WO2011154119A1

WO2011154119A1 - Preparation of a stabilized dry oral vaccine composed of a live attenuated virus

Info

Publication number: WO2011154119A1
Application number: PCT/EP2011/002760
Authority: WO
Inventors: Pierre Chouvenc; Alain Françon; Catherine Noel
Original assignee: Sanofi Pasteur SA
Current assignee: Sanofi Pasteur SA
Priority date: 2010-06-07
Filing date: 2011-06-06
Publication date: 2011-12-15
Anticipated expiration: 2012-12-07
Also published as: FR2960781B1; FR2960781A1

Abstract

The subject of the present invention is a method for preparing a dry oral vaccine, composed of a live attenuated virus, the stability of which has been improved compared with what has previously been obtained. Essentially, an aqueous composition containing not only the virus, but also a ballast agent, a thickener and a stabilizer, which are advantageously present at a concentration such that the dry weight of the composition is high, is dried. The drying can advantageously be continued so as to reach a residual moisture content of about 5% to 10%. Thus, the product which is obtained is a vaccine that can be administered orally without prior rehydration.

Description

Preparation of a Stabilized Dry Oral Vaccine Composed of a Live Attenuated Virus

The present invention relates to a method for preparing a dry oral vaccine, composed of a live attenuated virus of which the stability of the infectious titer over time is improved. According to one particular mode, a dry oral vaccine that can be used without prior rehydration is obtained.

Vaccines composed of live attenuated viruses are for the most part fragile and are generally provided in freeze-dried form, despite the drop in the infectious titer caused by the freeze-drying, which is particularly large in the case of the poliovirus. Various attempts to reduce this drop are reported in the prior art.

Application WO 89/06542, for example, teaches that the loss of infectious titer of a poliovirus type 3, dried at 37°C and stored for 7 days at this temperature, can be brought back from 6 to 3.7 logs by simply drying the stock solution of virus at 37°C in the presence of a stabilizing solution made up of 10% trehalose as sole protective agent. However, the drop in infectious titer remains considerable, and greater than that observed for the non-dried vaccine. This shows the difficulty in drying this virus. In addition, the dry virus thus prepared is not ready-to-use; it has to be rehydrated before it can be used.

An initiative was launched by the WHO between 1990 and 1995 (Children's vaccine initiative product development group on thermostable Oral Poliovaccine), in order to encourage the development of a liquid or freeze-dried, thermostable oral polio vaccine (OPV). This action was carried out in the context of the poliomyelitis eradication program, the instability of the OPV vaccine limiting the success of vaccination with this vaccine.

The only advantageous stabilization results obtained during this period were obtained by R. Crainic using deuterated water (D₂O) at a minimum concentration of 90%, in liquid form (Crainic R., Wu R., Otelea D. et al. The replacement of water with deuterium oxide significantly improves the thermal stability of the oral poliovirus. In Brown F, editor, New Approaches to Stabilization of Vaccines Potency Developments in Biologicals Standardization, 1996; vol. 87: 161-6; and patent application WO 94/21298: Stabilized pharmaceutical compositions and methods for preparing same). Nevertheless, the method of preparation using deuterated water is difficult to implement for administrative and safety reasons related to the use of deuterated water (heavy water).

Moreover, patent DD-A-299 213 A7 relates to a method for stabilizing a dry vaccine composed of the live measles virus. This method comprises a step of freezing this virus at -20° in a composition which must comprise arginine, dextran, trehalose or sorbitol and a phosphate buffer, followed by a step of desiccation for 48 hours at 22°C. According to this document, the loss of infectious titer of the virus is 0.81 log, but the method described in this document does not result in a dry vaccine in a form that can be administered orally and comprises a desiccation step, the relatively long duration of which has proved problematic for industrial exploitation. In addition, no mention is made of stability during a long storage period.

