JPH03503364A - Recombinant vaccinia virus for preventing diseases caused by flaviviruses - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Recombinant vaccinia virus to prevent diseases caused by bracken virus.

Technical field The present invention relates to the structure of a recombinant vaccinia virus. More specifically, the present invention is a bracken virus (flaviv-irus) such as dengue virus (dengue virus). virus), Japanese encephalitis virus (Japanese B en) ceptxaHtis virus) and tick-borne encephalitis virus (tick-b orne eneephalitis virus) Recombinant vaccinia virus useful in the production of vaccines for ant vaccfnia-virus) construction BACKGROUND OF THE INVENTION Certain diseases in public health services, such as dengue fever, SE) and certain types of encephalitis are caused by viruses belonging to the Brackenviridae family. It has been known. Dengue virus alone causes 100 million illnesses each year in tropical regions of the world It is estimated that this is caused by. However, such flaviviruses effective and immune system for controlling flavivirus disease. There are no preventive measures.

Summary of the invention Therefore, the purpose of the present invention is to infect a host infected with the following recombinant vaccinia virus. Immune response to specific bracken virus diseases ) To provide a recombinant vaccinia virus that induces

Other objects and advantages of the present invention will become apparent from the following detailed description of the invention. cormorant.

Brief description of the drawing These and other objects, features, and many advantages associated with the present invention are summarized in the attached document. If you consider it in relation to the drawing, you can understand it better by reading the detailed explanation below. There will be.

Figure 1 shows the schematic structure of the dengue virus-vaccinia virus recombinant plasmid. show. intermediate cloning vector vector) pscll is a transcription vector of the pH vaccinia virus promoter. Interrupted under key 1a (transcription control) thymidine kinase gene sequence 5 sequences) (TK” and TKL) and bacteria β- Contains the sidase gene (LacZ). The data located at the 5′-end of the viral genome A 4.1 kilobase (Kb) fragment of viral DNA For insertion, the vector DNA was inserted into the BamH1 region of the P7.5 promoter. In order to open the downstream (BamHI site downstream), BamHI is partially digested. This dengue cDNA fragment contains eight structural proteins [ capsid (C), Bremen Plan (pre-men+ brain) (play M) and envelope (en-velope) glycoprotein (E)] plus the first two downstream non-structural protein (down-stream@non-structural p transcription guidelines (roteins) dengue virus DNA insert) A recombinant plasmid containing ert) can be used to create a recombinant vaccinia virus. selected and used for.

Figures 2A and 2B are dengue viruses synthesized by recombinant vaccinia virus. shows the identification of

(Figure 2A:) (''S)-Methionine from recombinant vaccinia virus infected cells Immunoprecipitation of labeled decomposition products (i++ueno-precfpftation) ( Multiplicity of infection (10 PFU/cell) was performed with one of the following specific antibodies: Envero Monoclonal antibody IHIO specific for membrane glycoprotein (E), membrane glycoprotein precursor (membrane glycoprotein precursor M) monoclonal antibody specific for 5C9; and multivalent antibody (P). The labeled sediment is , analyzed on sodium dodecyl sulfate-12% polyacrylamide gels. den Multivalent antibody of (”S)-methionine-labeled decomposition product from virus-infected CV-1 cells Labeled dengue virus protein markers obtained by immunoprecipitation with There is.

(Figure 28:) jlI11! as above! labeled immunoprecipitate jmaunoprecipitates) is endoglycosidase H (en doglycosidase HX! Digest-ion and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. was analyzed. The numbers on the left side of the gel indicate protein size in kilodaltons (kDa).

Detailed description of the invention The above and various other objects and advantages of the present invention include the above-mentioned recombinant vaccinia virion. Expressing key specific protective brackenvirus antigens in cells infected with Rus achieved by a recombinant vaccinia virus containing the complete coding sequence for The above-mentioned antibody is used in the flavivirus in a recipient host (5usceptible host). Contains protective immunity against infection by Rus.

Unless otherwise defined, all technical and scientific terms used herein refer to the expresses the same meaning as commonly understood by ordinary specializations belonging to the field of light . Any methods and materials similar or equivalent to those described herein are also included in the invention. Preferred methods and materials that can be used in the practice or testing are now described.

All publications mentioned below are hereby incorporated by reference. Unless otherwise specified The techniques used herein are recognized by those of ordinary skill in the art (known standards). This is a quasi-method.

