MXPA98003198A - Methods to preserve microorganis - Google Patents

Abstract

Methods are provided to conserve microorganisms including viruses, so that the infectiousness is retained, as well as the use of such to prepare, for example, vaccination.

Description

METHODS TO PRESERVE MICROORGANISMS DESCRIPTION OF THE INVENTION The present invention relates to methods for preserving microorganisms, so that they retain their infectious capacity. In particular, the invention relates to methods for conserving viral particles. Storage / viability problems occur in relation to the storage of microorganisms. In particular, particular problems occur in relation to viral storage, where the virus particles are employed for uses such as: (a) viral vectors for use in, for example, gene therapy; (b) virus storage for general research progress, for example, in culture banks; (c) viruses to be used to be released into the environment for the control of agricultural pests; and (d) vaccines. Vaccines that comprise particles have been used for a number of years. However, it is essential that these vaccines can be stored, sometimes for long periods, without the viral com- ponent losing its capacity for infection. Common storage methods include freezing or freeze drying, the latter usually involving reconstitution using water in the last stage. Unfortunately, certain viruses exhibit reduced viability / infection capacity when subjected to these procedures. A virus, which is not properly stored, as described above, is the polio virus. This virus is easily degraded at room temperature in aqueous suspension, is stable only for 2 weeks at 0 ° C and is destroyed by lyophilization. For this particular virus, preferred storage methods involve freezing at -70 ° C or refrigeration at 4 ° C. However, such storage conditions are not particularly suitable for use in tropical countries or indeed in countries where the use of facilities and equipment required are scarce. The international application N o. PCT / G B94 / 02495 describes compositions comprising a hydrophilic species solubilized in a hydrophobic phase, as well as methods for their preparation. U.S. Application No. 9424901 .8 describes compositions as described in PCT / GB94 / 02495, which incorporate additional components, which aid in the retention of hydrophilic species in the hydrophobic phase. The request of the United Kingdom No. 9424902.6 disclose compositions as described in PCT / GB94 / 02495, which incorporate portions that assist in the formation of the composition. In addition, the U K patent application no. 9422990.3 describes immunogenic compositions, which comprise a solubilized, suspended or otherwise dispersed immunogen in a hydrophobic phase. The immunogen can be a virus and the compositions are useful as vaccines. It has now been found that microorganisms, particularly virus particles, such as polio virus particles, can be converted to a suitable form during long-term storage at room temperature, with retention of the infectivity capacity after reconstitution in an aqueous medium. In this way, said compositions have particular advantages for use in countries where the usual storage methods are less suitable, and provide effective means, through which said viruses can be transported and stored without the need for extreme freezing or prolonged refrigeration. . Thus, in a first aspect, the present invention provides a method for storing microorganisms, so as to maintain their infectious capacity, said method includes the steps of: (i) carrying the microorganisms in association with an amphiphile; and (ii) solve, suspend or otherwise disperse microorganisms in a hydrophobic phase. In a preferred embodiment, the microorganisms are virus particles, particularly particles of the polio virus.

Suitable methods for carrying out the above method are those described in PCT / G B94 / 02495, U K 9424901 .8, U K 9424902.6 and UK 9422990.3. The hydrophobic solvent can be, for example, a long chain fatty acid, a medium chain alcohol, a branched chain alcohol, a monoglyceride, a diglyceride, a medium chain triglyceride, a long chain triglyceride, a halogenated analogue ( for example, fluorinated) thereof, or a lipid containing polyoxyethylene. In particular embodiments, the hydrophobic solvent is a mono-, di- or triglyceride or oleic acid. In a preferred embodiment, the method comprises: (i) co-dispersing the microorganisms with an amphiphile in a liquid medium; (ii) removing the liquid medium to leave a disposition of amphiphilic molecules with their hydrophilic upper groups oriented towards the microorganism; and (iii) providing a non-aqueous solvent around the microorganism / amphiphile arrangement. The liquid medium can be water, and this can be removed through, for example, drying by freezing, drying by centrifugal vacuum or any other suitable method. Suitably, in the above methods, the amphiphile will be a phospholipid, for example, or not with a higher phosphatidylcholine group, for example phosphatidylcholine (PC), lysophosphatidylcholine (lyso-PC), sphingomyelin or a derivative of one of these, such as hexadecylcholine or an amphiphilic polymer containing phosphorylcholine. A bile salt, a glycolipid, a surfactant containing polyoxyethylene, a lipophilic sulfate, betaine, a surfactant agent containing sarcosine, Solulan C24, polyoxyethylene stearate 40, one of the Tween series of surfactants, one of the Span series of surfactants or a pegylated castor oil derivative, for example, Cremaphor EL35. Without wishing to be bound by the following, it is believed that in the methods described above, the microorganisms, e.g., virus particles, first form an arrangement with the amphiphilic molecules. This arrangement is in turn coated with the hydrophobic solvent. In this way, access to microorganisms by water is restricted, which in turn represent the improved storage properties when the preparation of the microorganism is reconstituted from the freeze-dried state. In a second aspect, the present invention provides a method for storing microorganisms, so as to retain their infectious capacity, said method includes the following steps: (i) bringing the microorganisms into association with an amphiphile in an aqueous phase; and (ii) remove the water. Preferably, the water is removed through freeze drying.