The stabilization of live attenuated vaccines remains a problem and laboratories continue to work on this subject. H. Shiomi et al. (Jpn. J. Infect. Dis., 56, 70 - 72, 2003) have obtained advantageous results regarding an OPV vaccine, just after drying by freeze-drying, the loss of infectious titer for serotypes 1 to 3 was respectively 0.52 log; 0.68 log; and 1.29 log. After drying for 7 days at 37°C, the loss of infectious titer for serotypes 1 to 3 was respectively 0.48 log; 0.57 log; and 0.38 log. These results, which represent a real improvement compared with the prior art, were obtained under conditions which require, however, rehydration before use.

US 2006/0127414 proposes a method of stabilizing poliovirus (IPV - inactivated polio vaccine) by drying, which makes it possible to obtain a highly viscous vaccine which requires rehydration. This method was not, however, applied to the oral vaccine (OPV).

The reemergence of cases of poliomyelitis in certain African countries in 2009, where the disease had been eradicated, shows the need for a stable vaccine that is easy to use (OPV or IPV).

It has now been found that a method of preparation which calls on a drying operation performed using a particular liquid composition makes it possible to significantly reduce the loss of infectious titer, not only after drying, but also after time spent at high temperatures. Furthermore, the use of this composition, combined with particular drying conditions, makes it possible to obtain a vaccine which can be administered by being taken directly, i.e. which can be used without prior rehydration.

Consequently, the subject of the present invention is a method for preparing a dry oral vaccine, comprising a live attenuated virus, by drying (desiccating) an aqueous composition comprising:

(i) said virus;

(ii) a ballast agent (filler), selected from the group made up of monosaccharides, oligosaccharides, polysaccharides and the sugar alcohols which are derived therefrom, and also mixtures thereof; (iii) a thickener; and

(iv) a stabilizer comprising a surfactant, amino acids and urea, in a buffered liquid medium.

The drying operation can be carried out according to conventional techniques. Freeze-drying and vacuum-drying are in particular mentioned.

According to one particularly advantageous embodiment, the drying is not continued beyond the time required to obtain a product which has a residual moisture content of between 5% and 10% (weight/weight), limits included. It is indicated that this residual moisture content can be conveniently determined according to the Karl Fischer method. To achieve the objective explained above by carrying out the procedure according to a conventional technique, the drying operation, for example the freeze-drying, is carried out in a few hours, advantageously in less than 24 hours, preferably in less than 12 hours, particularly preferably in less than 8 hours, which, from the industrial point of view, is of great advantage.

Still according to a particularly advantageous embodiment, the dry weight of the aqueous composition used in the method according to the invention and which is subjected to the drying operation is between 70% and 85% (weight/weight) limits included, of the total weight of the aqueous composition. The four components (i) to (iv) contribute thereto in various proportions, the greatest contribution thereto coming from the ballast agent and the minute contribution thereto coming from the virus.

Thus, the ballast agent (filler) is present in the aqueous composition intended to be dried, at a concentration between 80% and 00% (limits included) of the maximum solubility of the ballast agent in an aqueous medium between 25 and 35°C.

The thickener is present in the aqueous composition intended to be dried, at a concentration of between 0.08 and 0.12 g/100 ml, limits included.

The stabilizer is present in the aqueous composition intended to be dried, at a concentration of between 40% and 80%, advantageously 50% and 70%, preferably 55% and 60% (vol./vol.), limits included.

The method according to the invention can be carried out in order to obtain any dry oral vaccine containing a live attenuated virus as antigen. It is perfectly suitable for the preparation of vaccines composed of heat-sensitive viruses. The term "dry vaccine" is intended to mean a vaccine of solid, non-liquid consistency. The term "oral vaccine" means a vaccine intended to be administered orally. The virus may be any virus against which it is desirable to obtain a live attenuated vaccine intended to be administered orally. The poliovirus and the rotavirus are viruses against which a dry oral vaccine is entirely suitable. These viruses each exhibit strain variations such that a sub-classification applies. Thus, the poliovirus is at the current time divided up into three serotypes: 1 , 2 and 3; and the rotavirus is divided up into an even higher number of serotypes. For the rotavirus, serotypes G1 , G2, G3, G4, G8 and G9 are, for example, mentioned. The method according to the invention applies to all the serotypes of these viruses.