Like other members of the Flaviviridae family, extracellular dengue viruses are relatively simple It has a simple structure. Pilions (viral particles) are encoded by only 8 types of viruses. Proteins (virus-coded preteins): Designated Kabzid CC ) proteins, including membrane (M) proteins and envelope (E) glycoproteins. Also contagious Intracellular viruses that possess M lack M but contain another glycoprotein, precursor M (Bure M) ( M has ) which is derived by its division. Both C and M are internal proteins (in internal protein). Surface envelope glycoproteins have specific anti- Major site responsiveness for neutralization of infectivity by the body possible). Envelope glycoproteins also exhibit hemagglutination activity , causing adsorption to the cell surface. Some nonstructural proteins are also denguevirus confirmed in infected cells. One of them is the nonstructural protein NSI, which is a glycoprotein. In white matter, soluble complement-fixing antibodies (complement-fixing antibodies) igen). The similar NSI glycoprotein of yellow fever virus is protective antigen in mice and primates Since it has been found that NSI has a role in mediating immunity, it is believed that NSI plays a role in mediating immunity.

Furthermore, NSI of Dentsu type 2 virus has been found to be a protective antibody.

The Denji type 4 virus genome has a positive twist (pos) with a length of 10,644 nucleotides. tive-stranded) RN A molecules. Dentsu type 4 will A full-length cDNA copy of the gene was prepared and its complete nucleotide sequence determined. Ta. Such studies show that 96% of the dengue virus genome contains specific proteases. A code for polyproteins that are split by (class) to produce individual viral proteins. It is estimated that the code is Eight structural proteins C-M-E are located at the amino terminus. However, the nonstructural protein N5I-NS2a-NS2b-NS3-NS4a-NS4 b-NS5 is located at the carpoxy terminus. Dengue virus gene expression exp-ression) includes proteolytic fission of polyprotein (proteolytic fission). ttccleavage), so the E and The development of protective antibodies such as NSI and NSI is important for immunoprophylaxis. It is emphasized that one cannot intuitively infer that it is not useful for Should.

Such decisions can only be made through real-world testing. As 5 examples now Development of recombinant vaccinia flavivirus by using dengue virus genes Explain the creation.

Cotton senior virus is a live recombinant virus that expresses dengue virus genes. was used as a vector to create a vector. Dentsu type 4 virus cDNA Bgllll DNA fragment from 5' end (4,041 base pairs, 8 nucleotides) 8-4123) contains three structural proteins and the nonstructural proteins NSI and N32a. include. This fragment was dissected from the full-length dengue virus DNA copy and Insert into vaccinia intermediate vector It was done. Dengue virus DNA sequence is now available in vaccinia P7.5 early-late promoter early-1ate promoter am) B a m It was inserted into the H1 site (Figure 1).

During this construction, the dengue virus coding sequence was modified to vaccinia virus P7.5 early-late Substituted under the transcriptional control of the promoter. The vector is a bacterial Vaccinia lactosidase gene provides a visible selectable marker Contained under the control of the viral pH late promoter I did. The chimeric gene flanks the vaccinia virus thymidine kinase gene sequence. It was a monkey pre-infected with wild type vaccinia virus (WR strain). The following transfection of CV-1 cells of Subsequently, a set of homologous dengue virus sequences is found in the vaccinia virus genome. Dengue, which led to the homologous reeombinatfan A recombinant vaccinia virus with a viral DNA insert has been isolated and The protein was purified twice on thymidine kinase minus (TK) cells in selective media. Ta. Other dengue eDNA fragments (see below) were generated by the method just described. and inserted into the vaccinia recombinant virus. Similarly, other flavivirus vaccines Create a senior walla by following the steps described here for Dengue Virus This is done by inserting the viviral cDNA.

The dengue virus-specific protein synthesized during infection with the recombinant virus is initially Indirect fnm-unof 1uorescence ) was detected. CV-1 cells infected with the recombinant virus were infected with polyvalent viruses. Mouse antiserum highly immune to type 4 virus (hyperimmune mouse a) ntiserom), which shows fluorescent staining antigen in the cytoplasm. However, the staining intensity is less than that observed in dengue virus-infected cells. similar fluorescence When a monoclonal antibody specific to the E glycoprotein was used, the antibody was observed in CV-1 cells infected with the virus.