The amphiphile can be a phospholipid, for example, one with a higher phosphatidylcholine group, for example, phosphatidylcholine (PC), lysophosphatidylcholine (lyso-PC), sphingomyelin or a derivative of one of these, such as hexadecylphosphocholine or an amphiphilic polymer containing phosphatidylcholine. A bile salt, a glycolipid, a surfactant containing polyoxyethylene, a lipophilic sulfate, betaine, a surfactant agent containing sarcosine, Solulan C24, polyoxyethylene stearate 40, one of the Tween series of surfactants, one of the Span series of surfactants or a pegylated castor oil derivative, for example, Cremaphor EL35.

In a particularly preferred embodiment of this aspect, the amphiphile is Solulan C24, polyoxyethylene stearate 40, one of the Tween series of surfactants, one of the Span series of surfactants or a pegylated castor oil derivative, for example , Cremaphor EL35. In particularly preferred embodiments, the amphiphile is Solulan C24 or polyoxyethylene 40 stearate. It is possible that after removal of the water, the amphiphile / microorganism arrangement will be in an "open" form. In this way, after reconstitution, water can continue to have access to microorganisms and this will lead to the loss of infection capacity. Therefore, in another preferred embodiment of this aspect of the invention, the method also includes the step of raising the temperature of the mixture after the removal of the water. This ensures that the structure adopted by the amphiphile / microorganism arrangement is more condensed, which in turn results in more restricted access for water after reconstitution. When the heating step is employed, the amphiphile will be the one that remains solid after the water removal step, for example, it can be chosen from a phospholipid, for example, lecithin, a glycolipid, a surfactant containing polyoxyethylene, a sulfate lipophilic, betaine, a surfactant agent containing sarcosine, Solulan C24, polyoxyethylene stearate 40, one of the Tween series of surfactants, one of the Span series of surfactants or a pegylated castor oil derivative, for example, Cremaphor EL35 . In other aspects, the invention provides: i) a microorganism composition obtainable through any of the methods described herein, particularly a microorganism composition comprising virus particles, e.g., poliovirus particles; and ii) the use of a composition of the invention for the storage of virus particles. The preferred aspects of each aspect of the invention are to each other an aspect of mutatis mutandis. The invention will now be described with reference to the following example, which is not constructed as limiting the invention.EXAMPLE 1 A suspension of 109 particles of polio virus (Sabin strains, Types 1, 2, 3) per ml was diluted 1000 times in distilled water. 1 ml of the diluted suspension was mixed with 1 ml of a soy phospholipid dispersion with sound application (at a concentration of 100 mg / ml) in distilled water. A control bottle was prepared, which contained only the virus, without the addition of the phospholipid. The contents of both bottles were frozen as protection in liquid nitrogen and lyophilized overnight. The next day, 1 ml of oleic acid was added to the flask containing the virus and phospholipid, and the contents of the flask were then mixed in a roller mixer for several hours. A clear solution was obtained. A control bottle of polio virus was prepared, as was done previously. To this control bottle, containing only the virus, 1 ml of the culture medium was added. 10 μl of the oil / virus preparation was transferred to a fresh container, and 1 ml of a 2% solution of beef bile extract (containing predominantly sodium taurocholate) was added. The mixture was stirred well to disperse the oil in water, with the intention of releasing the particles into the aqueous phase. Serial dilutions were made 0 times in the culture medium, and 0.5 ml of each dilution was added to the monolayers of the Viro cell confluent, and incubated for four days, to test for the presence of intact virus. An identical procedure was followed for the contents of the control bottle. Growth was determined through visual observation of cell lysis induced by virus in each monolayer. Growth was recorded in the two series of dilutions as follows: Dilution of lyophilizate 10z 103 104 10 * 1 0 * Virus particles present 104 103 10z 10 (per ml) Oil-based lyophilizate + + free oil lyophilizate + + These results indicate that the method of the present invention clearly improves the viability of stored viral preparations, when compared only with lyophilization.

