BR112016026418B1 - BIOLOGICAL FREEZE DRYING AND REHYDRATION METHODS - Google Patents

Abstract

BIOLOGICAL FREEZE DRYING AND REHYDRATION METHODS. The present invention relates generally to the fields of immunology and vaccine technology. More specifically, the present invention relates to methods for lyophilizing biological preparations, including peptides, antigens, antibodies, and especially cells. It is important to note that the disclosed methods preserve the viability, infectivity and immunogenicity of cells of the Apicomplexa phylum, of the Sarcocystidae family and, in particular, of cells of the Toxoplasma genus.

Description

CROSS-REFERENCE TO RELATED ORDERS

[001] This patent application claims benefit from European patent application EP 14168327.6 which was filed on May 14, 2014, and which is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE

[002] All references cited herein are incorporated by reference in their entirety.

FIELD OF INVENTION

[003] The present invention generally relates to the fields of immunology and vaccine technology. More specifically, the present invention relates to methods for lyophilizing and rehydrating immunogenic and/or vaccine compositions which may comprise, inter alia, parasites, including protozoa, including Toxoplasma spp. The invention further relates to stabilized, freeze-dried immunogenic and/or vaccine compositions, for example T. gondii, which may contain these stabilizers. Other aspects of the invention are described in, or are obvious from the following description, and are within the scope of the invention.

BACKGROUND OF THE INVENTION

[004] Lyophilization, or the freeze-drying process, is a commonly used technique to remove water in the preparation of dehydrated products. Generally, "lyophilizing" an aqueous composition involves three steps. First, the aqueous composition is frozen under low temperature conditions. Second, the frozen water is removed by sublimation under conditions of reduced pressure and low temperature. At this stage, the composition usually contains about 15% water. Thirdly, residual water is further removed by desorption under conditions of reduced pressure and higher temperatures. At the end of the freeze-drying process, a freeze-dried product, also called a "lozenge" or "cake" is produced. The lyophilized product contains very low residual water (from about 0.5% to about 5% weight/weight) and dried material in an amorphous form. This specific state is qualified as "vitreous." Prior to use, powdered products must be rehydrated, which can significantly compromise the integrity of biological structures, especially in the case of whole cells/organisms. But damage to freeze-dried products, including loss of viability, decreased immunogenicity and impaired/abolished infectivity, can occur during any of the above recited steps (i.e. freeze-drying, storage, reconstitution and post-reconstitution storage). Therefore, experimentally determined stabilizers must be added to the compositions, before subjecting them to lyophilization, and the magnitude and kinetics of each step, in particular the rehydration step, must be precisely defined (Mil et al. ( 2003) Biotech Bioeng 83, 578 ). At the time of this submission, Applicants are aware of unsafe and effective stabilizers or freeze-drying methods to preserve the viability, infectiveness, and immunogenicity of parasitic protozoa, including T. gondii, during all stages of the freeze-drying/rehydration process.

[005] The stabilization of biological ingredients in dry form has typically involved the preservation of antitoxins, antigens and bacteria (Flosodort et al (1935) J. Immunol. 29, 389). However, a limitation in this process included the partial denaturation of the proteins when dried from an aqueous state at ambient temperatures. Drying from the frozen state helped reduce denaturation and led to better, though incomplete, preservation of biological ingredients, including bacteria and viruses (Stamp et al (1947) J. Gen. Microbiol 1, 251; Rightsel et al ( 1967) Cryobiology 3, 423; Crowe et al (1971) Cryobiology 8, 251). However freezing must be managed in order to maintain an optimal viability of such biological ingredients (Dumont et al. (2004) Appl. Env. Microbiol. 70, 268).

[006] Immunogenic compositions and vaccine compositions comprising biological ingredients, such as viruses, bacteria, parasites, fungi, proteins, polypeptides, glycoproteins, and especially live, attenuated microorganisms are markedly sensitive to the conditions under which they are prepared, formulated and stored. Such biological ingredients can be modified and degraded by chemical reactions (eg hydrolysis, deamination, Maillard reaction), many of which are mediated by water. Liquid water allows molecular movement and can result in modification of protein conformations in compositions comprising biological ingredients. By limiting access to water, or by removing water, an important factor of modification and degradation is reduced. This water transfer must be carefully regulated to avoid damage to the yeast (Gervais et al. (1992) Biotech. Bioeng. 40, 1435). Prior methods for imparting stability to biological ingredients have primarily involved freezing water or removing water by freeze drying.

[007] More recently, sugars such as sucrose, raffmose and trehalose have been added in various combinations as stabilizers prior to lyophilization of the virus. A large number of compounds have been tested for their ability to stabilize different vaccines containing live attenuated biological ingredients, in particular viruses. Such compounds include SPGA (sucrose, phosphate, glutamate and albumin; Bovarnick et al (1950) J. Bacteriol 59, 509-522; US Patent No. 4000256), bovine or human serum albumin, alkali metal salts of glutamic acid, aluminum salts, sucrose, gelatin, starch, lactose, sorbitol, Tris-EDTA, casein hydrolyzate, sodium and potassium lactobionate, and alkali metal or mono-dimetallic phosphate. Other compounds include, for example, SPG-NZ amine (eg US Patent No. 3783098) and polyvinylpyrrolidone (PVP) mixtures (eg US Patent No. 3915794). To preserve live attenuated flaviviruses, one group combined a complex mixture of several compounds, including sorbitol, sucrose, optionally trehalose and/or another disaccharide or trisaccharides, urea, and a specific combination of amino acids (US 2010/0015180A1, Sanofi Pasteur).

[008] With regard to preserving even more complicated biological structures, a reference describes a process for lyophilizing a mixture of Anaplasma (a bacterium), Toxoplasma cells and blood cells (WO 92/14360, to Cryopharm Corporation). This method keeps the parasites viable, but only in the context of their host cells. More recently, WO 2009/099075 (Snow Brand Milk Product Co., Ltd) disclosed a lyophilization method of preserving the viability of Bifidobacterium, lactobacillus, streptococcus and Lactococcus involving milk components. Even more recently, WO 2012/098358 (Biopharma Technology Ltd) describes a method for lyophilizing cell-based biological material while retaining some viability after reconstitution.

Review of other related literature references

[009] Marcotty et al. “Freeze and resuscitation of sporozoites of Theileria parva: preliminary experiments”. Vaccine, 22 (2003) 213-216. This reference disclosed the lyophilization of Theileria parva sporozoites and subsequent vaccination of cattle against East Coast fever. After the injection, several calves showed clinical symptoms and development of parasites (schizonts and piroplasms) was observed in biopsies. Only 0.1 to 1% of sporozoites appeared to survive freeze-drying.

[0010] Harp et al. “Protection of calves with a vaccine against Cryptosporidium parvum.” J. Parasitology, Vol. 81, No. 1 (Feb., 1995), p. 54. This reference presented data indicating that a vaccine containing lyophilized oocysts could reduce diarrhea and oocyst shedding after oral challenge with viable Cryptosporidium parvum oocysts. As there did not appear to be an increase in antibody titer (against C. parvum), the efficacy of the vaccine can be explained by a cellular, but not a humoral immune response. Viability of oocytes was destroyed by lyophilization.

[0011] Suzaki et al. “A simplified freeze-drying technology for protozoan cells.” J. Electron Microsc, Vol. 27, No. 2, 153-156, 1978. Although some surface preservation was achieved, as indicated by EM, the lyophilization method described failed to preserve viability or infectivity.

[0012] Gertrud E. and Kramer J. “The Preservation of Infective spores of Octosporea muscae domesticae in Phormia regina, of Nosema algerae in Anopheles stephensi, and of Nosema whitei in Tribolium castaneum by Lyophilization”. J. Invertebrate Pathology, 33, 300-306 (1979). Spores of three species of microsporidia (obligate intracellular parasites) were lyophilized "naked", or in host cells. The lyophilizates were then brought into contact with the target animals. Lyophilized "naked" parasites, formulated in a 50% glucose solution, were infectious to the target species.

[0013] Sherwood et al. “Cryptosporidiosis in Laboratory Experimental Mice”. Infection and Immunity, Nov. 1982, p. 471-475. This reference revealed that freezing and lyophilization failed to preserve the viability of Cryptosporidium sp.

[0014] Du Plessis et al. “The freeze-drying of Cowdria ruminantium". This reference revealed that freeze-dried parasites were infectious in mice and sheep within host cells. The precise form/state of the parasites was not evident.

[0015] Therefore, there continues to be a need for new formulation solutions and lyophilization processes to preserve the viability, immunogenicity and infectivity of protozoan parasites, particularly "brittle" and/or "naked" parasites in lyophilized form.

[0016] The citation or identification of any document in this patent application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

[0017] The present invention relates to a lyophilized composition containing lyophilized protozoa of an intracellular nature, said lyophilized composition is capable of reconstitution to restore the said state of viable and infectious protozoa.

[0018] A second aspect of the present invention is to provide a process for obtaining a lyophilized composition containing lyophilized protozoa of an intracellular nature and being capable of reconstitution to restore said protozoa to viable and infectious states comprising a freezing-drying step of a suspension of protozoa of an intracellular nature, devoid of host cells by protozoa, in an aqueous formulation solution.

[0019] The invention also relates to a lyophilized rehydrated composition comprising a lyophilized composition, as defined above, in a rehydration medium, said composition wherein said lyophilized product contains lyophilized protozoa of an intracellular nature, said composition re- being lyophilized hydrated, such that the protozoa contained therein are viable and infectious.

[0020] In one embodiment, the present invention relates to a lyophilized composition containing lyophilized protozoa of an intracellular nature, said lyophilized composition being devoid of protozoal host cells, containing a content of less than 12% moisture by weight and being capable of reconstituting and restoring said protozoa to viable and infectious states.

[0021] In an advantageous embodiment, the present invention relates to a lyophilized composition, as defined above, capable of reconstitution to restore said protozoa to viable and infectious states, said viable protozoa are responsible for more than 1% of lyophilized protozoa.

[0022] In an advantageous embodiment, the present invention relates to a lyophilized composition, as defined, being able to reconstitute and restore said protozoa to viable and infectious states after storage of said lyophilized composition for a period of time greater than two weeks at a temperature of -25 °C to 25 °C, in particular at a temperature from -25 °C to 2 °C, at a temperature of 2 to 8 °C, more particularly at a temperature of 2 to 6°C, or at a temperature from 8 to 25°C.

