Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 

 

 

Title:  Gene coding for the measles virus mutant antigen

United States Patent:  6,605,284

Issued:  August 12, 2003

Inventors:  Ueda; Shigeharu (Nishinomiya, JP); Watanabe; Michiko (Osaka, JP); Kawanish; Hitomi (Sakaide, JP)

Assignee:  The Research Foundation for Microbial Diseases of Osaka University (Osaka, JP)

Appl. No.:  873233

Filed:  June 5, 2001

Abstract

Disclosed is a measles virus mutant gene coding for a measles virus mutant H protein antigen, wherein said gene coding for a measles virus mutant H protein antigen is at least one member selected from the group consisting of the following genes (a) to (c): (a) a gene coding for an amino acid sequence of SEQ ID NO: 10; (b) a gene coding for an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 11; and (c) a gene coding for an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12. By the use of the measles virus mutant gene of the present invention, it has become possible to provide efficiently and economically a gene vaccine which is adapted for an epidemic strain of measles virus, and a diagnostic reagent capable of accurately detecting infections with an epidemic strain of measles virus.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the present invention, there is provided a measles virus mutant antigen, comprising at least one protein antigen selected from the group consisting of (I) a measles virus mutant H protein antigen and (II) a measles virus mutant F protein antigen,

the measles virus mutant H protein antigen (I) being at least one member selected from the group consisting of the following amino acid sequences (a) to (f) identified with the positional amino acid numbers of either SEQ ID NO: 2 or SEQ ID NO: 10:

(a) the whole sequence of the 1st to 617th amino acids;

(b) a fragmentary sequence of the 93rd to 616th amino acids;

(c) a fragmentary sequence of the 176th to 316th amino acids;

(d) fragmentary sequences of the 172nd to 178th amino acids, the 238th to 244th amino acids, the 277th to 282nd amino acids, and the 301st to 307th amino acids;

(e) fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences each comprise an amino acid selected from the group consisting of the 174th, 176th, 243rd, 279th and 302nd amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the fragmentary, contiguous sequences are exclusive of the fragmentary sequences (d); and

(f) fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences each comprise an amino acid selected from the group consisting of the 93rd, 157th, 169th, 175th, 211th, 252nd, 276th, 284th, 285th, 296th, 316th, 338th, 387th, 416th, 455th, 481st, 484th, 505th, 546th, 592nd, 600th, 603rd and 616th amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10 wherein the fragmentary, contiguous sequences are exclusive of the fragmentary sequences (d) and (e); and

the measles virus mutant F protein antigen (II) being at least one member selected from the group consisting of the following amino acid sequences (g) and (h) identified with the positional amino acid numbers of either SEQ ID NO: 18 or SEQ ID NO: 20:

(g) the whole sequence of the 1st to 550th amino acids; and

(h) fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 18 or SEQ ID NO: 20, wherein the sequences each comprise an amino acid selected from the group consisting of the 11th, 52nd, 107th, 165th, 398th, 417th and 523rd amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 18 or SEQ ID NO: 20.

For easy understanding of the present invention, the essential features and various preferred embodiments of the present invention are enumerated below.

1. A measles virus mutant antigen, comprising at least one protein antigen selected from the group consisting of (I) a measles virus mutant H protein antigen and (II) a measles virus mutant F protein antigen,

the measles virus mutant H protein antigen (I) being at least one member selected from the group consisting of the following amino acid sequences (a) to (f) identified with the positional amino acid numbers of either SEQ ID NO: 2 or SEQ ID NO: 10:

(a) the whole sequence of the 1st to 617th amino acids;

(b) a fragmentary sequence of the 93rd to 616th amino acids;

(c) a fragmentary sequence of the 176th to 316th amino acids;

(d) fragmentary sequences of the 172nd to 178th amino acids, the 238th to 244th amino acids, the 277th to 282nd amino acids, and the 301st to 307th amino acids;

(e) fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences each comprise an amino acid selected from the group consisting of the 174th, 176th, 243rd, 279th and 302nd amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the fragmentary, contiguous sequences are exclusive of the fragmentary sequences (d); and

(f) fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences each comprise an amino acid selected from the group consisting of the 93rd, 157th, 169th, 175th, 211th, 252nd, 276th, 284th, 285th, 296th, 316th, 338th, 387th, 416th, 455th, 481st, 484th, 505th, 546th, 592nd, 600th, 603rd and 616th amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the fragmentary, contiguous sequences are exclusive of the fragmentary sequences (d) and (e); and

the measles virus mutant F protein antigen (II) being at least one member selected from the group consisting of the following amino acid sequences (g) and (h) identified with the positional amino acid numbers of either SEQ ID NO: 18 or SEQ ID NO: 20:

(g) the whole sequence of the 1st to 550th amino acids; and

(h) fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 18 or SEQ ID NO: 20, wherein the sequences each comprise an amino acid selected from the group consisting of the 11th, 52nd, 107th, 165th, 398th, 417th and 523rd amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 18 or SEQ ID NO: 20.