Furthermore, to identify this and other dengue virus proteins, we developed recombinant virus proteins. The stained cells are radiolabeled with methionine, and the cell lysate is uniquely immunoprecipitated. produced by antibodies. on sodium dodecyl sulfate-polyacrylamide gel Analysis of label precipitation (Fig. 2A) shows that polyvalent hyperimmune mouse serum has a molecular size of 20.4 Three molecules estimated to be 0-46 and 55-60 kilodaltons (kDa) respectively It was shown that the main band was precipitated. Larger molecular size that appears to be non-specific A small band of was also observed. E glycoprotein-specific monoclonal antibody (I H20) was used, 50-60k, which matched the molecular size of E glycoprotein. D: The a band precipitated. Similarly, Denji type 2 virus N11-specific antiserum ( Dr. J, obtained from 5chlesi-Ber) precipitated to 40-46 kDa, It assumed the size of NS1 as a nonstructural glycoprotein. Denji type 4 virus The third major band precipitated by the hyperimmune antiserum is approximately 20 kDa, which is The size corresponds to that of extravesicular BreM glycoprotein. A similar band is a BreM-specific antibody (Kano, M., monoclonal antibody 2H2 or obtained from Gentry of WRA[R] was precipitated with 5C9).

In this way, the cloned DN A encodes in cells infected with the recombinant virus. The three glycoproteins identified were disrupted, similar to that observed during dengue virus infection. Can be modified by glycosylation in the manner of It became clear. This includes dengue virus structural proteins as well as NSI nonstructural proteins. In the absence of dengue virus function provided by polyprotein proteolysis, This suggests that processing is performed by fission. However However, as estimated by immunoprecipitation, the amount produced in recombinant virus-infected cells The amounts of Dengubule M, E and NSI glycoproteins detected in Dengubule type 4 infected cells were significantly less than that in the middle.

The glycosylation pattern of glycoproteins produced in recombinant virus-infected cells is Changes 1. as shown by its response to endoglycosidase H. , that's Ma splits into a North-rich carbohydrate core. Bre~1 protein band is glycosider It is completely sensitive to ZeH treatment, yielding a band of 17 kDa and a molecular size of 3 kDa decreases. On the other hand, an important portion of the carbohydrates of both E and 831 glycoproteins are Endoclicosidase I] was found to be resistant to digestion.

Each of the dengue virus glycoproteins, pre-M1E and NSI, serve as a signal. It precedes the spread of hydrophobic amino acids, which can serve as a catalyst. This is a cell signal Cellular stgnalase probably responds to proteolytic cleavage. This shows that it is responsive. Division mechanism that probably produces three types of glycoproteins also gives rise to the cabzid protein, which is located at the amino terminus of the BreM glycoprotein. .

As described hereinabove, initially three structural proteins [cabuzid (C), membrane (C), M) and envelope (B)] and non-cisternal structural proteins SI and N32a. 4. from the 5'-end of the coded cDNA. Recombinant vaccinia containing OKb sequence A was created. After that, we created another recombinant vaccine expressing only the three structural proteins. Nearviruses are created by deleting coding sequences for nonstructural proteins. It was. Protein analysis was performed on the first recombinant virus V (C-toB-NS1-NS2). Cells infected with m> were detected by radioimmunoprecipitation using specific antibodies. as well as producing authentic precursor jI (BreM), envelope (E) and NSI glycoproteins. We showed that this occurs. These dengue proteins are produced during dengue virus infection. showed a glycosylation pattern similar to that found for these proteins. . The second recombinant virus (C-FB) contains dengue virus BreM and E glycoproteins. was produced. As expected, NSI was not synthesized by the second recombinant. (Table 2).

A mouse model of dengue encephalitis was used to evaluate the protective immunity induced by these recombinants. It was used to value Recombinant vaccinia virus was administered to mice by intraperitoneal route. of 10' plaque forming units (pfu) were inoculated. Mice received the same amount after 2 weeks. The recombinant was reinoculated. One week after the second inoculation, 100LDi* was administered into the brain. A mouse was challenged by a virus. The animals were then examined for symptoms of encephalitis and death. Observed. Control vaccinia expressing HIV envelope glycoprotein after challenge. 91% of mice immunized with recombinant virus died (Tables 1 and 2). in addition On the other hand, vaccinia (C-M-B-NSI- NS2) 96% of mice immunized with recombinant survived. In addition, three types of structures Whole cells immunized with vaccinia (C-toB) recombinant virus that expresses only the protein. Fifteen animals were fully rescued. These results suggest that only structural proteins exert protective immunity. This shows that it is possible to induce Since C and M are internal proteins, , E expresses only the key elements involved in the protection conferred by the V(C-toB) recombinant. Complete resistance to intracerebral challenge induced by immunization with vaccinia recombinant will be supported. The data in Table 1 proves this. N-terminal hydrophobic signal sequence Or respiratory placental virus s:RS V) G glycoprotein (amino acids 1-7G)-E fusion protein or influenza Enzyme virus hemagglutination E containing N-terminal signal peptide-E fusion protein Recombinants expressing .