[0023] In one embodiment, the present invention relates to a lyophilized composition containing lyophilized protozoa of an intracellular nature, said lyophilized composition being devoid of protozoan host cells, containing a content of less than 12% moisture by weight and being capable of reconstituting and restoring said protozoa to viable and infectious states, said viable protozoa accounting for more than 1% of freeze-dried protozoa, in particular after storage of said freeze-dried composition for a period of time greater than two weeks at a temperature from -25 °C to 25 °C, in particular at a temperature from -25 °C to 2 °C, at a temperature from 2 to 8 °C, more particularly at a temperature from 2 to 6 °C, or at a temperature of 8 to 25°C.

[0024] In an advantageous embodiment, the invention relates to a lyophilized composition as defined above, wherein said lyophilized protozoa are fragile state protozoa.

[0025] As used herein, "fragile" state protozoa conform to at least one of the following descriptions (ie, in the absence of protective components and proper procedures): • are obligate intracellular protozoa, which are present outside of their host cells, for example, in the extracellular matrix; • they have a survival rate of less than 1 2 1% after 15 days cells at 4 ° C when stored; in a culture medium • they lose their infectivity during freezing; • they lose their infectivity during freeze-drying; • they lose their viability during freezing; • they lose their viability during freeze-drying; • they are freeze-dried parasites that lose their infectivity after 15 days of storage at +4°C; • they are freeze-dried parasites that lose their viability after 15 days of storage at + 4°C. In an advantageous embodiment, said fragile state is the tachyzoite state, the bradyzoite state, the sporozoite state, the promastigote state, the amastigote state, the epimastigote state or the trypomastigote state.

[0026] In another embodiment, said fragile state is the tachyzoite state, the bradyzoite state, the sporozoite state, the promastigote state, the amastigote state, the epimastigote state, the trypomastigote state, or the merozoite state.

[0027] In one embodiment, the invention relates to a lyophilized composition containing lyophilized protozoa of an intracellular nature, said lyophilized composition being devoid of protozoal host cells, containing a content of less than 12% moisture by weight and being capable of of reconstitution to restore said protozoa to viable and infectious states, wherein said lyophilized protozoa are protozoa of the fragile state, in particular, the tachyzoite state, the bradyzoite state, the sporozoite state, the promastigote state, the amastigote state, the epimastigote state, trypomastigote state or merozoite state.

[0028] In another embodiment, the invention relates to a lyophilized composition containing lyophilized protozoa of an intracellular nature, said lyophilized composition being devoid of protozoal host cells, containing a moisture content of less than 12% by weight and being capable of reconstitution to restore said protozoa to viable and infectious states, wherein said freeze-dried protozoa are fragility protozoa, in particular, of the tachyzoite state, of the bradyzoite state, of the sporozoite state, of the promastigote state, of the state amastigotes, the epimastigote state or the trypomastigote state.

[0029] In an advantageous embodiment, the present invention relates to a freeze-dried composition as defined above capable of being stored at a temperature comprised between -25°C to 10°C for at least 12 months before being reconstituted.

[0030] In an advantageous embodiment, the present invention relates to a lyophilized composition as defined above, capable of reconstitution to restore the immunogenic activity of said lyophilized protozoa.

[0031] In an advantageous embodiment, the present invention relates to a lyophilized composition as defined above, capable of reconstitution to restore the prophylactic activity of said lyophilized protozoa.

[0032] In another embodiment, the present invention relates to a freeze-dried composition, as defined above, in which the moisture content is less than 11% by weight, less than 10% > less than 9%) , less than 8%, less than 7%, less than 6%, less than 5% or less than 4%.

[0033] In an advantageous embodiment, the present invention relates to a lyophilized composition as defined above, comprising the elements of the culture medium of said protozoa and at least one cryoprotectant.

[0034] In another embodiment, the lyophilized composition, as defined above, further comprises at least one osmoprotectant and/or at least one antioxidant and/or at least one other additive.

[0035] In an advantageous embodiment, the present invention relates to a lyophilized composition, as defined above, comprising the elements of the culture medium of said protozoa, at least one cryoprotectant, at least one osmoprotector, at least one antioxidant and/or at least one other additive.

[0036] In one embodiment, said culture medium is chosen from DMEM, RPMI or PBS.

[0037] In one embodiment, said cryoprotectant agent is selected from DMSO, a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide or a mixture thereof.

[0038] In an advantageous embodiment, said cryoprotectant is chosen from among sucrose, trehalose, glucose, inulin or a mixture thereof.

[0039] In one embodiment, said osmoprotectant is ectoine.

[0040] In one embodiment, said antioxidant is selected from among GSH, EGCG, ascorbic acid or a mixture thereof.

[0041] In one embodiment, said other additive is chosen from polymers, copolymers, amino acids, in particular L-proline, peptides, proteins or a mixture thereof.

[0042] In an advantageous embodiment, the present invention relates to a lyophilized composition, as defined above, in which said culture medium is chosen from DMEM, RPMI or PBS, in which said cryoprotectant agent is selected from among sucrose, trehalose, glucose, DMSO, inulin or a mixture thereof, wherein said osmoprotectant is ectoin, wherein said antioxidant is selected from GSH, EGCG, ascorbic acid or a mixture thereof, and wherein said other additive is chosen from polymers, copolymers, amino acids, in particular L-proline, peptides, proteins or a mixture thereof.

[0043] In an advantageous embodiment, the lyophilized composition as defined above comprises: - DMEM, RPMI, or PBS components - The μmol to about 300 μmol of sucrose, in particular 0 μmol, 100 μmol or 150 μmol, - 0 μmol to about 1000 μmol of trehalose, in particular 0 μmol, 100 μmol, 200 μmol or 880 μmol, - 0 μmol to about 400 μmol (fructose equivalent) of inulin, in particular 0 μmol, 155 μmol or 309 μmol , - 0 μmol to about 400 μmol of ascorbic acid, in particular 0 μmol or 284 μmol, - 0 μmol to about 10 μmol of EGCG, in particular 0 μmol or 2 μmol, - 0 μmol to about 200 μmol of GSH , in particular 0 μmol or 100 μmol, - 0 μmol to about 150 μmol of proline, in particular 0 μmol or 87 μmol, - 0 μmol to about 150 μmol ectoine, in particular 0 μmol or 63 μmol,

[0044] said micromolar values being given per 1 ml of the suspension to be lyophilized,

[0045] provided that the amount of at least one of the components among sucrose, trehalose, inulin, ascorbic acid, EGCG, GSH, proline or ectoin is different from 0 μmol.

[0046] In another advantageous embodiment, the lyophilized composition as defined above comprises: - components of DMEM, RPMI, or PBS

[0047] - 0 μmol to about 300 μmol of sucrose, in particular, 0 μmol, 100 μmol or 150 μmol,

[0048] - 0 to about 1000 μmol trehalose, in particular 0 μmol, 100 μmol, 200 μmol μmol or 880 μmol, - 0 μmol to about 400 μmol inulin (fructose equivalent), in particular 0 μmol, 155 μmol or 309 μmol, - 0 μmol to about 400 μmol of ascorbic acid, in particular, 0 μmol or 284 μmol, - 0 μmol to about 10 μmol EGCG, in particular, 0 μmol or 2 μmol, - 0 μmol to about from 200 μmol GSH, in particular 0 μmol or 100 μmol, - 0 μmol to about 150 μmol proline, in particular 0 μmol or 87 μmol, - 0 to about 150 μmol ectoin, in particular 0 μmol or 70 µmol,

[0049] said micromolar values being given per 1 ml of the suspension to be lyophilized,

[0050] provided that the amount of at least one of the components among sucrose, trehalose, inulin, ascorbic acid, EGCG, GSH, proline or ectoin is different from 0 μmol.

[0051] In an advantageous embodiment, the present invention relates to a lyophilized composition as defined above, wherein said lyophilized protozoa are virulent protozoa, attenuated protozoa or avirulent protozoa.

[0052] In one embodiment, the present invention relates to a lyophilized composition as defined above, wherein said lyophilized protozoa are virulent protozoa, said lyophilized composition being capable of reconstitution to produce a lyophilized rehydrated composition of virulent protozoa .

[0053] In another embodiment, the present invention relates to a lyophilized composition as defined above, wherein said lyophilized protozoa are avirulent or attenuated protozoa, said lyophilized composition being capable of reconstitution to produce a vaccine composition , or an immunostimulant.

[0054] In one embodiment, said freeze-dried protozoan belongs to the subgroup of sporozoans or flagellates.

[0055] In one embodiment, said freeze-dried protozoa belong to the phylum Apicomplexa.

[0056] In another embodiment, said freeze-dried protozoa belong to the phylum Apicomplexa de or Euglenozoa.

[0057] In one embodiment, said freeze-dried protozoa belong to the Sarcocystidae family.

[0058] In another embodiment, said freeze-dried protozoa belong to the Sarcocystidae or Plasmodiidae or Trypanosomatidae family.

[0059] In one embodiment, said freeze-dried protozoa are live attenuated strains of Toxoplasma spp. or from Neospora spp.

[0060] In one embodiment, said freeze-dried protozoa are recombinant live attenuated strains of Toxoplasma spp., Neospora spp., Sarcocystis spp. or a combination of said orders.

[0061] In one embodiment, said lyophilized protozoa are live attenuated recombinant strains of Toxoplasma gondii, Neospora caniem um, Neospora hughesi, Sarcocystis neurona or a combination of said strains.

[0062] In one embodiment, said freeze-dried protozoa are live and/or attenuated recombinant strains of Leishmania spp.

[0063] In one embodiment, said lyophilized protozoa are live and/or attenuated recombinant strains of Leishmania donovani, of Leishmania infantum or a combination of said strains.

[0064] In one embodiment, said freeze-dried protozoa are live and/or attenuated recombinant strains of Plasmodium spp.

[0065] In one embodiment, said freeze-dried protozoa are live and/or attenuated recombinant strains of Plasmodium falciparum.

[0066] In one embodiment, said freeze-dried protozoa are live or attenuated strains of Toxoplasma spp. or from Neospora spp. or from Sarcocystis spp. or from Plasmodium spp. or Leishmania spp.

[0067] In one embodiment, said freeze-dried protozoa are live or attenuated recombinant strains of Toxoplasma spp., Neospora spp., Sarcocystis spp., Plasmodium spp., Leishmania spp. or a combination of said strains.