For the purpose of the present invention, the ballast agent may, for example, be dextran, maltodextran, glucose, xylose, galactose, arabinose, fructose, D-mannose, sorbose, maltose, sucrose, sucrose, lactose or trehalose; the latter being preferred. Typically, the trehalose can be used at a concentration of between 60 and 85 g/100 ml (limits included), advantageously of 0.7 g/ml (70% weight/vol). The ballast agent may also be a sugar alcohol such as sorbitol, mannitol, xylitol, glycerol, erythritol or arabitol. The ballast agent may also be a mixture of the compounds mentioned, for example a mannitol-sorbitol mixture.

For use in the method according to the invention, the thickener may, for example, be xanthan, carob, guargel, alginate and mucigel; xanthan being preferred since this thickener also has stabilizing effects on viruses. Typically, the xanthan can be used at a concentration of 0.1 % (weight/vol.).

The stabilizer is a mixture, in particular a liquid mixture, combining the following compounds: at least one surfactant, amino acids, urea and a buffer.

The active surfactant may be any surfactant. Mention is in particular made of sodium dodecyl sulfate (anionic surfactant) and also nonionic surfactants such as those of polyoxyethylenated sorbitan ester type. An example of the latter is the product known under the trade name Tween™.

A mixture of amino acids may be a mixture of essential amino acids or a mixture of nonessential amino acids. Both types of mixture are advantageously used in the stabilizing composition that is of use for the purposes of the present invention.

The buffer can be any saline or non-saline buffer. Phosphate buffers and the HEPES buffer are in particular mentioned. Advantageously, the stabilizer may also comprise (i) one or more polysaccharides such as dextran; and/or (ii) one or more sugar alcohols; in particular sorbitol and mannitol.

Thus, a mixture that is of use as a stabilizer can be made up:

of a surfactant (for example between 0.005% and 0.02% vol ./vol.);

of a mixture of amino acids (for example, as a concentrated mixture of between 30% and 45% vol./vol.);

of urea (for example between 0.5% and 1.5% weight/vol.);

of a buffer (for example between 15 and 40 mM);

of a sugar alcohol (for example between 5% and 10% weight/vol.); and of a polysaccharide (for example, between 1 and 2% weight/vol.).

By way of illustration, it is indicated that a liquid mixture having function of a stabilizer may, for example, be composed of the following products and mixtures (composition S1 ): Tween® 80 (Polysorbate 80, 0.01% weight/vol.), HEPES (20 mM), sorbitol (5% weight/vol.), mannitol (2.5% weight/vol.), arginine hydrochloride (Arg-HCI) (2.14% weight/vol.), cysteine-HCI (Cys-HCI, 0.00001% weight/vol.), BME essential amino acids mixture 100xc (20% vol./vol.), MEM nonessential amino acids mixture 100xc (20% vol./vol.), dextran 70 (1.5% weight/vol.), urea (0.9% weight/vol.).

To obtain this stabilizing mixture, the BME and MEM mixtures used are as follows:

BME (essential amino acids):

- Arginine hydrochloride

- Cystine

- Histidine

- Isoleucine

- Leucine

- Lysine hydrochloride

- Methionine - Phenylalanine 1.65 g/l

- Threonine 2.4 g/l

- Tryptophan 0.4 g/l

- Tyrosine 1.8 g/l

- Valine 2.35 g/l

MEM (nonessential amino acids):

- Alanine 0.89 g/l

- Asparagine 1.5 g/l

- Aspartic acid 1.33 g/l

- Glutamic acid 1.47 g/l

- Glycine 0.75 g/l

- Proline 1.15 g/l

- Serine 1.05 g/l

An aqueous composition that is of use for the purposes of the present invention can be obtained by dissolving a ballast agent, e.g. trehalose and a thickener, e.g. xanthan, in a liquid stabilizer comprising, e.g. at least one surfactant, a buffer, amino acids and urea; then by adding an appropriate amount of virus.