2. A measles virus mutant gene, comprising at least one gene selected from the group consisting of (I) a gene coding for a measles virus mutant H protein antigen and (II) a gene coding for a measles virus mutant F protein antigen,

the gene coding for a measles virus mutant H protein antigen (I) being at least one member selected from the group consisting of the following genes (a) to (f) identified with the positional amino acid numbers of either SEQ ID NO: 2 or SEQ ID NO: 10:

(a) a gene coding for the whole sequence of the 1st to 617th amino acids;

(b) a gene coding for a fragmentary sequence of the 93rd to 616th amino acids;

(c) a gene coding for a fragmentary sequence of the 176th to 316th amino acids;

(d) genes coding for fragmentary sequences of the 172nd to 178th amino acids, the 238th to 244th amino acids, the 277th to 282nd amino acids, and the 301st to 307th amino acids;

(e) genes coding for fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences each comprise an amino acid selected from the group consisting of the 174th, 176th, 243rd, 279th and 302nd amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the genes are exclusive of the genes (d); and

(f) genes coding for fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences each comprise an amino acid selected from the group consisting of the 93rd, 157th, 169th, 175th, 211th, 252nd, 276th, 284th, 285th, 296th, 316th, 338th, 387th, 416th, 455th, 481st, 484th, 505th, 546th, 592nd, 600th, 603rd and 616th amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the genes are exclusive of the genes (d) and (e); and

the gene coding for measles virus mutant F protein antigen (II) being at least one member selected from the group consisting of the following genes (g) and (h) identified with the positional amino acid numbers of either SEQ ID NO: 18 or SEQ ID NO: 20:

(g) a gene coding for the whole sequence of the 1st to 550th amino acids; and

(h) genes coding for fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 18 or SEQ ID NO: 20, wherein the sequences each comprise an amino acid selected from the group consisting of the 11th, 52nd, 107th, 165th, 398th, 417th and 523rd amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 18 or SEQ ID NO: 20.

Hereinbelow, the present invention is described in detail.

In the present invention, with respect to the nucleotide sequences, A represents adenine, C represents cytosine, G represents guanine and T represents thymine.

In the present invention, with respect to the amino acid sequences, Ala represents an alanine residue, Arg represents an arginine residue, Asn represents an asparagine residue, Asp represents an aspartic acid residue, Cys represents a cysteine residue, Gln represents a glutamine residue, Glu represents a glutamic acid residue, Gly represents a glycine residue, His represents a histidine residue, Ile represents an isoleucine residue, Leu represents a leucine residue, Lys represents a lysine residue, Met represents a methionine residue, Phe represents a phenylalanine residue, Pro represents a proline residue, Ser represents a serine residue, Thr represents a threonine residue, Trp represents a tryptophan residue, Tyr represents a tyrosine residue and Val represents a valine residue.

For making more clear the essential features of the present invention, the technical features of the present invention will be described in detail below by explaining how the present invention has been developed.

All of the conventional live measles vaccines are produced from virus strains which were obtained by attenuating the viruses which prevailed in the 1950's and 1960's. Therefore, the antigenicity of conventional vaccine strains corresponds to the antigenicity of virus strains which were epidemic half a century ago.

On the other hand, it has been found that the most recent epidemic strains and the relatively recent epidemic strains have mutations in the H protein gene and the F protein gene which are genes responsible for a virion to adsorb on and penetrate into cells to thereby cause an infection with the virus. Specifically, the mutation in the H protein gene causes substitution of 17 to 19 amino acids in a specific region in the whole amino acid sequence (consisting of 617 amino acids) of the H protein and, such a substitution changes the three-dimensional structure of the protein, so that an antigenic mutation occurs. This antigenic mutation is as large as the antigenic shift of the H protein, and important.

Further, the present inventors have found that the antigenic mutation of the epidemic strain is an important factor causing the above-mentioned secondary vaccine failure and modified measles.