[0068] In one embodiment, said lyophilized protozoa are recombinant live attenuated virus strains of T. gondii, Neospora caniem um, Neospora hughesi, Sarcocystis neurona, Plasmodium falciparum, Plasmodium vivax, Leishmania donovavi, Leishmania infantum or a combination of said strains.

[0069] In another aspect, the invention relates to a process for obtaining a lyophilized composition containing lyophilized protozoa of the intracellular nature, devoid of protozoan host cells, containing a content of less than 12% moisture by weight and being capable of reconstitution to restore said protozoa to viable and infectious states, said viable protozoa being responsible for more than 1% of the freeze-dried protozoa, comprising a freeze-drying step of a suspension of protozoa of an intracellular nature, devoid of of protozoan host cells, in an aqueous formulation solution.

[0070] In an advantageous embodiment, the invention relates to a process, as defined above, for obtaining a lyophilized composition capable of reconstitution to restore said protozoa to viable and infectious states after storage for a period of time. period of time longer than two weeks at a temperature of -25°C to +25°C, in particular at a temperature from -25°C to 2°C, at a temperature of 2 to 8°C, more particularly at a temperature of 2 to 6 ° C, or at a temperature of 8 to 25 ° C.

[0071] In one embodiment, the invention relates to a process for obtaining a lyophilized composition containing lyophilized protozoa of an intracellular nature, devoid of protozoan host cells, containing a content of less than 12% moisture by weight and being capable of of reconstitution to restore said protozoa to viable and infectious states, said viable protozoa accounting for more than 1% of the freeze-dried protozoa, in particular after storage for a period of time exceeding two weeks at a temperature of -25°C ° C to + 25 ° C, in particular at a temperature from - 25 ° C to 2 ° C, at a temperature from 2 to 8 ° C, more particularly at a temperature from 2 to 6 ° C, or at a temperature from 8 to 25 ° C, comprising a step of lyophilizing a suspension of protozoa of an intracellular nature, devoid of host cells by protozoa, in an aqueous formulation solution, said step of lyophilizing said suspension comprises:

[0072] a primary drying step of a frozen suspension to obtain a primary dry composition, in particular, said primary drying step is carried out at about -55°C, for about 20 h, and in particular said step primary drying is carried out at a pressure comprised between about 30 to about 80 Pa, from 0 to 7 h, and then at a pressure of less than 2 Pa for the remainder of the time, and a secondary drying step of primary dry composition to obtain a freeze-dried composition with a moisture content of less than 12% by weight, in particular, said secondary drying step is carried out at a pressure of less than 2 Pa and at an increasing temperature of -55° C to +5°C at a rate comprised between about 0.01°C/min to about 0.2°C/min, in particular at a rate of about 0.0625°C/min and, at in particular, said secondary drying comprises a temperature step of 1 h at each increase of 15 °C, said temperature being maintained at + 5 °C for 4 h when said temperature is reached.

[0073] In one embodiment, the invention relates to a process, as defined above, in which the step of lyophilizing said suspension comprises a step of freezing the suspension to obtain a frozen suspension.

[0074] In an advantageous embodiment, the freezing step is carried out for 4 to 23 h, at atmospheric pressure, by reducing the initial temperature of the protozoan suspension to a temperature comprised between about -40°C to about -80°C °C, under in particular about -75 °C, said initial temperature of the protozoan suspension comprised between about 15 °C to about 25 °C, in particular about 20 °C, and said lowering of the temperature to be carried out at a rate comprised between about -0.1°C/min to about -10°C/min, in particular at a rate of about -1°C/min.

[0075] In this embodiment, the freezing step starts after a period of time comprised between 0 and 2 h after the formation of the suspension.

[0076] In one embodiment, the invention relates to a process as defined above, comprising a step of primary drying of said frozen suspension to obtain a primary dry composition.

[0077] In an advantageous embodiment, said primary drying step is carried out at about -55°C, for about 20 h.

[0078] In an advantageous embodiment, said primary drying step is carried out at a pressure comprised between about 30 to about 80 Pa, from 0 to 7 h, and then at a pressure of less than 2 Pa during the rest of the time.

[0079] In one embodiment, the invention relates to a process as defined above, comprising a step of secondary drying of the primary dry composition to obtain a freeze-dried composition with a moisture content of less than 12% by weight.

[0080] In an advantageous embodiment, said secondary drying step is carried out at a pressure of less than 2 Pa and at an increasing temperature from -55°C to +5°C at a speed comprised between about 0.01°C / min at about 0.2°C/min, in particular at a rate of about 0.0625°C/min.

[0081] In an advantageous embodiment, said secondary drying comprises a temperature step of 1 h at each increase of 15 °C, said temperature being maintained at + 5 °C for 4 h when said temperature is reached.

[0082] In an advantageous embodiment, the invention relates to a process, as defined above, in which the freezing-drying step of said suspension comprises:

[0083] - a step of freezing the suspension to obtain a frozen suspension, in particular, said freezing step is carried out for 4 to 23 h, at atmospheric pressure, by reducing the initial temperature of the protozoan suspension to a temperature comprised between about -40 °C to about -80 °C, in particular about -75 °C, said initial temperature of the protozoan suspension comprised between about 15 °C to about 25 °C, in particular about 20°C, and said temperature lowering being carried out at a rate comprised between about -0.1°C/min to about -10°C/min, in particular at a rate of about -1 °C/min,

[0084] - A primary drying step of said frozen suspension to obtain a primary dry composition, in particular, said primary drying step is carried out at about -55°C, for about 20 h, and in particular said primary drying step is carried out at a pressure comprised between about 30 to about 80 Pa, from 0 to 7 h, and then at a pressure of less than 2 Pa for the remainder of the time, and

[0085] - A secondary drying step of the primary dry composition to obtain a freeze-dried composition with a moisture content of less than 12% by weight, in particular, said secondary drying step is carried out at a pressure of less than 2 Pa and at an increasing temperature from -55°C to +5°C at a rate comprised between about 0.01°C/min to about 0.2°C/min, in particular at a rate of about 0.0625 ° C / min and, in particular, said secondary drying comprises a temperature step of 1 h at each increase of 15 ° C, said temperature being maintained at + 5 ° C for 4 h when said temperature is reached .

[0086] In one embodiment, the invention relates to a process as defined above, which comprises an initial step of suspending protozoa of an intracellular nature and devoid of protozoan host cells in an aqueous formulation solution comprising a culture medium of said protozoa and at least one cryoprotectant to obtain a suspension of protozoa in an aqueous formulation solution.

[0087] In one embodiment, said aqueous solution formulation further comprises at least one osmoprotectant and/or at least one antioxidant and/or at least one other additive.

[0088] In one embodiment, said aqueous formulation solution comprises a culture medium for said protozoa, at least one cryoprotectant, at least one osmoprotectant, at least one antioxidant and/or at least one other additive.

[0089] In an advantageous embodiment, the culture medium, wherein said aqueous solution formulation is chosen from DMEM, RPMI or PBS.

[0090] In an advantageous embodiment, said cryoprotectant solution in the formulation is chosen from DMSO, a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide or a mixture thereof.

[0091] In an advantageous embodiment, said cryoprotectant agent solution in the formulation is chosen from sucrose, trehalose, glucose, inulin or a mixture thereof.

[0092] In an advantageous embodiment, said solution in osmoprotective formulation is ectoine.

[0093] In an advantageous embodiment, the antioxidant in said formulation solution is chosen from GSH, EGCG, ascorbic acid or a mixture thereof.

[0094] In an advantageous embodiment, the other additive in said formulation solution is chosen from polymers, copolymers, amino acids, especially L-proline, peptides, proteins or a mixture thereof. In an advantageous embodiment, the culture medium in which said aqueous formulation solution is chosen from DMEM, RPMI or PBS, in which the cryoprotectant in said formulation solution is chosen from sucrose, trehalose, glucose, DMSO, inulin or a mixture thereof, wherein the osmoprotectant in said formulation solution is ectoin, wherein the antioxidant in said formulation solution is chosen from GSH, EGCG, ascorbic acid or a mixture thereof, and wherein the other additive in said formulation solution is chosen among polymers, copolymers, amino acids, in particular L-proline, peptides, proteins or a mixture thereof.

[0095] In an advantageous embodiment, the present invention relates to a process, as defined above, in which said aqueous formulation solution comprises or consists of:

[0096] - DMEM, RPMI or PBS,

[0097] - 0 mM to about 3000 mM in DMSO, in particular 0 mM or 1405 mM,

[0098] - 0 mM to about 300 mM sucrose, in particular 0 mM, 100 mM or 150 mM,

[0099] - 0 mM to about 1000 mM trehalose, in particular 0 mM, 100 mM, 200 mM or 880 mM,

[00100] - 0 mM to about 400 mM (fructose equivalent) inulin, in particular 0 mM, 155 mM or 309 mM,

[00101] - 0 mM to about 400 mM ascorbic acid, in particular 0 mM or 284 mM,

[00102] - 0 mm to about 10 mm EGCG, in particular 0 and 2 mm,

[00103] - 0 to about 200 mM GSH mm, in particular 0 mM or 100 mM,

[00104] - 0 to about 150 mM proline mm, in particular 0 mM or 87 mM,

[00105] - 0 mM to about 150 mM ectoine, in particular 0 mM or 63 mM,

[00106] provided that the concentration of at least one of the components among DMSO, sucrose, trehalose, inulin, ascorbic acid, EGCG, GSH, proline or ectoine is different from 0 mm.

[00107] In another advantageous embodiment, the present invention relates to a process as defined above, in which said aqueous solution formulation B comprises or consists of:

[00108] - DMEM, RPMI or PBS,

[00109] - 0 mm to about 3000 mM in DMSO, in particular 0 mm or 1,280 mM,

[00110] - 0 to about 300 mM sucrose mm, in particular 0 mM, 100 mM or 150 mM,

[00111] - 0 mm to about 1,000 mm trehalose, in particular 0 mM, 100 mM, 200 mM or 880 mM,

[00112] - 0 mM to about 400 mM (fructose equivalent) inulin, in particular 0 mM, 155 mM or 309 mM,

[00113] - 0 mM to about 400 mM ascorbic acid, in particular 0 mM or 284 mM,

[00114] - 0 mm to about 10 mm EGCG, in particular 0 and 2 mm,

[00115] - 0 to about 200 mM GSH mm, in particular 0 mM or 100 mM,

[00116] - 0 to about 150 mM proline mm, in particular 0 mM or 87 mM,

[00117] - 0 mM to about 150 mM ectoine, in particular 0 mM or 70 mM,

[00118] provided that the concentration of at least one of the components among DMSO, sucrose, trehalose, inulin, ascorbic acid, EGCG, GSH, proline or ectoine is different from 0 mm.