According to one advantageous procedure, the drying is carried out using an aqueous composition divided up into samples such that, after drying, the dried samples are of a size suitable for oral administration, without prior rehydration. The dividing up into samples can be carried out in several ways: the aqueous composition can, for example, be divided up in cells, (e.g., cupules thermoformed from a PVC sheet) or in large drops on a plate. Under these conditions, the size of the cells and of the drops can be adjusted so as to correspond, in the end, to a vaccine dose. It is sufficient for the individual to take this dose as it is, to place it on the tongue, where it will disintegrate of its own accord.

According to an alternative method of dividing up, the composition is frozen in liquid or gaseous nitrogen, in the form of microbeads, in particular according to the technique described in WO 2010/003670. The drying of the microbeads is subsequently finished off. The dry microbeads are subsequently divided up into doses of identical weight, to be taken directly by the intended recipient.

The conventional drying methods apply irrespective of the selected method of dividing up; thus, once the dividing up has been carried out, a drying operation by freeze-drying can in particular be carried out. Freeze-drying is a drying method which is conventionally carried out in two stages: freezing and sublimation. By way of illustration, it is indicated that the aqueous composition, once divided up in cells or on a plate, can be frozen at approximately -70°C. The temperature is then brought back up to between -40 and -30°C, limits included, for example -35°C, at which temperature the sublimation is subsequently carried out at a pressure of 300 to 400 bar. The sublimation stage ends with the temperature being brought back up to between +5 and +15°C, limits included, for example +10°C. The cells are then sealed with aluminum foil. It is subsequently sufficient to pierce a cell, to take the vaccine dose that it contains and to apply it to the tongue. The dry doses produced on plates are simply collected in bottles.

When an aqueous composition divided up into microbeads (e.g. of 800 μιτι to 1 mm in diameter) by freezing, is used, the sublimation stage as described above can be carried out directly. After sublimation, the microbeads are divided up into a vaccine dose, for example in a single-dose hermetically sealed bottle.

Thus, the product obtained according to one particular embodiment of the method according to the invention does not need to be taken up extemporaneously with any solvent at the time of administration.

The method according to the invention makes it possible to preserve the infectious titer of the live attenuated virus and therefore the vaccinating power of the dry oral vaccine which contains it.

Specifically, just after the drying operation, the virus contained in the product obtained by means of the method shows a loss of infectious titer compared with that determined before drying using the aqueous composition, which loss is, surprisingly, very small: less than one or two log. Furthermore, after accelerated aging by heating and freeze-drying for 7 days at 45°C, a drop in infectious titer of the same order of magnitude is observed, which is a sign that the dry live vaccine obtained by means of the method according to the invention has an outstanding stability over time.

Consequently, the subject of the invention is also a dry oral vaccine comprising a live attenuated virus that can be obtained by means of a method according to the invention.

In particular, it has the characteristic of containing a live attenuated virus, the infectious titer of which is at most 1 or 2 log (log base 10) lower compared with that determined using the aqueous composition that is used in the method of production before it is subjected to the drying.

It can also have the characteristic of containing, after having been kept at 45°C for 7 days, just after it was obtained, a live attenuated virus, the titer of which is at most 2 log (log base 10), preferably at most 1 log, lower compared with that determined just after it was obtained.

It also has the characteristic of containing from 5% to 10% (weight/weight) of residual moisture.

According to one particular embodiment, the vaccine according to the invention is suitable for oral administration in its dry form (without prior rehydration).

The virus contained in the vaccine according to the invention may be any of those described as being able to be part of the aqueous composition for use in the method according to the invention. By way of example, it is indicated that a vaccine according to the invention can comprise (i) serotypes 1 , 2 and/or 3 of the poliovirus; or (ii) one or more serotypes of the rotavirus.