Based on these findings, the present inventors have succeeded in providing a viral genome of a measles virus mutant, particularly a mutant H protein gene and a mutant F protein gene, and the mutant antigens (not only the whole protein but also fragmentary peptides thereof) encoded by the genes.

In addition, the present inventors have successfully developed the following utilities (i) to (iii) of the above-mentioned genes, mutant antigens and their epitopes, and the like.

(i) Modification of a viral genome of a live vaccine strain: A recombinant virus is prepared by replacing the H protein gene of a conventional live vaccine strain with the H protein gene of an epidemic strain. By using this method, a live attenuated vaccine strain which is adapted for the antigenicity of the epidemic strain is obtained speedily. In other words, the recombinant virus obtained in the above-mentioned manner can be used as an active component of an excellent vaccine which is capable of effectively preventing infections with the epidemic strains. This method is also advantageous from an economical viewpoint. That is, the time, labor and costs necessary for attenuating a virus can be decreased to a large extent. As mentioned above, with respect to the production of conventional vaccines, there is no specific limitation on the method for attenuating viruses, and conventionally, the attenuation was conducted mainly by passage, which requires at least several years to about 10 years for establishing an attenuated strain for a live vaccine.

(ii) Preparation of an active component for a gene vaccine: A gene vaccine is prepared by inserting the H protein gene and the F protein gene of an epidemic strain into various vectors, such as a plasmid vector, a cosmid vector, a phage vector, a shuttle vector, a viral vector of a non-proliferating viral vector and the like.

For example, when a non-proliferating recombinant virus, which is prepared by inserting the cDNAs for the above-mentioned H protein gene and F protein gene into a non-proliferating viral vector, is used as an active component for a gene vaccine or DNA vaccine, such a vaccine is capable of inducing both humoral immunity and cellular immunity like a conventional live measles vaccine. A remarkable feature of this vaccine is that nasal injection is possible.

In addition, a cDNA fragment comprising the mutated region of the H protein gene of an epidemic strain can be inserted into, for example, a plasmid vector, to prepare a naked DNA. The thus prepared naked DNA can also be used as an active component for a DNA vaccine or gene vaccine for preventing measles.

(iii) Preparation of a suitable reagent for diagnosis of epidemic strains: PCR primers are synthesized so that the synthesized primers reflect the mutations in the H protein gene or F protein gene of the epidemic strains. The synthesized primers can be used as a reagent for gene diagnosis not only for identifying the epidemic strains, but also for differentiating a virulent strain from an attenuated strain, or vice versa.

Further, the mutant antigens (whole proteins or fragmentary peptides thereof) encoded by the above-mentioned genes are prepared, and their epitopes are chemically synthesized. The antigens and epitopes are provided as suitable antigens for diagnosis of epidemic measles.

An explanation is made below with respect to the preparation of a measles virus mutant antigen and a measles virus mutant gene of the present invention, and the use of the prepared antigens and genes as a vaccine and a diagnostic reagent.

[I] Preparation of Measles Virus Mutant Antigen and Measles Virus Mutant Gene

(1) Antigen analysis of various measles virus antigens: The antigenicity of the measles virus mutant antigen can be evaluated by a neutralization test, an HI (hemagglutination inhibition) test, a PA (passive agglutination) test, an enzyme immunoassay and a fluorescent antibody technique each using a monoclonal antibody, and the like. However, for determining the effectiveness of the virus antigen as antigen for a vaccine, it is requisite to evaluate the antibody titer by the neutralization test, and it can be performed in accordance with the modified Ueda method (Biken Journal, 14, 155-160, 1971) which employs microplates.

With respect to the antibodies used in the antigen analysis, sera, such as a serum from a measles patient and mouse immune sera against measles viruses as mentioned below, can be employed.

With respect to the antigens (challenge viruses) used in the antigen analysis, it is important to select different measles strains from the strains isolated in the past to the present. Representative examples of epidemic strains of the 1950's and 1960's (virulent strains of the past) include Tanabe strain and Edmonston strain; and examples of live vaccine strains established by attenuating the above-mentioned virulent strains (conventional attenuated strains) include CAM-70 strain and Edmonston B strain. As the recent epidemic strains (virulent strains), use can be made of the measles strains isolated in various countries in the 1990's. For example, the virus strains isolated from various resources by the present inventors, such as F-t strain (isolated in 1991 from throat swab of a reinfected patient), F-b strain (isolated in 1991 from blood of a reinfected patient), U-t strain (isolated in 1991 from throat swab of a non-vaccinated patient), U-b strain (isolated in 1991 from blood of a non-vaccinated patient), Momo strain (isolated in 1995 from a patient) and NA strain (isolated in 1996 from a patient) can be used as the recent epidemic strain.