[00119] In one embodiment, said aqueous solution formulation comprises or consists of:

[00120] - DMEM

[00121] - 20 mM to about 300 mM trehalose

[00122] - 20 mM to about 300 mM sucrose

[00123] - 20 mM to about 400 mM inulin (fructose equivalent equivalent) autoclaved

[00124] - 20 mm to about 200 mM GSH

[00125] - 20 mm to about 150 mM ectoine

[00126] - 20 mm to about 150 mM proline

[00127] and is adjusted to pH 7.4 with sodium hydroxide.

[00128] By "consists of" it is meant that in the above formulation, the following elements: DMSO, ascorbic acid and EGCG are all absent.

[00129] By "comprises", it is understood that

[00130] Or at least one of the following elements: DMSO, ascorbic acid and EGCG is absent, the other elements being in the concentrations defined above in formulation A or in formulation B different from 0 mm,

[00131] - Either all elements of DMSO, ascorbic acid and EGCG are present in the concentrations defined above in formulation A or in formulation B and are different from 0 mm.

[00132] In another embodiment, said aqueous solution formulation comprises or consists of:

[00133] - DMEM

[00134] - 20 mM to about 300 mM trehalose

[00135] - 20 mM to about 400 mM ascorbic acid and is adjusted to pH 7.4 with sodium hydroxide.

[00136] By "consists of" it is meant that in the above formulation, the following elements: DMSO, sucrose, inulin, EGCG, GSH, proline and ectoine are all absent.

[00137] By "comprises", it is understood that

[00138] either at least one of the following elements: DMSO, sucrose, inulin, EGCG, GSH, proline and ectoine is absent, the other elements being in the concentrations defined above in formulation A or in formulation B different from 0 mm,

[00139] or all elements of DMSO, sucrose, inulin, EGCG, GSH, proline and ectoine are present at the concentrations defined above in formulation A or in formulation B and are different from 0 mm.

[00140] In another embodiment, said aqueous solution formulation comprises or consists of:

[00141] - DMEM

[00142] - 20 mM to about 300 mM trehalose

[00143] - 20 mM to about 300 mM sucrose

[00144] - 20 mM to about 400 mM inulin (fructose equivalent).

[00145] By "consists of" it is meant that in the above formulation, the following elements: DMSO, ascorbic acid, EGCG, GSH, proline and ectoine are all absent.

[00146] By "comprises", it is understood that

[00147] - Or, at least, one of the following elements: DMSO, ascorbic acid, EGCG, GSH, proline and ectoine is absent, the other elements being in the concentrations defined above in formulation A or in formulation B different from 0 mm,

[00148] or all elements of DMSO, ascorbic acid, EGCG, GSH, proline and ectoine are present at the concentrations defined above in formulation A or in formulation B and are different from 0 mM.

[00149] In another embodiment, said aqueous solution formulation comprises or consists of:

[00150] - DMEM

[00151] - 20 mM to about 300 mM trehalose

[00152] - 20 mM to about 300 mM sucrose

[00153] - 20 mM to about 200 mM GSH

[00154] - 20 mM to about 150 mM ectoine

[00155] - 20 mM to about 150 mM of proline and is adjusted to pH 7.4 with sodium hydroxide.

[00156] By "consists of" it is meant that in the above formulation, the following elements: DMSO, inulin, ascorbic acid and EGCG are all absent.

[00157] By "comprises", it is understood that

[00158] either at least one of the following elements: DMSO, inulin, ascorbic acid and EGCG is absent, the other elements being in the concentrations defined above in formulation A or in formulation B different from 0 mM,

[00159] or all elements of DMSO, inulin, ascorbic acid and EGCG are present at the concentrations defined above in formulation A or in formulation B and are different from 0 mM.

[00160] In another embodiment, said aqueous solution formulation comprises or consists of:

[00161] - DMEM

[00162] - 200 mM and about 3000 mM DMSO.

[00163] By "consists of" it is meant that in the above formulation, the following elements: trehalose, sucrose, inulin, ascorbic acid, EGCG, GSH, proline and ectoine are all absent.

[00164] By "comprises", it is understood that

[00165] either at least one of the following elements: trehalose, sucrose, inulin, ascorbic acid, EGCG, GSH, proline and ectoine is absent, the other elements, the concentrations being defined above in formulation A or in formulation B and different from 0 mM,

[00166] or all elements of trehalose, sucrose, inulin, ascorbic acid, EGCG, GSH, proline and ectoine are present at the concentrations defined above in formulation A or in formulation B is other than 0 mM.

[00167] In another embodiment, said aqueous solution formulation comprises or consists of:

[00168] - DMEM

[00169] - 20 mM to about 300 mM trehalose

[00170] - 0.1 mM to about 10 mM EGCG.

[00171] By "consists of" it is meant that in the above formulation, the following elements: DMSO, sucrose, inulin, ascorbic acid, GSH, proline and ectoine are all absent.

[00172] By "comprises", it is understood that

[00173] either at least one of the following elements: DMSO, sucrose, inulin, ascorbic acid, GSH, proline and ectoine is absent, the other elements being in the concentrations defined above in formulation A or in formulation B different from 0 mm,

[00174] or all elements of DMSO, sucrose, inulin, ascorbic acid, GSH, proline and ectoine are present, with the above mentioned concentrations, with formulation A or in formulation B and are different from 0 mM.

[00175] In another embodiment, said aqueous solution formulation comprises or consists of:

[00176] - PBS

[00177] - 20 mM to about 300 mM trehalose

[00178] - 20 mM to about 300 mM sucrose

[00179] - 20 mM to about 400 mM autoclaved inulin (fructose equivalent)

[00180] - 20 mM to about 200 mM GSH

[00181] - 20 mM to about 150 mM ectoine

[00182] - 20 mM to about 150 mM of proline and is adjusted to pH 7.4 with sodium hydroxide.

[00183] By "consists of" it is meant that in the above formulation, the following elements: DMSO, ascorbic acid and EGCG are all absent.

[00184] By "comprises", it is understood that

[00185] either at least one of the following elements: DMSO, ascorbic acid and EGCG is absent, the other elements being at the concentrations defined above in formulation A or in formulation B other than 0 mm,

[00186] - Either all elements of DMSO, ascorbic acid and EGCG are present in the concentrations defined above in formulation A or in formulation B are different from 0 mm.

[00187] In one embodiment, said aqueous solution formulation comprises or consists of:

[00188] - DMEM

[00189] - Trehalose 0.1 M

[00190] - Sucrose 0.1 M

[00191] - 2.5% autoclaved inulin

[00192] - H GSH 0.1

[00193] - 1% ectoine

[00194] - 1% proline

[00195] and adjusted to pH 7.4 with sodium hydroxide.

[00196] By "consists of" it is meant that in the above formulation, the following elements: DMSO, ascorbic acid and EGCG are all absent.

[00197] By "comprises", it is understood that

[00198] either at least one of the following elements: DMSO, ascorbic acid and EGCG is absent, the other elements being at the concentrations defined above in formulation A or in formulation B are different from 0 mM,

[00199] or all elements of DMSO, ascorbic acid and EGCG are present at the concentrations defined above in formulation A or in formulation B and are different from 0 mm.

[00200] In another embodiment, said aqueous solution formulation comprises or consists of:

[00201] - DMEM

[00202] -0.1 M trehalose

[00203] - 5% Ascorbic Acid

[00204] and is adjusted to pH 7.4 with sodium hydroxide.

[00205] By "consists of" it is meant that in the above formulation, the following elements: DMSO, sucrose, inulin, EGCG, GSH, proline and ectoine are all absent.

[00206] By "comprises", it is understood that

[00207] - Or, at least, one of the following elements: DMSO, sucrose, inulin, EGCG, GSH, proline and ectoine is absent, the other elements being in the concentrations defined above in formulation A or in formulation B different from 0 mm,

[00208] or all elements of DMSO, sucrose, inulin, EGCG, GSH, proline and ectoine are present at the concentrations defined above in formulation A or in formulation B and are different from 0 mm.

[00209] In another embodiment, said aqueous solution formulation comprises or consists of:

[00210] - DMEM

[00211] - Trehalose 0.1 M

[00212] - Sucrose 0.1 M

[00213] - 5% inulin.

[00214] By "consists of" it is meant that in the above formulation, the following elements: DMSO, ascorbic acid, EGCG, GSH, proline and ectoine are all absent.

[00215] By "comprises", it is understood that

[00216] either at least one of the following elements: DMSO, ascorbic acid, EGCG, GSH, proline and ectoine is absent, the other elements being in the concentrations defined above in formulation A or in formulation B different from 0 mm,

[00217] or all elements of DMSO, ascorbic acid, EGCG, GSH, proline and ectoine are present at the concentrations defined above in formulation A or in formulation B and are different from 0 mM.

[00218] In another embodiment, said aqueous solution formulation comprises or consists of:

[00219] - DMEM

[00220] - Trehalose 0.2 M

[00221] - Sucrose 0.15 M

[00222] - H GSH 0.1

[00223] - 1% ectoine

[00224] - 1% proline

[00225] and is adjusted to pH 7.4 with sodium hydroxide.

[00226] By "consists of" it is meant that in the above formulation, the following elements: DMSO, inulin, ascorbic acid and EGCG are all absent.

[00227] By "comprises", it is understood that

[00228] either at least one of the following elements: DMSO, inulin, ascorbic acid and EGCG is absent, the other elements being in the concentrations defined above in formulation A or in formulation B different from 0 mm,

[00229] or all elements of DMSO, inulin, ascorbic acid and EGCG are present at the concentrations defined above in formulation A or in formulation B and are different from 0 mM.

[00230] In another embodiment, said aqueous solution formulation comprises or consists of:

[00231] - DMEM

[00232] - 10% DMSO.

[00233] By "consists of" it is meant that in the above formulation, the following elements: trehalose, sucrose, inulin, ascorbic acid, EGCG, GSH, proline and ectoine are all absent.

[00234] By "comprises", it is understood that

[00235] either at least one of the following elements: trehalose, sucrose, inulin, ascorbic acid, EGCG, GSH, proline and ectoine is absent, the other elements, being the concentrations defined above in formulation A or in formulation B and different from 0 mm,

[00236] or all elements of trehalose, sucrose, inulin, ascorbic acid, EGCG, GSH, proline and ectoine are present at the concentrations defined above in formulation A or in formulation B and is different from 0 mm.