The seroresponse of animals immunized with these recombinants was , tested by radioimmunoprecipitation of labeled antigen. V expressing three types of structural proteins (C-M-B-NSI-NS2) Each animal immunized with recombinant NSI antibody , but the amount of antibodies to E was low or undetectable. E antibody response Low levels further reduce virus neutralization (vfrus neut-ralization). ) and other serological tests such as ELISA. In addition, three types of structural proteins Only white matter expressed or undetectable amounts of E antibodies were generated.

The nonstructural protein NSI expressed by various vaccinia recombinant viruses also has its antigen. The most promising purified products have been evaluated for their protective properties in mice. We evaluated its effectiveness. A total of six NS1 vaccinia recombinants were studied.

These recombinants: (i) NSI precedes the structural protein sequence (C-M-8); (ff) the NSI is preceded only by its intermediate upstream hydrophobic sequence, or (iii) NS1 contains an insert that is not preceded by a structural gene or its intermediate upstream hydrophobic sequences. It contained. Each of these inserts contains the complete N52a sequence or the N52a sequence. It terminated at 15% of the N-terminus. V (C-M-E-NSI-NS2a) and V ( NSI-NS2a) recombinant produces authentic NS1, while N-terminal hydrophobic Other recombinants lacking sex signals or lacking the complete downstream N52a sequence are different to produce NSI products of molecular size and/or glycodilation as shown by gel analysis. It was not standardized. These studies have shown that the N-terminal signal sequence of NSI It was determined that appropriate processing and proteolytic cleavage were required.

The N31 recombinant was then tested for its preventive potential in mouse protection studies as described above. It was evaluated for its power. The results showed that vaccinia expressing structural proteins and NS1 ( C-M-B-NSI-NS2a) All but one of the 28 animals inoculated with the recombinant Mice survived dengue challenge (Table 1). Significantly, the absence of structural proteins A recombinant expressing authentic NSI, namely V (NSI-NS2), is a Dengwi induced complete resistance to lethal attack in Rus (Table 2). Only abnormal protection guided. Thus, (i) the upstream N-terminal R8V G sequence fused to NSI (amino acids 1-70) or (if) expressing NS1 + NS2a 15% Vaccination with recombinant vaccinia is a partial only induced protection.

These tests demonstrate that dengue envelope (E) glycoprotein and nonstructural protein NSI Separated into multiple protective antigens, each of which induces encephalitis in mouse models of dengue disease. Leading to complete resistance to. These results have now shown that humans are safe against Dentsu type 4 virus. Vaccines expressing E and/or NS1 glycoproteins as a vaccine to protect between Allows development of senior recombinants. Therefore, vaccines contain immunogenic amounts of recombinant It consists of vaccinia virus, which is infected with the recombinant vaccinia virus mentioned above. induces protective immunity against specific flaviviruses in the host. Conventional pharmaceutically acceptable Compatible carriers or excipients such as non-toxic buffers, saline, etc. may optionally assist. can be used with agents and reinforcing inoculations. Of course, the vaccine should be administered once as directed. or may be administered in multiple doses.

for C, M and E controlled by the pH vaccinia virus promoter. A vaccinia-Detsuji type 2 virus recombinant containing the Denzo sequence was recently developed by Dube et al. Deubee et al.) in 1988 (J. Gen. Viro L, 69:1921-1929) and protect monkeys against dengue fever. It is important to point out here that this has failed. On the other hand, the original Ming's recombinant is effective in protecting test animals against viral infection.

Genome organization and replication method of all flaviviruses (replfcat-ion str ategy) and gene expression are similar, prophylaxis against other flaviviruses The recombinant vaccinia virus with the characteristic characteristics is the vaccinia virus shown in this specification. It is created in a manner similar to the creation of a

The recombinant virus was deposited on August 19, 1988, at 2085 Maryland, USA. 2. American Tata, 12301 Burklawn Drive, Rockville. IP Culture Collection (ATCC) under accession number VR2228. It was held in

The deposit shall be made for a period of 30 years from the date of deposit or from the last date on which a deposited sample was requested. It will remain viable for the longer of five years and will be removed if it dies. and be made publicly available without any restrictions as provided by law. . The Commissioner of Patent and Trademark Offices may obtain deposits upon request.

The examples and specific descriptions herein are for illustrative purposes only, and and various easy improvements or modifications thereto have been suggested by those skilled in the art. is understood to be within the spirit and power of the invention and within the scope of the appended claims.

Table 1 Waxes expressing Denzo (E) glycoprotein with or without non-structural NSI glycoprotein Mice infected with Nia recombinants resist lethal challenge with dengue virus develops Denzo protein (S) expression infection infection Comb □ Mouse Luz 10”LD.