Hereinafter, the following strains will be frequently referred to as indicated in the parentheses: Tanabe (Tana) strain, Edmonston (Edmo) strain, CAM-70 (CAM) strain and Momo (MO) strain.

(2) Determination of the mutated regions in the nucleotide sequence of a gene, and translation of the gene into an amino acid sequence: The analysis of the viral genome of each of the measles strains mentioned in item (1) above is carried out as follows. First, the viral RNA genome is extracted and the cDNA is prepared using primers. The nucleotide sequence of the prepared cDNA is determined by the direct sequencing method which employs PCR method (hereinafter, simply referred to as "PCR-direct sequencing method"). The search for DNA sequence homology between different measles virus strains is performed while determining the nucleotide sequence of the genes, to thereby specify the mutated regions within the genes.

Next, each of the above-specified mutated regions are translated into amino acid sequence in accordance with the universal code, and the deductive analyses of the amino acid sequences are performed as follows. Analysis of the hydrophobicity pattern and determination of the secondary structure of a protein by ChouFasman analysis are performed by computer using the computer software "DNASIS-Mac (version 3.6)" (manufactured and sold by Hitachi Software Engineering Co., Ltd., Japan). Epitopes can be identified, for example, by computer using the computer software "Epitope Advisor" [manufactured and sold by Fujitsu Kyushu System Engineering (FQS) Ltd., Japan].

(3) Measles virus mutant antigens and genes coding for the same: Based on the antigen analyses mentioned in item (1) above and the studies on the nucleotide and amino acid sequences mentioned in item (2) above, the present inventors have conducted comparative analyses between the strains of recent epidemic measles, the virulent strains of the past and the conventional strains for a live attenuated measles vaccine, and they identified the respective regions in the H protein and the F protein which contain amino acid substitutions. Further, the present inventors specified the antigens useful for the vaccine or the reagent for diagnosis of epidemic strain of measles virus. The measles virus mutant antigen of the present invention is the whole protein or a fragmentary peptide of the H protein and F protein of the attenuated measles virus CAM-70 strain or the epidemic measles virus NA strain. Each of the amino acid sequences is disclosed for the first time by the inventors of the present invention. Specifically, the measles virus mutant antigen of the present invention is an antigen comprising at least one protein antigen selected from the group consisting of (I) an H protein antigen of a measles mutant and (II) an F protein antigen of a measles mutant.

The measles virus mutant H protein antigen (I) is at least one member selected from the group consisting of the following amino acid sequences (a) to (f) identified with the positional amino acid numbers of either SEQ ID NO: 2 or SEQ ID NO: 10:

(a) the whole sequence of the 1st to 617th amino acids;

(b) a fragmentary sequence of the 93rd to 616th amino acids;

(c) a fragmentary sequence of the 176th to 316th amino acids;

(d) fragmentary sequences of the 172nd to 178th amino acids, the 238th to 244th amino acids, the 277th to 282nd amino acids, and the 301st to 307th amino acids;

(e) fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences each comprise an amino acid selected from the group consisting of the 174th, 176th, 243rd, 279th and 302nd amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the fragmentary, contiguous sequences are exclusive of the fragmentary sequences (d); and

(f) fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the sequences each comprise an amino acid selected from the group consisting of the 93rd, 157th, 169th, 175th, 211th, 252nd, 276th, 284th, 285th, 296th, 316th, 338th, 387th, 416th, 455th, 481st, 484th, 505th, 546th, 592nd, 600th, 603rd and 616th amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the fragmentary, contiguous sequences are exclusive of the fragmentary sequences (d) and (e).

The measles virus mutant F protein antigen (II) is at least one member selected from the group consisting of the following amino acid sequences (g) and (h) identified with the positional amino acid numbers of either SEQ ID NO: 18 or SEQ ID NO: 20:

(g) the whole sequence of the 1st to 550th amino acids; and

(h) fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 18 or SEQ ID NO: 20, wherein the sequences each comprise an amino acid selected from the group consisting of the 11th, 52nd, 107th, 165th, 398th, 417th and 523rd amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 18 or SEQ ID NO: 20.

