United States Patent:  6,039,958

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

Filed:  October 28, 1998

A stabilized live vaccine containing a varicella virus and a stabilizer, wherein the vaccine is substantially free of Ca²⁺ ions and Mg²⁺ ions is described. This stabilized live vaccine is excellent in storage stability and heat resistance. Also described is an improved stabilizer for a live varicella vaccine, comprising at least gelatin or hydrolyzed gelatin, each being substantially free of Ca²⁺ ions and Mg²⁺ ions. The stabilizer can advantageously be used to stabilize a live vaccine containing a varicella virus. The substantial freedom of Ca²⁺ ions and Mg²⁺ ions can be attained by masking Ca²⁺ ions and Mg²⁺ ions present in a live vaccine containing a varicella virus and a stabilizer, with a chelating reagent, or by using as a stabilizer gelatin and/or a gelatin derivative after being purified to remove Ca²⁺ ions and/or Mg²⁺ ions contained therein.

SUMMARY OF THE INVENTION

In these situations, the present inventors have made extensive and intensive studies to solve the above-mentioned difficult problems in order to provide a stabilized live vaccine comprising, as a virus component, an attenuated live varicella virus. As a result, they have unexpectedly found that when a chelating reagent, such as ethylenediaminetetraacetic acid (EDTA), is added to a live varicella vaccine which has conventionally been poor in stability irrespective of the presence or absence of gelatin and/or a gelatin derivative such as hydrolyzed gelatin (which have been conventionally regarded as being useful for stabilizing other types of live virus vaccines), the stability of the varicella virus-containing vaccine is greatly improved. The present inventors have further studied the reason why the improved stability can be attained by the addition of EDTA. As a result, they have unexpectedly found that the stability of a live varicella virus vaccine depends upon the presence or absence of Ca²⁺ ions and Mg²⁺ ions and that the essential requirement for the stabilization of a live varicella virus vaccine is to render the varicella vaccine substantially free of Ca²⁺ and Mg²⁺. This is very surprising in view of the fact that the stability of various live vaccines, e.g., poliovirus vaccine, is rather improved by the presence of Ca²⁺ ions and Mg²⁺ ions.

In many cases, Ca²⁺ ions and Mg²⁺ ions are introduced when a cell culture is prepared for the multiplication of a virus. Also it is known that gelatin, which is widely used in recent years as a representative stabilizer component for various vaccines, contains about 0.1% or less of Ca²⁺ ions and that commercially available hydrolyzed gelatin contains about 0.1% Ca²⁺ ions and about 0.01% Mg²⁺ ions.

The exact mechanism in which such Ca²⁺ ions and Mg²⁺ ions impair the stability of a live vaccine containing varicella virus has not yet been elucidated. It is presumed, however, that the inactivation of live varicella virus by heat would disadvantageously be promoted by the action of these specific ions, thus lowering the infectivity titer of the vaccine and that the coagulation of the varicella virus particles would be induced by the action of these specific ions, thus causing the vaccine to become unstable.

EDTA has not been considered to be a favourable additive for use in a vaccine because EDTA has stinging properties. However, during the study by the present inventors, it has been unexpectedly found that by the addition of EDTA to a live vaccine containing a varicella virus and a stabilizer, the stability of the vaccine can be greatly enhanced. The reason for this is believed to reside in that EDTA acts as a chelating reagent on Ca²⁺ ions and Mg²⁺ ions derived from cultured cells and a stabilizer, to thereby mask the ions.

Based on the above-mentioned novel findings, the present invention has been completed.

It is, therefore, an object of the present invention to provide a live vaccine containing an attenuated varicella virus and a stabilizer, which has high stability.

It is another object of the present invention to provide a stabilized live vaccine having excellent stability, which comprises attenuated live varicella virus and, as a stabilizer, at least one member selected from the group consisting of gelatin and a gelatin derivative such as hydrolyzed gelatin, the stabilizers having been considered to be extremely effective for the stabilization of live vaccines other than a live varicella virus vaccine, but considered to have no effect for the stabilization of a live varicella vaccine but rather have an adverse effect therefor.

It is a further object of the present invention to provide a stabilizer for a live vaccine comprising a varicella virus, which comprises at least one member selected from the group consisting of a treated gelatin and a treated gelatin derivative which are respectively obtained by subjecting commercially available gelatin and a commercially available gelatin derivative such as hydrolyzed gelatin (the gelatin and gelatin derivative having not been actually utilized as a stabilizer for a live vaccine containing varicella virus), to a treatment for masking or removing Ca²⁺ ions and Mg²⁺ ions contained therein.

Claim 1 of 10 Claims

1. A stabilized live vaccine comprising a virus component comprising: (i) at least one varicella virus selected from the group consisting of an attenuated live varicella virus and an attenuated recombinant varicella virus and (ii) a stabilizer comprising

(a) from 0.5 to 10%(w/v) of sucrose or a mixture of from 0.5 to 10%(w/v) of lactose, from 0.2to 6.0%(w/v) of sorbitol and from 0.02to 1.0%(w/v) of cysteine;

(b) from 0.01 to 5.0%(w/v) of sodium glutamate;

(c) from 0.02 to 1.0%(w/v) of gelatin;

(d) from 0.5 to 10%(w/v) of hydrolyzed gelatin; and

(e) from 0.001 to 0.1%(w/v) of a chelating reagent; in terms of the concentration in the stabilized live vaccine; so that said vaccine is substantially free of Ca²⁺ ions and Mg²⁺ ions to the extent that Ca²⁺ ions and Mg²⁺ ions are substantially not detected by a colorimetric titration method using a chelating reagent.

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