Finally, the subject of the invention is also an aqueous composition for use in the method according to the invention, comprising:

(i) a live attenuated virus; (ii) a ballast agent (filler), selected from the group comprising monosaccharides, oligosaccharides, polysaccharides, the sugar alcohols which are derived therefrom, and also mixtures thereof;

(iii) a thickener; and

(iv) a stabilizer comprising a surfactant, amino acids, urea and a buffer; and, optionally,

(v) a sugar alcohol and/or a polysaccharide. Experimental data

A - Preparation of a dry oral polio vaccine. An aqueous composition is prepared by dissolving, in a stabilizing liquid preparation S1 , S2 or S3, xanthan of a final concentration of 0.7 g/ml (70%) and a filler, the final concentration of which is given in table I hereinafter. Approximately 7 CCID 50/d of the poliovirus made up of serotypes 1 , 2 and 3 (CCID 50/d: viral dose which infects 50% of cell cultures) and prepared by propagation on cell cultures according to the recommendations given by the European Pharmacopoeia Ed.: 6.1 , are then incorporated.

The stabilizing preparations S1 to S3 are as follows:

S1 : Tween® 80 (Polysorbate 80, 0.01 % weight/vol.), HEPES (20 mM), sorbitol (5% weight/vol.), mannitol (2.5% weight/vol.), Arg-HCI (2.14% weight/vol.), Cysteine-HCI (Cys-HCI, 0.00001 % weight/vol.), BME essential amino acids mixture 100xc (20% vol ./vol.), MEM nonessential amino acids mixture 100xc (20% vol./vol.), dextran 70 (1.5% weight/vol.) and urea (0,9% weight/vol.).

S2: Sorbitol (5% weight/vol.), mannitol (2.5% weight/vol.), Arg-CI (2.14% weight/vol.), Cysteine-HCI (Cys-Cys-HCI, 0.00001 % weight/vol.), BME essential amino acids mixture 100xc (40% vol./vol.), MEM nonessential amino acids mixture 100xc (40% vol./vol.), Dextran 70 (1.5% weight/vol.) and urea (0.9% weight/vol.). S3: BME essential amino acids mixture 100xc (10% vol./vol.) and MEM nonessential amino acids mixture 100xc (10% vol./vol.).

The aqueous composition is then divided up into the 0.3 ml cells of a PVC thermoformed sheet. The sheet with the cells is frozen at -70°C. Sublimation is carried out at - 35°C for 2 hours, and then at + 10°C for 6 hours, at a pressure of 350 pbar throughout.

The cells are then sealed with aluminum foil. The dry product obtained is in the form of a pastille formed by the cells. To the eye, it has a homogeneous white color. Its percentage residual moisture is between 7% and 8%. Its resistance/ coherence is sufficient to perforate the aluminum foil which seals the cells. It disintegrates rapidly on contact with water or saliva.

B - Infectious titer measurement

The infectious titer of the virus before and after freeze-drying and also after freeze- drying followed by storage at 37°C or 45°C for 7 days, is determined by the CCID50 technique (determination of the titer infecting 50% of cell cultures) described in the European Pharmacopoeia Ed.: 6.1 , page 3588, and specified hereinafter.

The viral preparation to be titered is diluted from -1 to - 6 or -7 (in log) in 96-well plates in a proportion of 50 μΙ per well with a 1xc MEM culture medium supplemented with 2% (vol./vol.) of fetal calf serum (not containing anti-polio antibodies), 4% (vol./vol.) of a 5.6% sodium bicarbonate solution and 0.2% (vol./vol.) of a 400xc solution of penicillin-didromycin antibiotics.