Among the protein antigens included in the measles virus mutant antigens of the present invention, the protein antigens as defined in items (a) and (g) above are H protein and F protein, respectively, and the protein antigens as defined in items (b) to (f) and (h) above are peptides (fragmentary sequences). Further, the four fragmentary sequences as defined in item (d) above, namely, the fragmentary sequences of the 172nd to 178th amino acids, the 238th to 244th amino acids, the 277th to 282nd amino acids, and the 301st to 307th amino acids, identified with the positional amino acid numbers of either SEQ ID NO: 2 or SEQ ID NO: 10, are epitopes of the H protein which are disclosed for the first time by the inventors of the present invention. With respect to the protein antigens as defined in items (a) to (d) and (g) above, the specific sequences are shown in the Sequence Listing. Each of the antigens of the present invention can be chemically synthesized, based on the sequences shown in the Sequence Listing (see Example 5).

The measles virus mutant antigen of the present invention comprises at least one protein antigen selected from the group consisting of the above-mentioned whole proteins and fragmentary peptides, and the protein antigen can be chosen, based on the intended utility of the measles virus mutant antigen. Occasionally, several protein antigens can be used in combination.

In a further aspect of the present invention, a gene coding for the above-mentioned measles virus mutant antigen is provided. Specifically, the measles virus mutant gene comprising at least one gene selected from the group consisting of (I) a gene coding for an H protein antigen of a measles mutant and (II) a gene coding for an F protein antigen of a measles mutant is provided.

The gene (I) coding for a measles virus mutant H protein antigen is at least one member selected from the group consisting of the following genes (a) to (f) identified with the positional amino acid numbers of either SEQ ID NO: 2 or SEQ ID NO: 10:

(a) a gene coding for the whole sequence of the 1st to 617th amino acids;

(b) a gene coding for a fragmentary sequence of the 93rd to 616th amino acids;

(c) a gene coding for a fragmentary sequence of the 176th to 316th amino acids;

(d) genes coding for fragmentary sequences of the 172nd to 178th amino acids, the 238th to 244th amino acids, the 277th to 282nd amino acids, and the 301st to 307th amino acids;

(e) genes coding for fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein said sequences each comprise an amino acid selected from the group consisting of the 174th, 176th, 243rd, 279th and 302nd amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the genes are exclusive of the genes (d); and

(f) genes coding for fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein said sequences each comprise an amino acid selected from the group consisting of the 93rd, 157th, 169th, 175th, 211th, 252nd, 276th, 284th, 285th, 296th, 316th, 338th, 387th, 416th, 455th, 481st, 484th, 505th, 546th, 592nd, 600th, 603rd and 616th amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 2 or SEQ ID NO: 10, wherein the genes are exclusive of the genes (d) and (e).

The gene (II) coding for measles virus mutant F protein antigen is at least one member selected from the group consisting of the following genes (g) and (h) identified with the positional amino acid numbers of either SEQ ID NO: 18 or SEQ ID NO: 20:

(g) a gene coding for the whole sequence of the 1st to 550th amino acids; and

(h) genes coding for fragmentary, contiguous sequences of at least 6 amino acids in either SEQ ID NO: 18 or SEQ ID NO: 20, wherein the sequences each comprise an amino acid selected from the group consisting of the 11th, 52nd, 107th, 165th, 398th, 417th and 523rd amino acids, and neighboring amino acids of the selected amino acid in either SEQ ID NO: 18 or SEQ ID NO: 20.

With respect to the gene coding for the measles virus mutant antigen of the present invention, there is no particular limitation as long as the gene codes for the whole protein or a fragmentary peptide of the measles virus mutant antigen. Therefore, the gene is not limited to the nucleotide sequence of the genomic RNA of CAM-70 strain or NA strain. As the measles virus mutant gene, use can be made of the cDNAs shown in SEQ ID NOs: 1, 9, 17 and 19, or the gene can be prepared by synthesizing a nucleotide sequence on the basis of an amino acid sequence of a measles virus mutant antigen.

The measles virus mutant gene of the present invention comprises at least one gene selected from the group consisting of the above-mentioned genes, and the gene can be chosen, based on the intended utility of the measles virus mutant gene. Like the measles virus mutant antigen of the present invention, the measles virus mutant gene of the present invention comprises both the genes of the attenuated strain and the genes of the epidemic strain. Based on the disclosure of the present invention, for example, a live vaccine effective for preventing the infection with the epidemic strains can be produced {see the below-mentioned item [II](1), and Examples 2 and 3}. When several genes are used in combination, they can also be used in such a form as ligated to each other {see the below-mentioned item [II](2) and Example 4}.