[00237] In another embodiment, said aqueous solution formulation comprises or consists of:

[00238] - DMEM

[00239] - 0.1 M Trehalose

[00240] - 1 mg / mL of EGCG.

[00241] By "consists of" it is meant that in the above formulation, the following elements: DMSO, sucrose, inulin, ascorbic acid, GSH, proline and ectoine are all absent.

[00242] By "comprises", it is understood that

[00243] - Or, at least, one of the following elements: DMSO, sucrose, inulin, ascorbic acid, GSH, proline and ectoine is absent, the other elements being in the concentrations defined above in formulation A or in formulation B different from 0 mM ,

[00244] or all elements of DMSO, sucrose, inulin, ascorbic acid, GSH, proline and ectoine are present at the concentrations defined above in formulation A or in formulation B and are different from 0 mm.

[00245] In another embodiment, said aqueous solution formulation comprises or consists of:

[00246] - PBS

[00247] - 0.1 M Trehalose

[00248] - 0.1 M Sucrose

[00249] - 2.5% autoclaved inulin

[00250] - GSH at 0.1 M

[00251] - 1% ectoine

[00252] - 1% proline

[00253] and is adjusted to pH 7.4 with sodium hydroxide.

[00254] By "consists of" is meant that in the above formulation, the following elements: DMSO, ascorbic acid and EGCG are all absent.

[00255] By "comprises", it is understood that

[00256] either at least one of the following elements: DMSO, ascorbic acid and EGCG is absent, the other elements being at the concentrations defined above in formulation A or in formulation B other than 0 mm,

[00257] or all elements of DMSO, ascorbic acid and EGCG are present at the concentrations defined above in formulation A or in formulation B and are different from 0 mm.

[00258] In an advantageous embodiment, the present invention relates to a process as defined above, comprising the steps of:

[00259] - Suspending the same protozoan in an aqueous solution comprising the formulation

[00260] - DMEM, RPMI or PBS

[00261] - 0 mM to about 3000 mM in DMSO, in particular from 0 mm or 1405 mM,

[00262] - 0 mM to about 300 mM sucrose, in particular 0 mM, 100 mM or 150 mM,

[00263] - 0 mM to about 1000 mM trehalose, in particular 0 mM, 100 mM, 200 mM or 880 mM,

[00264] - 0 mM to about 400 mM inulin (fructose equivalent), in particular, 0 mM, 155 mM or 309 mM,

[00265] - 0 mM to about 400 mM ascorbic acid, in particular, 0 mM or 284 mM,

[00266] - 0 V to about 10 mm EGCG, in particular, 0 and 2 mm,

[00267] - 0 mM to about 200 mM GSH, in particular, 0 mM or 100 mM,

[00268] — 0 mM to about 150 mM proline, in particular, 0 mM or 87 mM,

[00269] - 0 mM to about 150 mM ectoine, in particular, 0 mM or 63 mM,

[00270] provided that the concentration of at least one of the components among DMSO, sucrose, trehalose, inulin, ascorbic acid, EGCG, GSH, proline or ectoine is different from 0 mm, to obtain a suspension of protozoa in which the said aqueous solution formulation.

[00271] - Freezing said suspension for 4 to 23 h, at atmospheric pressure, by reducing the initial temperature of the protozoan suspension to a temperature ranging from about -40 ° C to about - 80 ° C, in particular about of -75 °C, said initial temperature of the protozoan suspension comprised between about 15 °C to about 25 °C, in particular about 20 °C, and said lowering of the temperature to be carried out at a rate comprised between about from -0.1°C/min to about -10°C/min, in particular at a rate of about -1°C/min, to obtain a frozen suspension.

[00272] - Subject said frozen suspension to primary drying at -55 ° C for about 20 hours by hand at a pressure ranging from about 30 to about 80 Pa for 0-7 hours, then at a pressure of less than 2 Pa for the rest of the time to obtain a primary dry composition.

[00273] - Submitting said primary dry composition to secondary drying at a pressure of less than 2 Pa and at an increasing temperature from -55 ° C to + 5 ° C at a speed comprised between about 0.01 ° C / min at about 0.2°C/min, in particular at a rate of about 0.0625°C/min, said secondary drying comprising a temperature step of 1h at each increase of 15°C and said temperature maintained at + 5 °C for 4 h when said temperature is reached, to obtain said lyophilized composition.

[00274] In another advantageous embodiment, the present invention relates to a process as defined above, comprising the steps of:

[00275] - Suspending the same protozoan in an aqueous solution comprising the formulation

[00276] - DMEM, RPMI or PBS

[00277] - 0 mM to about 3000 mM in DMSO, in particular from 0 mm or 1,280 mM,

[00278] - 0 mM to about 300 mM sucrose, in particular 0 mM, 100 mM or 150 mM,

[00279] - 0 mM to about 1000 mM trehalose, in particular, 0 mM, 100 mM, 200 mM or 880 mM,

[00280] - 0 mM to about 400 mM inulin (fructose equivalent), in particular 0 mM, 155 mM or 309 mM,

[00281] - 0 mM to about 400 mM ascorbic acid, in particular, 0 mM or 284 mM,

[00282] - 0 mM to about 10 mM EGCG, in particular, 0 mM and 2 mM,

[00283] - 0 mM to about 200 mM GSH, in particular, 0 mM or 100 mM,

[00284] - 0 mM to about 150 mM proline, in particular, 0 mM or 87 mM,

[00285] - 0 mM to about 150 mM ectoine, in particular, 0 mM or 70 mM,

[00286] provided that the concentration of at least one of the components among DMSO, sucrose, trehalose, inulin, ascorbic acid, EGCG, GSH, proline or ectoine is different from 0 mm, to obtain a suspension of protozoa in which the said aqueous solution formulation.

[00287] - Freezing said suspension for 4 to 23 h, at atmospheric pressure, by reducing the initial temperature of the protozoan suspension to a temperature ranging from about -40 ° C to about - 80 ° C, in particular about of -75 °C, said initial temperature of the protozoan suspension comprised between about 15 °C to about 25 °C, in particular about 20 °C, and said lowering of the temperature to be carried out at a rate comprised between about from -0.1°C/min to about -10°C/min, in particular at a rate of about -1°C/min, to obtain a frozen suspension.

[00288] - Subject said frozen suspension to primary drying at -55 ° C for about 20 hours by hand at a pressure ranging from about 30 to about 80 Pa for 0 - 7 h, then at a pressure of less than 2 Pa for the rest of the time to obtain a primary dry composition.

[00289] - Submitting said primary dry composition to secondary drying at a pressure of less than 2 Pa and at an increasing temperature from -55 ° C to + 5 ° C at a speed comprised between about 0.01 ° C / min at about 0.2°C/min, in particular at a rate of about 0.0625°C/min, said secondary drying comprising a temperature step of 1h at each increase of 15°C and said temperature maintained at + 5 °C for 4 h when said temperature is reached, to obtain said lyophilized composition.

[00290] In another aspect, the invention relates to a freeze-dried composition as obtained by the process as defined above.

[00291] In another aspect, the invention relates to a lyophilized rehydrated composition comprising a lyophilized composition as defined above, in a rehydration medium, the composition wherein said lyophilized product contains lyophilized protozoa of an intracellular nature, is devoid of host cells by protozoa and contains a moisture content of less than 12% by weight, said rehydrated lyophilized composition being such that the protozoa contained therein are viable and infectious and said viable protozoa account for more than 1% of the protozoa lyophilized.

[00292] In an advantageous embodiment, the invention relates to a rehydrated lyophilized composition as defined above, in which the protozoa are viable and infectious after storage, before reconstitution, of said lyophilized composition for a period of time greater than two weeks at a temperature of -25 to 25 °C, in particular at a temperature from -25 °C to 2 °C, at a temperature of 2 to 8 °C, more particularly at a temperature of 2 to 6 °C, or at a temperature of 8 to 25 ° C.

[00293] In an advantageous embodiment, the present invention relates to a lyophilized rehydrated composition as defined above, wherein said rehydration medium is a culture medium for said protozoa.

[00294] In an advantageous embodiment, said rehydration medium is the same culture medium as the culture medium in the aqueous formulation composition in which the protozoa are suspended before lyophilization.

[00295] In an advantageous embodiment, said rehydration medium is DMEM.

[00296] In an advantageous embodiment, the present invention relates to a rehydrated lyophilized composition as defined above, comprising

[00297] - DMEM, RPMI or PBS

[00298] - 0 mM to about 300 mM sucrose, in particular 0 mM, 100 mM or 150 mM,

[00299] - 0 mM to about 1000 mm trehalose, in particular 0 mM, 100 mM, 200 mM or 880 mM,

[00300] - 0 mM to about 400 mM inulin (fructose equivalent), in particular 0 mM, 155 mM or 309 mM,

[00301] - 0 mM to about 400 mM ascorbic acid, in particular 0 mM or 284 mM,

[00302] - 0 mM to about 10 mM of EGCG, in particular, 0 and 2 mm,

[00303] - 0 mM to about 200 mM GSH, in particular, 0 mM or 100 mM,

[00304] - 0 mM to about 150 mM proline, in particular, 0 mM or 87 mM,

[00305] - 0 mM to about 150 mM ectoine, in particular, 0 mM or 63 mM,

[00306] provided that the concentration of at least one of the components among sucrose, trehalose, inulin, ascorbic acid, EGCG, GSH, proline or ectoine is different from 0 mm.

[00307] In another advantageous embodiment, the present invention relates to a rehydrated lyophilized composition as defined above, comprising

[00308] - DMEM, RPMI or PBS

[00309] - 0 mM to about 300 mM sucrose, in particular 0 mM, 100 mM or 150 mM,

[00310] - 0 mM to about 1000 mM trehalose, in particular 0 mM, 100 mM, 200 mM or 880 mM,

[00311] - 0 mM to about 400 mM inulin (fructose equivalent), in particular, 0 mM, 155 mM or 309 mM,

[00312] - 0 mM to about 400 mM ascorbic acid, in particular, 0 mM or 284 mM,

[00313] - 0 mM to about 10 mM EGCG, in particular, 0 and 2 mm,

[00314] - 0 mM to about 200 mM GSH, in particular, 0 mM or 100 mM,

[00315] - 0 mM to about 150 mM proline, in particular, 0 mM or 87 mM,

[00316] - 0 mM to about 150 mM ectoine, in particular, 0 mM or 70 mM,

[00317] provided that the concentration of at least one of the components among sucrose, trehalose, inulin, ascorbic acid, EGCG, GSH, proline or ectoine is different from 0 mm.

