Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-08-15
2003-11-25
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S236000
Reexamination Certificate
active
06653069
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for quality control of an attenuated varicella live vaccine. More particularly, the present invention relates to a method for quality control of an attenuated varicella live vaccine, which comprises subjecting the genomic DNA of a sample varicella vaccine virus to sequence analysis and confirming that the genomic DNA of the sample varicella vaccine virus conserves the specific nucleotides without suffering mutation. By the use of the method of the present invention, it has become possible to determine very accurately the qualification of an attenuated varicella virus as an active ingredient of an attenuated varicella live vaccine and, consequently, to conduct an exact quality control of the vaccines.
2. Prior Art
As is well known, attenuated varicella live vaccines used today are produced from a seed strain of varicella virus which is derived from the attenuated varicella virus Oka strain (see Examined Japanese Patent Application Publication No. 53-41202 and U.S. Pat. No. 3,985,615), and the attenuated live vaccines are used widely throughout the world (Requirements for Varicella Vaccine (Live) Adopted 1984; Revised 1993: WHO Technical Report Series, No. 848, pp. 22-38, 1994). To ensure the safety and effectiveness of the vaccine, the number of passages of a virus used for producing the vaccine is restricted under the control of a seed lot system, taking into consideration the potential genetic mutation which is likely to occur during the passage. That is, the manufacturers are under an obligation to produce varicella vaccines only from the virus derived from the approved seed virus for the live varicella vaccine, wherein the number of passages of the virus is not more than 10 as counted from the approved seed virus which is counted as 0 passage. In other words, the quality control and quality assurance of the attenuated varicella live vaccine rely upon the fulfillment of the seed lot system by the manufacturers, and such a method for the quality control and quality assurance is not a method which can be traced and analyzed by a person skilled in the art.
Further, from the viewpoint of epidemiology which involves a tracing of the effects of the varicella vaccine and a post-market surveillance (PMS), the virological difference between the fresh wild-type strains isolated from the naturally infected varicella patients and the vaccine virus strains derived from the above-mentioned Oka strain needs to be determined, and various analyses, such as those utilizing immunological techniques and genetic engineering techniques, have been attempted for determination of the virological difference. For example, the following analyses have been reported: the difference in DNA sequence between the various VZV strains (Journal of virology, 59, 660-668, 1986; Journal of General virology, 67, 1759-1816, 1986), the difference in the absence or presence of a restriction enzyme
Pst
I cleavage site (Japanese Journal of Experimental Medicine, 59, 233-237, 1989), the difference in RFLP (restriction fragment length polymorphism) of the PCR (polymerase chain reaction) product (Journal of virology, 66, 1016-1020, 1992), and the difference in the absence or presence of a restriction enzyme
Pst
I restriction site which is taken in combination with the difference in RFLP of the PCR product (Journal of Clinical Microbiology, 33, 658-660, 1995). However, all of these analyses only propose criteria which can be used for differentiating a fresh wild-type strain from a vaccine strain derived from the Oka strain, and such analyses lack reliability and exactness. In addition, a method for identifying the attenuated varicella virus Oka strain by using gene 14 region (U.S. Pat. No. 6,093,535) and a method for identifying the attenuated varicella live vaccine virus by using gene 62 region (International Patent Application Publication No. WO 00/50603) have been known. Both of these methods enabled a determination of the differences among the varicella virus Oka strain (virulent parental strain), a vaccine strain derived therefrom (attenuated Oka strain) and a varicella virus strain other than the Oka strain, but neither of these methods was satisfactory as a standard for the quality control and quality assurance of the attenuated varicella live vaccine.
As mentioned above, at present, the quality of the attenuated varicella virus used as an active ingredient of an attenuated varicella live vaccine is controlled by the fulfillment of the seed lot system by the manufacturers. In other words, a method which can be traced and analyzed by a third party for evaluating and confirming the effectiveness of the vaccine, such as a method utilizing a direct and quantitative genetic analysis of the genomic DNA of a seed virus or a vaccine virus, has not been used for the quality control of the vaccine and, thus, the exactness of the quality control is incomputable and vague. Therefore, an improvement in the exactness of the quality control and quality assurance is critically important for assuring the effectiveness, safety and uniformity of the attenuated varicella live vaccine. However, as mentioned above, a reliable method for the quality control has not been established, and a development of such a method has been earnestly desired in the art.
SUMMARY OF THE INVENTION
In the above situation, the present inventors have made extensive and intensive studies with a view toward developing a novel method for accurately and quantitatively conducting the quality control of an attenuated varicella live vaccine. Specifically, the present inventors determined the whole genomic nucleotide sequence of the attenuated varicella virus Oka strain containing more than 120,000 nucleotides, conducted a comparative analysis between the determined nucleotide sequence of the attenuated Oka strain and the whole genomic nucleotide sequences of the virulent strain and the parental Oka strain (virulent strain), and identified the genetic mutations of the attenuated varicella virus Oka strain. As a result, they have found that, by evaluating and determining whether or not a varicella virus strain conserves the below-mentioned specific nucleotides, a virus strain conserving the specific nucleotides can be determined accurately as a virus strain capable of functioning as an attenuated varicella vaccine virus. The present invention has been completed, based on this novel finding.
Therefore, it is an object of the present invention to provide a novel method for the quality control of an attenuated varicella live vaccine.
It is another object of the present invention to provide an attenuated varicella live vaccine which is quality-controlled by the above-mentioned method.
It is a further object of the present invention to provide a vaccine strain capable of functioning as an attenuated varicella vaccine virus, which is identified by a method used in the above-mentioned method.
The foregoing and other objects, features and advantages of the present invention will be apparent to those skilled in the art from the following detailed description and the appended claims taken in connection with the accompanying sequence listing and drawings.
SEQUENCE LISTING FREE TEXT
SEQ ID NOs: 3 and 4 are of PCR primers used for detecting a mutation of the 560th nucleotide of a varicella vaccine virus.
SEQ ID NOs: 5 and 6 are of PCR primers used for detecting a mutation of the 5,745th nucleotide of a varicella vaccine virus.
SEQ ID NOs: 7 and 8 are of PCR primers used for detecting a mutation of the 26,125th nucleotide of a varicella vaccine virus.
SEQ ID NOs: 9 and 10 are of PCR primers used for detecting a mutation of the 94,167th nucleotide of a varicella vaccine virus.
SEQ ID NOs: 11 and 12 are of PCR primers used for detecting mutations of the 105,356th, 105,544th, 124,353rd and 124,541st nucleotides of a varicella vaccine virus.
SEQ ID NOs: 13 and 14 are of PCR primers used for detecting mutations of the 105,705th, 106,262nd, 123,635th and 124,192nd nucleotides of a varicell
Gomi Yasuyuki
Sunamachi Hiroki
Takahashi Michiaki
Yamanishi Koichi
Mosher Mary E.
The Research Foundation for Microbial Diseases of Osaka Universi
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