The antigens and genes coding for the same of the present invention, which respectively comprise the above-mentioned sequences, are effective as a marker for identifying a virulent strain or an attenuated strain, and are also important and advantageous for improving conventional vaccines and developing diagnostic reagents.

[II] Use of Measles Virus Mutant Antigen and Measles Virus Mutant Gene of the Present Invention as Vaccine and Diagnostic Reagent

(1) Preparation of an effective live vaccine for epidemic measles strains: A recombinant virus is prepared by replacing a gene of a live vaccine strain with a corresponding gene of an epidemic strain. With respect to the live vaccine strain, various strains mentioned under "Prior Art" of the specification can be used, but preferably, use is made of a strain which has been employed as an active component of a live vaccine in various countries for at least 10 years. That is, a strain having approved safety and effectiveness as an active component for a vaccine, such as CAM-70 strain, is preferred.

With respect to the epidemic strain used for preparing a live vaccine, the epidemic strain is selected so that when the selected strain is compared with a live vaccine strain, the epidemic strain has a marked, broad antigenic mutation due to the genetic mutation thereof. Specifically, a preferred epidemic strain is a recent epidemic strain which is being isolated at high frequency and is widely prevailing, and which has a universal antigenic mutation (that is, an antigenic mutation which is not peculiar to a particular strain), for example, MO strain or NA strain isolated by the inventors of the present invention in 1995 to 1996.

The recombinant virus can be produced by the method of Radecke et al. (EMBO Journal, Vol. 14, No. 23, pp. 5773-5784, 1995) which is a method for genetic recombination of a non-segmented negative-strand RNA viral (mononegaviral) genome, or by the modified method of Radecke et al., which has been developed by the inventors of the present invention.

The method of Radecke et al. (frequently referred to as "reverse genetics") will be explained below. First, the cells of 293 cell line (American Type Culture Collection, Accession No. ATCC CRL-1573) were transfected with a recombinant vector containing genes coding for T7 RNA polymerase and measles virus N protein and P protein, thereby obtaining transfectants (i.e., helper cells) capable of expressing T7 RNA polymerase, N protein and P protein. Next, an expression vector capable of expressing L protein (polymerase) of the measles virus under the control of T7 promoter is constructed (hereinafter, the constructed expression vector is simply referred to as "V1"). Further, a cDNA for the (+) sense RNA of the whole genome of CAM-70 strain is prepared, and a DNA fragment coding for a region in the H protein which contains the above-mentioned amino acid substitutions is cleaved and removed from the cDNA for CAM-70 strain by means of restriction enzymes. Then, the DNA sequence of the corresponding region of the viral genome of epidemic MO strain or NA strain is prepared therefrom and inserted into the restriction site of the cDNA for CAM-70 strain, to thereby obtain a recombinant cDNA. The obtained recombinant cDNA is inserted into plasmid pBluescript SK or KS (manufactured and sold by Stratagene Co., Ltd., England), thereby obtaining an expression vector (hereinafter, the obtained expression vector is simply referred to as "V0"), wherein the expression vector is prepared so that the recombinant cDNA is capable of transcription by T7 RNA polymerase. V0 and V1 are co-transfected to the helper cells prepared above, and the desired recombinant measles virus can be obtained by subsequently culturing the transfected cells. The proliferation of the recombinant virus in the transfected cells can be confirmed by detecting the occurrence of CPE (cytopathic effect), wherein the transfected cells generate syncytia, or by conducting a microscopic observation using a fluorescent antibody technique with a monoclonal antibody against the epitope of the protein encoded by the replaced gene.

The recombinant attenuated measles virus of CAM-70 strain, in which the gene coding for the 176th to 316th amino acids of the H protein of CAM-70 strain (SEQ ID NO: 4) is replaced by the gene coding for the 176th to 316th amino acids of the H protein of MO strain or NA strain (SEQ ID NO: 12), is obtained by using the above-mentioned method.