[00318] In another aspect, the present invention relates to a therapeutic composition or vaccine comprising the rehydrated lyophilized composition as defined above.

[00319] In another aspect, the present invention relates to a process for the reconstitution of a lyophilized composition, as defined above, that said lyophilized composition containing lyophilized protozoa of an intracellular nature, being devoid of host cells by protozoa and containing a moisture content of less than 12% by weight, which comprises a step of adding a rehydration medium to said lyophilized composition to obtain a lyophilized rehydrated composition in which said protozoa are viable and infectious, said Viable protozoa accounting for more than 1% of freeze-dried protozoa.

[00320] In an advantageous embodiment, the present invention relates to a process, as defined above, for obtaining a lyophilized rehydrated composition in which said protozoa are viable and infectious after storage, before reconstitution, of said composition lyophilized for a period of time longer than two weeks at a temperature of -25 to 25 °C, in particular at a temperature from -25 °C to 2 °C, at a temperature of 2 to 8 °C, more particularly at a temperature of 2 to 6 ° C, or at a temperature of 8 - 25 ° C.

[00321] In one embodiment, the present invention relates to a process as defined above, in which the rehydration medium is a culture medium for said protozoa.

[00322] In an advantageous embodiment, the rehydration medium is the same culture medium as the culture medium in the aqueous solution in which said formulation of protozoa are suspended before lyophilization.

[00323] In an advantageous embodiment, the rehydration medium is DMEM.

[00324] In an advantageous embodiment, the present invention relates to a process as defined above, in which said step of adding the rehydration medium is carried out at a rate comprised between about 25 μl, per second up to about 1000 μl per second, in particular at a rate of about 250 μl per second.

[00325] In another aspect, the present invention relates to a suspension of protozoa of an intracellular nature and devoid of host cells in a protozoal solution aqueous formulation, aqueous solution comprising said formulation:

[00326] - DMEM, RPMI or PBS

[00327] - 0 mM to about 3000 mM in DMSO, in joint, from 0 mm or 1.405 mM,

[00328] - 0 mM to about 300 mM sucrose, in particular 0 mM, 100 mM or 150 mM,

[00329] - 0 mM to about 1,000 trehalose, in particular, 0 mM, 100 mM, 200 mM or 880 mM,

[00330] - 0 mM to about 400 mM inulin (fructose equivalent), in particular, 0 mM, 155 mM or 309 mM,

[00331] - 0 mM to about 400 mM ascorbic acid, in particular, 0 mM or 284 mM,

[00332] - 0 mM to about 10 mM EGCG, in particular, 0 mM and 2 mM,

[00333] - 0 mM to about 200 mM GSH, in particular, 0 mM or 100 mM,

[00334] - 0 mM to about 150 mM proline, in particular, 0 mM or 87 mM,

[00335] - 0 mM to about 150 mM ectoine, in particular, 0 mM or 63 mM,

[00336] provided that the concentration of at least one of the components among DMSO, sucrose, trehalose, inulin, ascorbic acid, EGCG, GSH, proline or ectoine is different from 0 mm.

[00337] In another embodiment, the present invention relates to a suspension of protozoa of an intracellular nature and devoid of host cells in a protozoal solution aqueous formulation, aqueous solution comprising said formulation:

[00338] - DMEM, RPMI or PBS

[00339] - 0 mM to about 3000 mM in DMSO, in particular from 0 mM or 1280 mM,

[00340] - 0 mM to about 300 mM sucrose, in particular 0 mM, 100 mM or 150 mM,

[00341] - 0 mM to about 1000 trehalose, in particular 0 mM, 100 mM, 200 mM or 880 mM,

[00342] - 0 mM to about 400 mM inulin (fructose equivalent), in particular, 0 mM, 155 mM or 309 mM,

[00343] - 0 mM to about 400 mM ascorbic acid, in particular, 0 mM or 284 mM,

[00344] - 0 mM to about 10 mM of EGCG, in particular, 0 and 2 mm,

[00345] - 0 mM to about 200 mM GSH, in particular, 0 mM or 100 mM,

[00346] - 0 mM to about 150 mM proline, in particular, 0 mM or 87 mM,

[00347] - 0 mM to about 150 mM ectoine, in particular, 0 mM or 70 mM,

[00348] provided that the concentration of at least one of the components among DMSO, sucrose, trehalose, inulin, ascorbic acid, EGCG, GSH, proline or ectoine is different from 0 mm.

BRIEF DESCRIPTION OF THE DRAWINGS

[00349] This order does not contain a drawing.

DETAILED DESCRIPTION OF THE INVENTION

[00350] In this specification, "comprises", "comprising", "containing" and "having" and the like, may have the meaning assigned to them in US Patent law and may mean "includes", "including" , and the like; "Consisting essentially of, or "consisting essentially of" also has the meaning assigned in US Patent law and the term is open-ended, allowing the presence of more than what is recited, provided that the basic or novel features of what is recited is unaltered by the presence of more than what is recited, but excludes prior art embodiments.

[00351] A "subject" in the context of the present invention may be a vertebrate, such as a mammal, bird, reptile, amphibian or fish; more advantageously, a human, a domesticated or companion animal; the production of food or animal feed; livestock, game, racing or sport animals such as, but not limited to, bovine, canine, feline, caprine, ovine, swine, equine and poultry. Preferably, the vertebrate is a canine.

[00352] An "antigen" is a substance that is recognized by the immune system and induces an immune response. The antigen may comprise an entire organism, dead, attenuated, or alive; a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; a piece of nucleic acid or a fragment thereof capable of eliciting an immune response against presentation to a host animal; a protein, a polypeptide, a peptide, a glycoprotein, an epitope, a hapten, a carbohydrate, a sugar, or any combination thereof. Alternatively, the antigen may comprise a toxin or antitoxin. A similar term used interchangeably in this context is "immunogen". A "pathogenic agent" refers to a specific disease-causing agent, such as a bacterium, fungus, protozoan, parasite, or a virus.

[00353] As used herein, the terms "immunogenic composition" and "immunological composition" and "immunogenic or immunological composition" encompass any composition that elicits an immune response against the antigen or immunogen of interest expressed from vectors; for example, upon administration to a subject, elicits an immune response against the target immunogen or antigen of interest. The terms "vaccine composition" and "vaccine" and "vaccine composition" include any composition that elicits a protective immune response against the antigen of interest, or that effectively protects against the antigen; for example, upon administration or injection into the subject, induces a protective immune response against the target antigen or immunogen or provides effective protection against the antigen or immunogen expressed from vectors.

[00354] As used herein, the term "multivalent" means an immunogenic composition or vaccine composition containing more than one antigen, whether from the same species, from different species, or an immunogenic composition or vaccine composition containing a combination of antigens from different genera.

[00355] An "active immunogenic component" in the context of the present invention includes live attenuated pathogens, such as live attenuated, live attenuated viruses, bacteria, fungi or parasites. Also encompassed by the present invention are recombinant heterologous immunogens or antigens derived from or originating from one or more pathogens described herein, which may be contained and expressed in, inter alia, viral vectors, bacterial vectors, fungal vectors, and plasmid vectors. The invention also comprises the epitopes of heterologous immunogens or antigens derived from one or more pathogens, immunomodulators, such as cytokines, therapeutic agents, toxins, antibodies, antigen-binding fragments of an antibody, adjuvants, or other species, such as RNAs antisense, catalytic RNA, small interference RNAs, among others.

[00356] The term "veterinary composition" means any composition comprising a vector for veterinary use that expresses a therapeutic protein such as, for example, erythropoietin (EPO) or for an immunomodulatory protein, such as, for example, interferon (IFN). Likewise, the term "pharmaceutical composition" means any composition comprising a vector for expressing a therapeutic protein.

[00357] The compositions and methods of the present invention can be suitably applied for the stabilization of any pharmaceutical/veterinary substance/agent or combination of biological substance/biological agent plus agent. "Biologics" include, but are not limited to, immunomodulators, such as cytokines, therapeutic agents, toxins, antibodies, antigen-binding fragments of an antibody, adjuvants, or other species, such as antisense RNAs, catalytic RNAs, small interfering ANNs among others. After reconstitution of the vitrified materials/substances, these compounds can be used for disease prevention as prophylactic immunization or provide relief against disease symptoms as therapeutic immunization.

[00358] The invention encompasses a method for freeze-drying intracellular protozoan parasites, including parasites, from the Apicomplexa phylum and the Sarcocystidae family, and including T. gondii. Prior to freeze-drying, the protozoa can be combined with at least one stabilizer, eg sugars or antioxidant compounds.

[00359] In some embodiments, freeze-drying stabilizers may optionally comprise at least one non-reducing oligosaccharide and/or at least one bulking agent and/or at least one sugar alcohol. These stabilizers can preserve or help retain the immunogenicity, infectivity and viability of biological ingredients, including, but not limited to, viruses, bacteria, fungi, parasites, proteins, polypeptides, among others. Stabilizers used in the freeze-drying methods of the invention can have a good appearance, including, for example, uniform shape and color, and are safe for administration to a subject.

[00360] In some embodiments, the protozoan parasites are combined in a suspension comprising various safeners, which are present in the suspension to preserve the viability, infectiveness and immunogenicity of the parasites before, during and after freeze-drying.

[00361] "Non-reducing oligosaccharides" in the context of the present invention are sugars comprising from two to ten saccharide units and are incapable of reducing another compound during oxidation-reduction reactions. In the present invention, the non-reducing oligosaccharide may be a non-reducing disaccharide or a non-reducing trisaccharide, advantageously comprising trehalose, sucrose, or raffinose. The stabilizers of the invention can also comprise a mixture of at least two non-reducing oligosaccharides.

[00362] "Inulin" is a polysaccharide composed of chains of fructose units linked by bonds (fructosyl D-fructose) of various lengths with a glucose molecule at the end of each fructose chain. Inulin oligosaccharides (FOS) have been shown to protect the membrane from bacteria during freeze-drying and increase the survival of freeze-dried bacteria. However, until the present disclosure, it was not known whether eukaryotic T. gondii parasites could be protected by inulin during freezing.

[00363] "Polyethylene glycol (PEG)" are linear low molecular weight polyether polymers made from ethylene glycol monomers. It has been reported that associated with a mixture of sugar (trehalose, lactose or mannitol), 1% PEG maintains the activity of lyophilized enzymes (LDH) and phosphofructokinase (Prestrelski et al., 1993) PEG can be used in the practice of published lyophilization methods.