Then, either 50 μΙ of diluting medium (measurement of overall titer); or 50 μΙ of one of the following mixtures are added:

- anti-polio type 2 antiserum plus anti-polio type 3 antiserum diluted in MEM medium, in order to neutralize the polioviruses type 2 and poliovirus type 3, contained in the viral preparation (measurement of type 1 titer); anti-polio type 1 antiserum plus anti-polio type 3 antiserum diluted in MEM medium, in order to neutralize the poliovirus type 1 and poliovirus type 3, contained in the viral preparation (measurement of type 2 titer);

anti-polio type 1 antiserum plus anti-polio type 2 antiserum diluted in MEM medium, in order to neutralize the poliovirus type 1 and poliovirus type 2, contained in the viral preparation (measurement of type 3 titer).

A negative control column is formed with 100 μΙ of MEM medium per well.

The plates are covered and agitated for 1 hour at 36°C.

After this contact time, 100 μΙ of Hep-2 (Cincinnati) cell suspension, at 50 000 cells/ml, are added to each well. The plates are then placed in an incubator at 36°C, for 9 days without agitation.

The cytopathogenic effects are read in each well after having verified the integrity of the cell controls.

For each of the dilutions, the number of positive wells is determined. The titer is calculated using a linear regression of the number of positive wells (transformed using the "arcsine square root" function) as a function of the dilutions (transformed by logarithm transformation).

The loss of infectious titer before/after freeze-drying and also that directly related to the storage conditions (after freeze-drying/after freeze-drying+storage) is expressed in log (log base 10) in table I below.

Table I

The stability of the dry oral polio vaccine containing trehalose, xanthan and the S1 preparation as described above (Lyoc vaccine) is compared to that observed with (i) a liquid oral polio vaccine obtained according to the D2O method (the method of R. Crainic) and (ii) a commonly used liquid oral polio vaccine based on MgCI₂, 1 M (T vaccine). The infectious titer of each of the three serotypes is determined during a storage period of 3 months at 25°C, for 3 weeks at 37°C or of 7 days at 45°C.

The loss of titer directly related to the conditions of storage at 25°C (after freeze- drying/after freeze-drying + storage) is expressed in log (log base 10) in table II below. The total infectious titers during the storage at 37°C and 45°C can be directly read from figure I (Stability after 3 weeks at 37°C) and figure II (Stability after 7 days at 45°C) hereinafter.

Table II: Stability after 3 months at 25°C

It is noted, according to table II and the stability curves, that the stabilization obtained for OPV is surprising. This is because the loss of infectious titer, of about 1 log in 7 days at 45°C, is very small when the method according to the invention is carried out, in comparison with a prior art method in the presence of MgCI₂, a stabilizer considered to be powerful, which results in a loss of about 3.4 logs under the same storage conditions. It follows that the method according to the invention unexpectedly increases the known stability of the overall infectious titer of the mixture of the three serotypes of the poliovirus. This stabilization is surprising and greater than that obtained with the method of R Crainic (D₂0), and greater than that obtained by H. Shiomi et al., while at the same time providing easy administration for OPV.

Claims

What is claimed is

A method for preparing a dry oral vaccine, comprising a live attenuated virus, by drying an aqueous composition comprising:

(i) said virus;

(ii) a ballast agent (filler), selected from the group made up of monosaccharides, oligosaccharides, polysaccharides, and the sugar alcohols which are derived therefrom, and also mixtures thereof;

(iii) a thickener; and

The method as claimed in claim 1 , wherein the drying is not continued beyond the time required to obtain a product which has a residual moisture content of between 5% and 10% (weight/weight), limits included.

The method as claimed in claim 1 or 2, wherein the dry weight of the aqueous composition is between 70% and 85% (weight/weight), limits included, of the total weight of the aqueous composition.

The method as claimed in one of claims 1 to 3, wherein the ballast agent (filler) is present in the aqueous composition intended to be dried, at a concentration of between 80% and 100% (limits included) of the maximum solubility of the ballast agent in an aqueous medium at between 25 and 35°C.