Further, the modified method of Radecke et al. is explained below. This modified method is such that the helper cells are not required, and any desired permissive cells can be used as host cells for the recombinant virus. With respect to the host cells employed, cells which are ensured to be safe as a culture host for the live vaccine strains and are approved as the host cells therefor, such as, MRC-5 cells and WI-38 cells are preferably used to prevent an introduction of an unidentified factor, a carcinogen and the like into the virus. First, the expression vectors for each of the genes coding for N, P, and L proteins of CAM-70 strain are individually prepared using plasmids pcDNA3.1(+) or pcDNA3.1(-) (manufactured and sold by Invitrogen Co., Ltd., Canada). For example, each of the genes encoding N, P and L proteins of CAM-70 strain is individually inserted into an appropriate restriction site of pcDNA3.1(-), thereby constructing the expression vectors. For the expression of T7 RNA polymerase, recombinant MVA (hereinafter, simply referred to as "recMVA"; FEBS Letter, vol. 371, no. 1, pp. 9-12, 1995) can be used. The above-prepared three expression vectors and the expression vector V0 mentioned in connection with the method of Radecke et al. are co-transfected to either the MRC-5 cells or WI-38 cells which have already been transfected with recMVA. The desired recombinant attenuated measles virus is obtained by culturing the transfected cells at about 35o to 38 oC. The proliferation of the virus can be confirmed by detecting the occurrence of CPE or by conducting the microscopic observation using the fluorescent monoclonal antibody technique mentioned above. Further, the antigenicity and immunogenicity of the obtained recombinant virus can be qualified in accordance with the antigen analysis mentioned in item [I](1) above.

(2) Preparation of an active component of a gene vaccine: The non-proliferating recombinant adenovirus can be prepared by inserting a gene of an epidemic measles virus into a non-proliferating adenoviral genome. The prepared recombinant adenovirus is effective as an active component of a gene vaccine. For preparing the recombinant virus, COS-TPC method developed by Saito et al. [Cell Technology (Saibo Kogaku), vol. 13, no. 8, pp. 757-763, 1994] can be employed. In this method, DNA-TPC (viral DNA-Terminal Protein Complex) of the genome of human adenovirus 5, and a cassette cosmid carrying almost all of the whole genome of the non-proliferating adenovirus (cassette cosmid pAdex1; U.S. Pat. No. 5,700,470) are used. The non-proliferating adenovirus is derived from human adenovirus 5 and it lacks E1A and E1B genes which are essential for viral proliferation, and therefore, this virus is incapable of proliferation in cells other than the 293 cells which constantly express E1A and E1B genes. Further, this virus lacks gene coding for E3 protein, a protein which antagonizes the recognition of viral antigens by CTL (cytotoxic T lymphocytes). Due to this contrived design of the adenovirus, cellular immunity induced by CTL is expected to develop even in the presence of this virus.

With respect to a measles virus gene used for preparing the recombinant virus, the gene can be selected from the genes coding for the antigens mentioned in item [I](3) above, and the genes can be used individually or in combination. However, for improving the immunogenicity which is necessary for providing a protection against the viral infection (that is, adsorption and penetration of a measles virus to a cell), it is preferred to use in combination the gene coding for the whole H protein mentioned in item (a) (SEQ ID NO: 2 or SEQ ID NO: 10) and the gene coding for the whole F protein mentioned in item (g) (SEQ ID NO: 18 or SEQ ID NO: 20).

Specifically, the cDNAs for the above mentioned H protein gene and F protein gene (for example, the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 19) are prepared [when the cDNAs are ligated, they are ligated in the order of H protein--F protein (HF) or F protein--H protein (FH) in the direction of from the 5' end to the 3' end], and the prepared cDNAs are inserted into the E1A.multidot.E1B deletion site of the cassette cosmid pAdex1, to thereby obtain recombinant cosmid pAdex1/HF or pAdex1/FH. On the other hand, DNA-TPC is extracted from the parent adenovirus strain, and the DNA-TPC is digested with the restriction enzyme Eco T22I (manufactured and sold by Takara Shuzo Co., Ltd., Japan), to thereby obtain digestion product DNA-TPC/Eco T22I. Subsequently, pAdex1/HF or pAdex1/FH, and DNA-TPC/Eco T22I are co-transfected to the 293 cells by calcium phosphate method. As a result of the co-transfection, homologous recombination between the transfected DNAs occurs in the cells, and a non-proliferating recombinant adenovirus containing measles virus H protein gene and F protein gene is obtained. The presence of measles virus H and F proteins in the non-proliferating recombinant adenovirus can be confirmed by testing Hela cells infected with the obtained adeno-virus by fluorescent antibody technique using the monoclonal antibodies against the measles proteins.