[00364] Polyvinylpyrrolidone "PVP-40" is a water-soluble polymer made from N-vinylpyrrolidone monomer. This polymer was used to protect proteins against freeze-drying damage. One study reported that the addition of PVP (40 kDa) and BSA to lactate dehydrogenase (LDH) resulted in stabilization of the enzyme during the freezing step by inhibiting the dissociation enzyme during freezing. (Wang W. 2000. UP. Lyophilization and the development of solid protein drugs.)

[00365] "Pluronic F68" is a non-ionic surfactant used to lower the surface tension of cells.

[00366] During the freezing process, the formation of an ice-water interface can cause protein denaturation, thus the presence of a surfactant in the solution will reduce protein adsorption or aggregation with the ice-water interface. The combination of PLURONIC F68 with other excipients such as sugars (trehalose) helped maintain the viability and infectivity of F. tularensis (intracellular bacteria) after foam drying (Ohtake S. et al., 2011).

[00367] "Gelatin" is a heterogeneous mixture of high average molecular weight, water-soluble, collagen-derived proteins and peptides. It is used in the pharmaceutical industry as a stabilizer and encapsulating agent. Gelatin (Vaccipro) was used by Ohtake S. et al. in a Francisella tularensis (intracellular bacteria) live vaccine formulation. (Satoshi O. et al., 2011. J Pharm Sci).

[00368] "Ectoin" is a cyclic organic tetrahydropyrimidine osmolyte discovered in halophilic bacteria. It has been characterized as an osmoprotectant and stabilizer for cells and biomolecules, and appears to preserve enzymes and whole cells against adverse growth conditions such as freezing, drying or heating. A combination of ectoine and proline was used to optimize a cryopreservation protocol for human cells. (Freimark D. et al. 2011. Cryobiology).

[00369] An "acidic antioxidant" compound is defined as a chemical compound that reacts with and neutralizes oxidants, free radicals (ie, molecules with unpaired electrons), or chemicals that release free radicals. In the context of the present invention, the antioxidant compound may be in acid form. Acid antioxidants include, but are not limited to, ascorbic acid and/or acidic amino acids, such as aspartic acid and glutamic acid. Combinations of antioxidant compounds with more than one acid are suitable components of lyophilized preparations according to the methods of the present description.

[00370] Thickening agents are also suitable components of vitrified compositions according to the present disclosure. Bulking agents can be pharmaceutically or veterinarily acceptable polymers, such as, but not limited to, dextran, maltodextrin, polyvinylpyrrolidone (PVP), crospovidone and hydroxyethyl starch. Other non-limiting examples of starch derivatives include microcrystalline cellulose, methyl cellulose, carboxy methyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose and hydroxypropyl methyl cellulose. Bulking agents increase the compositions' biological T'g value, which allows the use of higher temperatures during freezing. The "T'g value" is defined as the glass transition temperature, which corresponds to the temperature below which the frozen composition becomes glassy. The filler can help provide the good looks seen in the glazed doughs of this disclosure, that the doughs have the general appearance of light, fluffy, cotton candy.

[00371] Some components, including stabilizers, of biological preparations may not be readily soluble. However, it is well within the reach of one skilled in the art to substitute appropriately analogous components (e.g., selecting a more soluble component) and/or to adapt the amounts or amounts of the insoluble component present in the stabilizer for the purpose of obtaining a soluble stabilizer. . The solubility of a component can easily be checked by a visual solubility test. A solubility test comprises the steps of adding all of the stabilizer components at a temperature of about 55°C, and mixing for about 30 minutes. After approximately 24 hours at room temperature and without any agitation, the stabilizer can be visually checked for the appearance of precipitates. If the stabilizer is transparent or clear then all components of the stabilizer are soluble.

[00372] In the context of the present specification, the term "bulk vaccine composition" is intended to mean a composition coming out of the final stage of antigen production, purified or unpurified, monovalent, or after multivalent mixing. The term "a dry vaccine composition" is intended to mean a composition of which the residual water content is less than or equal to about 12%, for example about 4%, or about 3%, and which is ready to be be reconstituted with an aqueous solution in order to be used as a vaccine or directly in the form of dry particles. The vaccine composition can also be dried and milled and formulated with appropriate excipients, including binders, to produce suitable oral dosage units, for example tablets and pills.

[00373] The active immunogenic component can be selected from protozoa and their antigens, including, but not limited to, Plasmodium species, Trypanosome species, Giardia species, Boophilus species, Babesia species, Entamoeba species, Eimeria species, Leishmania species, Schistosoma species, Brugia species, Fascida species, Dirofilaria species, Wuchereria species, Onchocerea species, Treponema species, Toxoplasma species, Cryptococcus species, Coccidia species, Histomoniasis species, Hexamidos species, Giardia species, among others ; nematodes. Methods for preparing protozoan-derived immunogens are known in the prior art.

[00374] In the present invention, the active immunogenic component may also comprise a therapeutic agent, a cytokine, a toxin, an immunomodulator, a protein, a peptide, an antibody, an antigen-binding fragment of an antibody, an adjuvant or any another DNA-encoded molecule desired for delivery to an animal or animal cell or tissue.

[00375] The cooling step (b) can occur at temperatures lower than about -40°C (water freezing step). Drying of the stabilized immunogenic suspensions or solution by sublimation of ice under low pressure (c) can occur at, for example, pressure lower than or equal to about 80 Pa.

[00376] For its use and administration to a subject, the immunogenic composition or lyophilized vaccine composition can be reconstituted by rehydration with a solvent. The solvent is typically water, such as distilled or demineralized water, water-for-injection, but may also comprise physiological solutions or buffers, such as, for example, phosphate buffer solution (PBS), or adjuvants including, but not limited to , water-in-oil emulsions, Corynebacterium parvum, Bacillus Calmette Guerin, aluminum hydroxide, glucan, dextran sulfate, iron oxide, sodium alginate, Bacto-Adjuvant, certain synthetic polymers such as polyethylene amino acids and amino acid copolymers, saponin , "REGRESSIN" (Vetrepharm, Athens, Ga.), "AVPJDINE" (N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine), paraffin oil, muramyl dipeptide and the like. Other specific examples of adjuvants and adjuvant compositions are detailed herein.

[00377] Suitable adjuvants include fMLP (N-formyl-methionyl-leucyl-phenylalanine; US Patent No. 6017537) and/or acrylic acid or a polymer of methacrylic acid and/or a copolymer of maleic anhydride and alkenyl derivative. Acrylic acid or methacrylic acid polymers can be cross-linked, for example, with polyalkenyl ethers of sugars or polyalcohols. These compounds are known under the term "carbomer" (Pharmeuropa, Vol. 8, No. 2, June 1996). One skilled in the art may also refer to US Patent No. US 2,909,462 (incorporated by reference), which discusses such acrylic polymers crosslinked with a polyhydroxy compound containing at least 3 hydroxyl groups; a polyhydroxy compound contains not more than 8 hydroxyl groups; As another example, the hydrogen atoms of at least 3 hydroxyls are replaced with unsaturated aliphatic radicals containing at least 2 carbon atoms. The radicals can contain from about 2 to 4 carbon atoms, about, for example, vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. Products sold under the name Carbopol (Noveon Inc., Ohio, USA) are particularly suitable for use as adjuvants. They are crosslinked with an allyl sucrose or allylpentaerythritol with which mention is made of the products Carbopol 974P, 934P, 971P e.g.

[00378] As for maleic anhydride and alkenyl derivative copolymers, mention is made of EMA® products (Monsanto), which are maleic anhydride and ethylene copolymers, which can be linear or crosslinked, for example, crosslinked with ether divine. In addition, reference may be made to US Patent No. 6,713,068 and Regelson, W. et al, 1960; (Incorporated by reference).

[00379] Cationic lipids containing a quaternary ammonium salt are described in US Patent No. 6,713,068, whose contents are incorporated by reference, can also be used in the methods and compositions of the present invention. Among these cationic lipids, preference is given to DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propane ammonium; WO 96/34109), advantageously associated with a lipid neutral, advantageously DOPE (dioleoyl-phosphatidylethanolamine; Behr JP et al, 1994), to form DMRIE-DOPE.

[00380] The total content of reconstituted components ready-to-inject immunogenic compositions or vaccine compositions of the invention can be used to provide an injection with an isotonic concentration, for example, within the range of about 100-1200 mOsm, generally within about 250-600 mOsm, and preferably about 330 mOsm.

[00381] The dosages of live pathogens, namely T. gondii, of immunogenic compositions or stabilized vaccine composition, or in the reconstitution of immunogenic compositions or ready-to-inject vaccine compositions can vary from about 102 to about 107 DICC50 lyophilization / dose.

[00382] The reconstituted ready-to-use immunogenic vaccine compositions or compositions can be administered to an animal by injection via the parenteral or mucosal route, preferably intramuscularly and subcutaneously. However, administration of such ready-to-use immunogenic compositions or reconstituted vaccine compositions may also comprise intranasal, epicutaneous, topical or oral administration. The volume of an injection dose can be from about 0.1 ml to about 2.0 ml, and preferably about 1.0 ml.

[00383] The invention will now be further described by way of the following non-limiting examples, given by way of illustration of various embodiments of the invention and are not intended to limit the present invention in any way.

EXAMPLES

[00384] For the examples that follow, "Toxo KO" means an attenuated Toxoplasma parasite ("KO" means "knockout") and "Neo KO" means an attenuated Neospora parasite which can be found in the detailed instructions for strain production of attenuated/mutated parasites in the following documents: US 7964185 B2 (to CNRS et al), which describes the construction of attenuated Toxoplasma gondii having MICL and/or MIC3 gene deletions (referred to as: Toxo MICL KO, Toxo mic3 KO, and Toxo MICL-3 KO), WO 2014/020291 (to Vitamfero et al), which describes the construction of attenuated Neospora caniem having ncMICl and/or ncMIC3 gene deletions (referred to as: Neo ncmicl KO, Neo ncmic3 KO, and neo-ncmicl 3 KO) and WO 2014/020290 A2 (to VitamFero et al), which describe the construction of Neo ncmic3 KO, neo ncmicl KO and ncmicl Neo-3 KO strains. Unless otherwise specified, "Toxo KO" refers to Toxo MICL-3 KO strain and "Neo KO" refers to Neo ncmicl-3 KO.

