Preservation of viruses

Details Preservation of viruses Preservation of viruses

1989-11-20

1992-09-22

Robinson, Douglas W.

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Preserving or maintaining micro-organism

435235, 435948, C12N 700, C12N 104, C12N 100

Patent

active

051496534

DESCRIPTION:

BRIEF SUMMARY
This invention relates to the preservation of live viruses and in particular to the drying of live viruses in a stable form from which they can be reconstituted while retaining their immunogenic or other useful activity.
Live viruses have a number of important uses. The most important is the use of viruses, either intact or attenuated, as immunogenic vaccines. Two good examples are polio virus and measles virus. However, live virus vaccines are extremely difficult to maintain under storage. They cannot, at the moment, be dried and reconstituted without losing their immunogenic effect. Similarly, they cannot be frozen, for the same reason. Consequently, virus vaccines are required to be kept in aqueous media under cool sterile conditions, for example in refrigerators. One of the diseases mentioned above, measles, is of great epidemiological importance, especially in the third world. Each year in Africa alone millions of children die of measles. This is largely because the vaccines necessary for prevention cannot be distributed in the majority of countries concerned which do not have the infrastructure or wealth to provide point-of-use refrigeration.
There is thus a desperate need worldwide for a simple means of preserving viruses in an intact immunogenic form which can be kept without refrigeration or other elaborate control, and which can be reconstituted simply with water just before use.
Other viruses have considerable importance in other fields. For example, Epstein Barr virus (EBV) is of considerable importance in the preparation of human B-cell lines to produce monoclonal antibodies and for studies on human molecular genetics. EBV at present has to be stored in aqueous media under refrigeration.
Similarly, bacteriophages such as phage Lambda derived from E. coli, are of importance in manipulation of DNA and the production of gene libraries in the so-called genetic engineering. Again, this virus must be kept under aqueous conditions under refrigeration.
While it is well-known that drying of unstable biological products, either at room temperature or in the frozen state (lyophilisation) can be aided by adding stabilizing gents to the product in question, there has been no report to our knowledge of the successful stabilization of live viruses.
Thus, for example, UK patent application 2009198A describes the stabilization of meningococcal polysaccharides under lyophilisation, by combining them with various sugars including sucrose, raffinose, glucose and trehalose. In this case, of course, the antigenic component is not a live virus. Similarly. UK Patent application 2126588A describes the stabilization of Tumor Necrosis Factor in the presence of certain sugars and sugar acids.
In European Patent application 140489A1, rubella virus antigen is stabilized by immersion in certain sugars. Again, however, no possibility of stabilizing the virus itself is mentioned.
Our own earlier British patent application 2187191A (WO87/00196) and the corresponding U.S. Pat. No. 4,891,319 describes and claims the preservation of various proteins and other macromolecules at ambient temperature, by drying in the presence of trehalose. Dead virus vaccines are mentioned, but there is no indication that the immunogenic and other functions of live viruses can be preserved in this way.
We have now found that the presence of trehalose in the viral medium during drying either frozen or at ambient temperature, enables live viruses to be preserved and subsequently reconstituted while retaining substantially all their immunogenic properties or other useful properties, including viability. Thus, for the first time, the possibility is provided of having stable dry formulations of vaccines such as polio and influenza viruses.
According to the present invention there is provided a method of preserving live viruses comprising subjecting an aqueous system containing the virus to drying either in the frozen state or at ambient temperature, in the presence of trehalose.
In general, the more trehalose added the better, although in practice benefi

REFERENCES:
patent: 3674864 (1972-07-01), Angelucci
patent: 4380582 (1983-04-01), Orlando et al.
patent: 4891319 (1990-01-01), Roser
patent: 5026566 (1991-06-01), Roser

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