinant replicon or a DEN-VLP, and pharmaceutically acceptable carrier.
In a fourth another aspect, the present invention also provides use of a DEN recombinant replicon or a DEN-VLP for prophylactic and therapeutic use as a drug for tumor and viral infection.
In a fifth aspect, the present invention also provides cells for a packaging system encapsulating DEN recombinant replicons into VLP. These packaging cells are selected from:
In a sixth 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 vector with an NS3 deletion and a Tet-regulated vector.
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. The preferred release element selects from nucleotide sequence encoding Foot-And-Mouth virus hydrolytic enzyme as SEQ ID NO: 3 showed and nucleotide sequence encoding signal peptide hydrolytic enzyme substrate as SEQ ID NO:44 showed or their combination. Usually there is only one “release element”, but there are many “release element” occasionally.
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.
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.
Based on the experiments, the DEN recombinant replicon will have the following characteristics.
The DEN virus that can be 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 a property of being bound by dendritic cells, which can enhance vaccine 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 immunization. The ADE phenomena can be used for exogenous protein expression systems, which not only avoid 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.
Exogenous genes used in the present invention have no special limitation. It can be any exogenous coding sequence, such as an aptamer, siRNA, 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, an immunoregulator gene and so on. The representative antigens include (but not limited to): human HPV antigen (such as the E6 or E7 protein of the 16 and 18 subtype), HIV antigen, HBV antigen, HCV antigen, EBV antigen, HTLV-1 antigen, MAGE, 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. 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 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 HPV 16 or HPV 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 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.
Construction of said replicon can be processed as follows:
1. Construction of a full-length DEN genome sequence.
2. Deletion of parts of one or more DEN structural genes.
1. Insertion of an exogenous gene coding sequence.
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, and 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, 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 pharmaceutic arts, “Remington: Pharmacology and Pharmacological Practice” 19th ed. (1995) Mack Publishing Co.
Pharmaceutical compositions can be made into a variety of forms, such as injection, solid forms, such as grains, tablets, pills, suppositories and capsules, other liquid forms, such as a suspension, a spray etc. The pharmaceutical grade organic or inorganic vectors and/or attenuants suitable to oral or local use can be used for dispensing of various compositions including therapeutically active compounds. 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, the VLP can be put into dispensing through familiar methods of this field and suitable pharmaceutical or transforming vehicles. The suitable vehicles are sterile saline. Other aqueous or inaqueous iso-osmotic sterile injections and sterile suspensions also can be used (pharmaceutically available vehicles, also familiar to technicians of this field).
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 vaccine administering pathway or modeling pathogen infection pathway. The pharmaceutically available vehicles can be used when administrating vaccine compositions.
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.
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.
4. Low cost:
The invention now is exemplified in the following representative examples. Clearly, these examples only clarify and don't limit this invention. The experimental methods taught herein, many of which are known in the art, 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.
The resulting three DEN cDNA fragments are 5484 bp, 2530 bp and 2922 bp, respectively, in length.
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 a 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.
Groups 2 and 3 of eight mice each were inoculated in the flank with 105 JHU-1 HPV cells. After fourteen days, groups 3 and 4 of eight mice each were inoculated ip with 107 PFU HPV-VLP and after seven days they were inoculated with 107 PFU HPV-VLP again.
1. To examine immunity of HPV VLP:
2. Results:
If a tumor is larger than 25 mm, the mouse was sacrificed.
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-VPL. 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-VPL 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.
b) The Effects of HPV VLP on E6-E7-Specific CD8+ T Cells
The effects of the HPV VPL on E6-E7-specific CD8 T cells were examined by cytoplasm staining. The HPV VPL induced CD8+ T cells to secrete IFN-γ and TNF-α.
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 NSI 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 |
| CN2004000088 | Jan 2004 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11192923 | Jul 2005 | US |
| Child | 12129908 | US |