EXAMPLE 2 A virus suspension (Sabin strains, Types 1, 2, 3) containing 5 x 108 particles / ml (rotation to remove protein from contamination) was diluted 50-fold through the addition of 200 μl of the suspension to 9.9 ml of distilled water, producing a concentration of 107 particles / ml. The suspension was divided into four equal aliquots of 2.5 ml, and supplied to glass jars capped with a 7 ml screw. An aliquot was used in the experiment described above, while two were used in the experiment described in Example 3. 2.5 ml of the phospholipid dispersion with sound application (1000 mg / ml) was added to the aliquot of the particles. viruses diluted with moderate mixing. 200 μl of this mixture was filled into 20 flasks dried by freezing, and the rest was transferred, in 100 μl aliquots, into other tubes as "pre-drying" controls. The controls were stored overnight at + 4 ° C. The bottles were placed in the centrifugal rotor of the freeze dryer and lyophilized overnight. The next day, 1 μl of the culture medium was added to the contents of ten of the bottles dried by freezing, overnight, while 1 00 μl of oleic acid (B. P.) was added to the other ten. The groups were marked as "M" and "O", respectively. 1 μl of the samples of the tubes marked with the letter "M" were transferred to fresh 1 ml containers, and 1 ml of the 0. 1 M bicarbonate solution containing 25 mg / ml of sodium taurocholate were added and They mixed well. Under these conditions, the oil dispersed well to give a clear solution. 4 x 20 μl were transferred to the samples of the pre-dried control group sample stored overnight at + 4 ° C in fresh 1 ml containers. Two of these containers were added with 1 ml of medium, while the other two were added with 1 ml of a 0.1 M solution of bicarbonate, containing 25 mg / ml of sodium tau lactic acid. The contents of the jars mixed very well. The suspensions previously prepared were used to perform 10-fold dilutions in Vero cell monolayer cultures, in order to measure the viability of the polio virus present. The results were expressed as the highest dilution, at which cytopathic effects were observed in 50%.

Nature of the Highest Dilution to the It shows that 50% of CPE was observed Control not drying in medium 10"4/10" 5 Control not dried in taurocholate 10"3/10" 3 Free oil lyophilate in the medium 10"1/10 ° Free Liofilato oil in taurocholate 10"1/10" 1 Oil-based lyophilate in taurocholate 10"6/10" 6 EXAMPLE 3 2.5 ml of distilled water was added to an aliquot of virus particles prepared as described in Example 2, and this group was marked with the letter "W." 2.5 ml of Solulan were added.

C24 (100 mg / ml) to another aliquot and mixed moderately. This group was marked with the letter "S". 200 μl of each preparation were filled in 10 bottles dried by freezing, and the rest in aliquots of 100 μl in other tubes as "pre-drying" controls. The controls were stored overnight at + 4 ° C. The freeze-dried bottles were placed in the centrifugal rotor of the freeze dryer and freeze-dried overnight. The next day 100 μl of the culture medium was added to each bottle in the "W" group and mixed modernly. The flasks in the "S" group were sealed and heated to 60 ° C in a hot water bath for 5 seconds to melt the Solulan C24, which resulted in a clear solution. After cooling to room temperature, this material solidified. 90 μl was added to the bottles of the "S" group to make a total volume of up to 100 μl. Then 10 μl of the sample was transferred from each of the "S" and "W" groups to fresh 1 ml containers and 1 ml of the medium was added to each and mixed well. To fresh 1 ml containers were added 4 x 20 μl of samples from each of the pre-dried groups and 1 ml of the medium was added to each. The contents of each bottle were mixed well.