Near recombinant IJ) Number of authentic brain attacks B NSI (IOPFU) Survival number V (C-M-E-NSI-NS2.) YBS YES 28 27 (96%) V(C-M-B-NSI-N S2. A)” YBS NO1515 (100%) V (C-M-E) 　　　　　　　　　　-1919(100%)V(Eri) 　　　　 YES -1515 (100%) 99V (R8V G-8)” YBS -1515 (100%) 90V (FLtl HA-E)” YBS -1515 (100%) V (HIV GP160)"" --464 (8.7%)" 85% deletion of the terminal sequence of N52A.

′　　Surviving mice with remission of encephalitis.

” Expression of RSVG (amino acids 1-70) +E N-terminal fusion protein.

Expression of a fusion protein of O influenza A virus N-terminal jig T Rubebti F1E.

◆◆Control vaccinia recombinant expressing the envelope glycoprotein of φH[V.

Table 2 Mice infected with a vaccinia recombinant expressing only the dengue nonstructural protein NSI The virus is a recombinant strain that develops resistance to lethal attack by the dengue virus. (IOPFU) Survival number V (NSI-NS2.) YE! S i5 27 (100%) V (NSI-NS2.’)” No 30 15 (67%) V (R8V G-NSI)” NO1515 (54%) V (HIV GP160)” 4 6　　　4(8.7%) *85% deletion of the terminal sequence of N52A.

” R3V G (7 amino acids 1-70) + NS 1 (7) Fusion protein 1@L ″′″ Control vaccinia recombinant expressing HIV envelope glycoprotein, FIG.1 FIG, 2A FIG, 2B p”　　　ε　　NSl End H-÷-+-÷ Submission of translation of written amendment (Patent Law No. 184-8) February 20, 1996 "l"Fj? :itm　　　　　　　　　　　　Prison 1, Display of patent application PCT/US 89108589 2. Name of the invention Recombinant vaccinia virus for preventing diseases caused by flaviviruses 3. Patent applicant Name United States of America 4. Agent (〒101) Address: 6, Kanda Surugadai 1, Chiyoda-ku, Tokyo July 13, 1990 The scope of the claims ! , the viral protein is obtained from bracken virus, V(C-M-E-NSI ), V (C-M-E, V (B), V (RSV G-8), V (FLU A recombinant selected from the group consisting of HA-8) and V (NSI-NS2A) Vaccinia virus.

2. The viral protein is obtained from flavivirus, and the carboxy terminus of N52A Substantially C, M, E, NSI, and N S 2 with 85% of the sequence deleted A recombinant vaccinia virus consisting of the coding sequence for the a protein.

3. The virus according to claim 1, wherein the flavivirus is Dentsu type 4 virus.

4. The virus according to claim 2, wherein the flavivirus is Dentsu type 4 virus.

5. The flavivirus mentioned above is Japanese encephalitis B virus or tick-borne encephalitis virus. The virus according to claim 1.

6. The flavivirus mentioned above is Japanese encephalitis B virus or tick-borne encephalitis virus. The virus according to claim 2.

7. containing an immunogenic amount of the virus according to claim 1 in a pharmaceutically acceptable carrier; A pharmaceutical composition comprising:

8. containing an immunogenic amount of the virus according to claim 2 in a pharmaceutically acceptable carrier; A pharmaceutical composition comprising:

9. Injecting a small amount of an immunogenic amount of the virus according to claim 1 into a flavivirus recipient host. (Includes single-dose vaccination to induce protective immunity against bracken virus disease.) How to make it emit.

10. Injecting an immunogenic amount of the virus according to claim 2 into a flavivirus recipient host in a small amount. To provide protective immunity against flavivirus diseases, consisting of at least a single dose of vaccination. How to induce it.

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Claims (6)

[Claims] 1. A recombinant virus containing the complete coding sequence for the expression of flavivirus antigenic proteins. Cushina virus. 2. The virus according to claim 1, wherein the flavivirus is dengue type 4 virus. 3. The above flavivirus is Japanese encephalitis B virus or tick-borne encephalitis virus. The virus according to claim 1. 4. The above antigenic proteins are envelope glycoproteins, capsid proteins, and prematric proteins. NS1, NS2 nonstructural proteins, or a part or whole of a combination thereof The virus according to claim 1, which is selected from the group consisting of: 5. comprising an immunogenic amount of the virus of claim 1 in a pharmaceutically acceptable carrier. Pharmaceutical composition. 6. Claims of susceptible hosts and immunological doses of flaviviruses during single or combined administration Protective immunity against flavivirus disease comprising inoculating with the virus described in item 1 induction method.