The method as claimed in one of claims 1 to 4, wherein the ballast agent (filler) is chosen from the group made up of mannitol, sorbitol, trehalose, sucrose, xylose, maltose and lactose or a mixture thereof. The method as claimed in claim 5, wherein the ballast agent (filler) is trehalose.

The method as claimed in one of claims 1 to 6, wherein the thickener is present in the aqueous composition intended to be dried, at a concentration of between 0.08 and 0.12 g/100 ml, limits included.

The method as claimed in one of claims 1 to 7, wherein the thickener is xanthan.

The method as claimed in one of claims 1 to 8, wherein the stabilizer is present in the aqueous composition intended to be dried, at a concentration of between 40% and 80%, advantageously 50% and 70%, preferably 55% and 60% (vol./vol.), limits included.

The method as claimed in one of claims 1 to 9, wherein the stabilizer is a composition comprising a nonionic surfactant, a buffer, sugar alcohols, a mixture of amino acids, a polysaccharide and urea.

The method as claimed in one of claims 1 to 10, wherein the drying is carried out using an aqueous composition divided up into samples such that, after drying, the dried samples are of a size suitable for oral administration, without prior rehydration.

The method as claimed in one of claims 1 to 11 , wherein the drying is carried out by freeze-drying which is a method comprising a freezing stage and a sublimation stage.

13. The method as claimed in claim 12, wherein the sublimation stage of the freeze-drying is first carried out at a temperature between -40 and -30°C, limits included, and then at a temperature of between +5°C and +15°C, limits included, at a pressure of 300 to 400 pbar and for an appropriate period of time.

14. The method as claimed in claim 13, wherein the drying is carried out using an aqueous composition divided up into samples in cells or on plates. 15. The method as claimed in one of claims 1 to 10, wherein the drying is carried out using an aqueous composition frozen in liquid or gaseous nitrogen, in the form of microbeads.

16. The method as claimed in claim 15, wherein the drying is carried out by sublimation. 17. The method as claimed in one of claims 1 to 16, wherein the virus is the poliovirus, composed of at least one serotype.

18. The method as claimed in claim 17, wherein the poliovirus is composed of two serotypes.

19. The method as claimed in claim 17, wherein the poliovirus is composed of three serotypes.

20. The method as claimed in one of claims 1 to 16, wherein the virus is the rotavirus composed of at least one serotype.

21. A dry oral vaccine comprising a live attenuated virus, that can be obtained by means of a method as claimed in one of claims 1 to 20. 22. The dry oral vaccine as claimed in claim 21 , wherein the infectious titer of the live attenuated virus is at most 2 log (log base 10), preferably at most 1 log, lower compared with that determined using the aqueous composition on which the method is carried out.

23. The dry oral vaccine as claimed in claim 21 or 22, wherein, after a vaccine dose has been kept at 45°C for 7 days just after it has been obtained, the infectious titer of the live attenuated virus is at most 2 log (log base 10), preferably at most 1 log lower, compared with that determined using a vaccine dose just after it has been obtained.

24. The dry oral vaccine as claimed in one of claims 21 to 23, containing from 5% to 10% (weight/weight) of residual moisture.

25. The vaccine as claimed in one of claims 21 to 24, for oral administration in its dry form (without prior rehydration).

26. The vaccine as claimed in one of claims 21 to 25, comprising one or more of serotypes 1 , 2 and 3 of the poliovirus.

27. The vaccine as claimed in one of claims 21 to 25, comprising one or more serotype(s) of the rotavirus.

28. An aqueous composition for use in the method as claimed in one of claims 1 to 20, comprising:

(i) a live attenuated virus;

(ii) a ballast agent (filler), selected from the group comprising a monosaccharide, an oligosaccharide, a polysaccharide, the sugar alcohols which are derived therefrom, and also mixtures thereof;

(iii) a thickener; and

(iv) a stabilizer comprising a surfactant, amino acids, urea, and a buffer; and, optionally,

(v) a sugar alcohol and/or a polysaccharide.