The suspensions previously prepared were used to carry out 10-fold dilutions in Vero cell cultures to measure the viability of the poliovirus present. The results were expressed as the highest dilution, at which cytopathic effects were observed at 50%.

Nature of the Highest Dilution to the Shows that 50% of CPE Control was observed without drying + water 10/10"Control without drying + Solulan C24 10" 5/10"Control dried by freezing + water 10" 2/10" Freeze-dried control + Solulan C24 10"6/10" 8

Claims (9)

1. - A method for storing microorganisms, in a manner that retains the ability to infect, the method includes the steps of: (i) carrying the microorganisms in association with an amphiphile; and (ii) solubilizing, suspending or otherwise dispersing the microorganisms in a hydrophobic phase.

2. A method according to claim 1, wherein the microorganisms are virus particles.

3. A method according to claim 2, wherein the virus particles are particles of poliovirus.

4. A method according to any of claims 1 to 3, wherein the solvent is a long chain fatty acid, a medium chain alcohol, a branched long chain alcohol, a monoglyceride, a diglyceride, a triglyceride of medium chain, a long chain triglyceride, a halogenated analogue thereof (eg, fluorinated), or a lipid containing polyoxyethylene.

5. A method according to claim 4, wherein the hydrophobic solvent is mono-, di- or tri-glyceride.

6. A method according to claim 4, wherein the hydrophobic solvent is oleic acid.

7. A method according to any of claims 1 to 6, which comprises: (i) associating the microorganisms with an amphiphile in a liquid medium; (ii) removing the liquid medium to leave an arrangement of amphiphilic molecules with their hydrophilic upper groups oriented towards the microorganism; and (iii) providing a non-aqueous solvent around the microorganism / amphiphile arrangement.

8. A method according to any of claims 1 to 7, wherein the amphiphile is a phospholipid, a bile salt, a glycol lipid, a surfactant containing polyoxyethylene, a lipophilic sulfate, betaine, a surfactant containing sarcosine, Solulan C24, polyoxyethylene stearate 40, one of the Tween series of surfactants, one of the Span series of surfactants or a pegylated castor oil derivative, for example, Cremaphor EL35.

9. A method for storing microorganisms, so as to retain the capacity for infection, said method includes the following steps: (i) bringing the microorganisms into association with an amphiphile in an aqueous phase; and (ii) remove the water. 1 - A method according to claim 10, wherein the water is removed by freeze drying. 1 - A method according to claim 10, wherein the mixture of amphiphile and microorganisms is converted to a condensed form by raising the temperature of the mixture after the removal of the water. 13. A method according to any of claims 10 to 12, wherein the microorganisms are virus particles. 14. A method according to claim 1, wherein the virus particles are particles of poliovirus. 15. A method according to any of claims 10 to 14, wherein the amphiphile is a phospholipid, a glycolipid, a surfactant containing polyoxyethylene, a lipophilic sulfate, betaine, a surfactant containing sarcosine, Solulan C24, polyoxyethylene stearate 40, one of the Tween series of surfactants, one of the Span series of surfactants or a pegylated castor oil derivative, for example, Cremaphor EL35. 16. A method according to claim 1, wherein the amphiphile is Solulan C24, polyoxyethylene stearate 40, one of the Tween series of surfactants, one of the Span series of surfactants or an oil derivative of pegolado castor, for example, Cremaphor EL35. 17. A method according to claim 16, wherein the amphiphile is polyoxyethylene stearate 40. 18. - A method according to claim 16, wherein the amphiphile is Solulan C24. 9. A microorganism composition obtainable through the method defined in any of claims 1 to 18. 20. A microorganism composition according to claim 19, which comprises virus particles. 21. A microorganism composition according to claim 20, comprising virus particles. 22. The use of a composition according to claim 20 or claim 21 for the storage of virus particles. 23. The use of a composition according to any of claims 19 to 21 to induce an im response in a subject. 24. The use of a composition according to any of claims 19 to 21 to prepare an agent capable of inducing an im response in a subject.