(3) Production of a measles vaccine: The live attenuated measles vaccine can be produced by using the recombinant attenuated measles virus mentioned in item [II](1) above as a seed virus. For example, the recombinant virus is proliferated in permissive cells, such as chicken embryo cells, thereby obtaining a virus suspension. The cells are removed from the obtained virus suspension by low-speed centrifugation, thereby obtaining a supernatant. Then, the obtained supernatant is subjected to filtration to thereby prepare a bulk vaccine solution. The prepared bulk vaccine solution is diluted with a medium, such as BME medium (Eagle's Basal Medium), so as to obtain a vaccine solution comprising the virus in a sufficient antigenic amount, for example, not less than 5,000 TCID50 (Median Tissue Culture Infective Dose) per 0.5 ml of vaccine solution. A stabilizer for stabilizing the virus can be added to the vaccine solution when the bulk vaccine solution is being diluted. Subsequently, the diluted vaccine solution is dispensed into suitable containers, such as 1 to 20 ml volume vials, and then, the containers are sealed hermetically, and the sealed vaccine is provided as a vaccine preparation. The vaccine preparation can be provided as a liquid preparation or as a lyophilized preparation obtained by lyophilizing the vaccine after dispensation. Prior to the use of the vaccine preparation, it is requisite to subject the vaccine preparation to various tests on effectiveness and safety to assure its quality as a vaccine. The tests are conducted in accordance with Pharmaceutical Affairs Law (the Law No. 145 established in 1960) and a provision entitled "Dried Attenuated Measles Virus Live Vaccine" in the Notification No. 217 of the Japanese Ministry of Health and Welfare: Seibutsugakuteki Seizai Kijun (Minimum Requirements for Biological Products) established in 1993. With respect to the manner of administration, for example, the vaccine preparation is administrated by subcutaneous injection in an amount of 0.25 to 0.5 ml per dose.

The non-proliferating recombinant virus mentioned in item [II](2) above can be produced in large yield using the 293 cells. The recombinant virus can be prepared from the liquid culture of 293 cells in substantially the same manner as mentioned above for preparing the vaccine preparation, so that the final virus content of a liquid or lyophilized preparation is 106 to 108 PFU (plaque-forming unit) per 1 ml of preparation. Such virus preparation can be provided as an active component for a gene vaccine. With respect to the manner of administration, the gene vaccine can be administered by subcutaneous, intramuscular or nasal injection in an amount of 0.25 to 0.5 ml per dose, and from the viewpoint of ease in injection procedure, nasal injection is especially preferred.

(4) Preparation of a diagnostic reagent: The antigens mentioned in item [I](3) above (whole protein or fragmentary peptide thereof) can be used individually or in combination as an antigen for diagnosis. When using several antigens in combination, the antigens containing different epitopes are preferably used to broaden the spectrum of reactivity with antibody. The antigen of the present invention can be provided as an antigen to be used in various diagnoses, such as diagnosis using precipitation reaction, agglutination reaction, neutralization reaction, fluorescent antibody technique, enzyme immunoassay, and radioimmunoassay. Further, the antigens can be inoculated intraperitoneally, subcutaneously or intramuscularly to an animal, such as rabbit, guinea pig and mouse, to prepare an immune serum, antibody or the like. The thus prepared antibody can be also provided as an antibody for detecting antigens in various diagnoses.

The antigen or antibody of the present invention is diluted so as to prepare a diagnostic reagent containing the antigen or antibody in an amount sufficient to cause an antigen-antibody reaction.

Further, the genes coding for the antigen mentioned in item [I](3) above can be used individually or in combination, for example, as a probe reagent for gene diagnosis and a reagent for identifying the measles virus strains. PCR primers can be designed, based on the amino acid sequences of the H protein and F protein of the attenuated strain and epidemic strain (for example, SEQ ID NOs: 2, 10, 18 and 20) and based the nucleotide sequences of the genes coding for the same (for example, SEQ ID NOs: 1, 9, 17 and 19) which are disclosed in the present specification. Such PCR primers can be provided as a reagent for diagnosis using the PCR method.

Claim 1 of 4 Claims

What is claimed is:

1. A measles virus mutant gene consisting of a gene coding for a measles virus mutant H protein antigen, wherein said gene coding for a measles virus mutant H protein antigen is at least one member selected from the group consisting of the following genes (a) to (c):

(a) a gene coding for an amino acid sequence of SEQ ID NO: 10;

(b) a gene coding for an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 11; and

(c) a gene coding for an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 12.


____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]