Example 1: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution of formulation Fl

[00385] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e., Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, 0.1M autoclaved 2.5% inulin sucrose, 0.1M GSH (glutathione), 1% ectoin, 1% proline adjusted to pH 7.4 with sodium hydroxide (FL formulation). The concentration of the parasites is 1.107/ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. The samples were kept frozen at atmospheric pressure for 23h at a temperature lowered from 20°C to -75°C, according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4°C. Four batches of freeze-dried composition, also called a "plunge" or "cake" were produced on separate days. They contained between 4.78 and 4.97% of waste water.

Example 2: Reconstitution of the lyophilized composition of Example 1 after storage for 7 to 22 days at 4°C, measurement of viability and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine

[00386] Prior to use, lyophilized samples from the 4 batches produced in the example above were brought back to atmospheric pressure by gently opening glass vials after 7 to 22 days of storage at 4 °C. Dry cakes were resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of the resuspended tachyzoites, determined by flow cytometry, was between 3.5 and 67.5%.

[00387] An in vivo study was performed to evaluate the effectiveness of 4 batches of lyophilized tachyzoites as a vaccine against toxoplasmosis. For each batch, 3 to 6 mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites and a control mouse received the freeze-dried formulation bio-parasite free only (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased by 333%) to 372% for the mice inoculated with the lyophilized tachyzoites compared to the placebo group. Following a challenge with the RH strain of T. gondii on day 60, the mouse survival rate ranged from 83% to 100%) on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis.

Example 3: In vivo study of the infectivity of tachyzoites from the rehydrated lyophilized composition of Example 2

[00388] An in vivo study was performed to evaluate the infectivity of a batch of lyophilized tachyzoites produced in the example above. Two mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites and sacrificed on day 12. The spleen was removed and crushed. Splenocytes were extracted and added to cultured human foreskin fibroblast cells. After 9 days, parasites were observed in the culture medium for the 2 mice, confirming the infectivity of freeze-dried tachyzoites.

Example 4: Reconstitution of the lyophilized composition of Example 1 after storage for 60 days at 4°C, measurement of viability and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine.

[00389] A batch of freeze-dried samples produced in the example above were tested after 60 days of storage at 4 °C. The glass vials were brought back to atmospheric pressure by gently opening the glass vials after 60 days of storage . Residual water (water content) was 4.2%. Dry cakes were resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of resuspended tachyzoites, determined by flow cytometry, was 9.4%.

[00390] An in vivo study was performed to evaluate the effectiveness of this batch of lyophilized tachyzoites as a vaccine against toxoplasmosis. Six mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 freeze-dried tachyzoites and 2 control mice received the freeze-dried formulation bio-parasite free only (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased in 270% of the mice inoculated with the freeze-dried tachyzoites compared to the placebo group. Following a challenge with the T. gondii RH strain on day 60, the mouse survival rate was 50% on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis after 60 days of storage at 4 °C.

Example 5: Reconstitution of the lyophilized composition of Example 1 after storage for 60 days at -20°C, measurement of viability and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine.

[00391] A batch of freeze-dried samples produced in the above example were tested after 60 days of storage at -20°C. The glass vials were brought back to atmospheric pressure by gently opening glass vials after 60 days of storage. storage. Residual water (water content) was 7.0%. Dry cakes were resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of the resuspended tachyzoites, determined by flow cytometry, was 10.1%.

[00392] An in vivo study was performed to evaluate the effectiveness of this batch of lyophilized tachyzoites as a vaccine against toxoplasmosis. Six mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites and a control mouse received the lyophilized parasite-free bioformulation alone (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased in 483% of the mice inoculated with the freeze-dried tachyzoites compared to the placebo group. Following a challenge with the T. gondii RH strain on day 60, the mouse survival rate was 67% on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis after 60 days of storage at -20 °C.

Example 6: Lyophilization of dead Toxo KO tachyzoites suspended in the aqueous solution of the Fl formulation (negative control)

[00393] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e., Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, 0.1M autoclaved 2.5% inulin sucrose, 0.1M GSH (glutathione), 1% ectoine, 1% proline adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1 x 107 / ml. This parasite suspension was then placed in a water bath and kept at 56 °C for 90 minutes to kill the parasites. An aliquot of this suspension was then added to Vera cultured cells to assess the infectivity of the parasites. After 5 days, no parasitic vacuoles or plaque-forming units were observed confirming the death of the parasites. One ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C. The freeze-dried composition, also called a "lozenge" or "cake" contained 4.85% residual water.

Example 7: Reconstitution of the lyophilized composition from Example 6 and in vivo study of the effectiveness of the rehydrated lyophilized composition as a vaccine (negative control.

[00394] Prior to use, lyophilized samples were brought back to atmospheric pressure by gently opening glass vials. Dry cakes were resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second.

[00395] An in vivo study was performed to evaluate the effectiveness of batch freeze-dried dead tachyzoites as a vaccine against toxoplasmosis. Six mice were inoculated by intraperitoneal injection on day 0 with a dose of 4106 freeze-dried dead tachyzoites. The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased by 206%. Following a challenge with the RH strain of T. gondii on day 60, 5 out of 6 mice died leading to a 17% survival rate on day 90. These results confirm the inability of freeze-dried killed tachyzoites to effectively vaccinate and protect mice against toxoplasmosis.

Example 8: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution of formulation F2

[00396] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e., Dulbecco's Modified Eagle Medium) supplemented with 0.1 M trehalose and 5% ascorbic acid adjusted to pH 7.4 with sodium hydroxide ( formulation F2). The concentration of the parasites is 1 x 107 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C. Two batches of freeze-dried composition, also called a "pellet" or "cake" were produced on separate days. They contained between 9.6 and 9.8% residual water.

Example 9: Reconstitution of the lyophilized composition of Example 8, after storage for 7 to 8 days at 4°C, measurement of viability and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine

[00397] Prior to use, freeze-dried samples of the 2 batches produced in the above example were brought back to atmospheric pressure by gently opening glass vials after 7 to 8 days of storage at 4°C. Dry cakes were resuspended in 1 ml of DMEM (re-hydration medium) introduced at a flow rate of 250 μl/second. The viability of the resuspended tachyzoites, determined by flow cytometry, was between 17% and 66.3%.

[00398] An in vivo study was performed to evaluate the effectiveness of 2 batches of lyophilized tachyzoites as a vaccine against toxoplasmosis. For each batch, 3 to 6 mice were inoculated by intraperitoneal injection on day 0 with a dose of 4 x 106 lyophilized tachyzoites and a control mouse received the freeze-dried formulation bio-parasite free only (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased from 256% to 286% for the mice inoculated with the freeze-dried tachyzoites compared to the placebo group. Following a challenge with the RH strain of T. gondii on day 60, the mouse survival rate ranged from 67% to 100%) on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis.

Example 10: Reconstitution of the lyophilized composition of Example 8, after storage for 60 days at 4°C, measurement of viability and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine

[00399] A batch of freeze-dried samples produced in the example above were tested after 60 days of storage at 4 °C. The glass vials were brought back to atmospheric pressure by gently opening the glass vials after 60 days of storage . Residual water (water content) was 10.7%. Dry cakes were resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of resuspended tachyzoites, determined by flow cytometry, was 9.8%.

[00400] An in vivo study was performed to evaluate the effectiveness of this batch of lyophilized tachyzoites as a vaccine against toxoplasmosis. Six mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 freeze-dried tachyzoites and 2 control mice received the freeze-dried formulation bio-parasite free only (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased in 410% of the mice inoculated with the freeze-dried tachyzoites compared to the placebo group. Following a challenge with the T. gondii RH strain on day 60, the mouse survival rate was 83% on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis after 60 days of storage at 4 °C.

Example 11: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution formulation of Formulation F3

[00401] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1,500g. The supernatant was removed and the pellet resuspended in an aqueous formulation consisting of DMEM (i.e. Dulbecco's modified eagle medium) supplemented with 0.1 M trehalose, 0.1 M sucrose, 5% inulin (formulation F3) . The concentration of the parasites is 1 X 107 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa for the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and the freeze dryer shelf temperature was increased from -55 °C to +5 °C at a rate of 0.0625 °C per minute with a plateau lh upon reaching each 15 °C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C. Two batches of freeze-dried composition, also called a "pellet" or "cake" were produced on separate days. They contained between 5.0 and 6.9% residual water.

Example 12: Reconstitution of the lyophilized composition of Example 11 after storage for 7 to 8 days at 4°C, measurement of viability and in vivo study of the effectiveness of the rehydrated lyophilized composition as a vaccine.

[00402] Prior to use, the lyophilized samples from 2 batches produced in the example above were brought back to atmospheric pressure by gently opening glass vials after 7 to 8 days of storage at 4 °C. Dry cakes were resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of the resuspended tachyzoites, determined by flow cytometry, was between 18.9% and 72.5%.

[00403] An in vivo study was performed to evaluate the effectiveness of 2 batches of lyophilized tachyzoites as a vaccine against toxoplasmosis. For each batch, 3 to 6 mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites and a control mouse received the freeze-dried formulation bio-parasite free only (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased by 101% to 246% for the mice inoculated with the freeze-dried tachyzoites compared to the placebo group. Following a challenge with the RH strain of T. gondii on day 60, the mouse survival rate ranged from 67% to 100%) on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis.

Example 13: Reconstitution of the lyophilized composition of Example 11 after storage for 60 days at 4°C, measurement of viability and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine

[00404] A batch of freeze-dried samples produced in the example above was tested after 60 days of storage at 4 °C. The glass vials were brought back to atmospheric pressure by gently opening the glass vials after 60 days of storage . Residual water (water content) was 7.0%. Dry cakes were resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of resuspended tachyzoites, determined by flow cytometry, was 5.2%.

[00405] An in vivo study was performed to evaluate the effectiveness of this batch of lyophilized tachyzoites as a vaccine against toxoplasmosis. Six mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites and 2 control mice received the parasite-free lyophilized bioformulation alone (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased in 255% of the mice inoculated with the freeze-dried tachyzoites compared to the placebo group. Following a challenge with the T. gondii RH strain on day 60, the mouse survival rate was 67% on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis after 60 days of storage at 4 °C.

Example 14: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution formulation of Formulation F4

[00406] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e., Dulbecco's Modified Eagle Medium) supplemented with 0.2M trehalose, 0.15M sucrose, 0.1M GSH (glutathione), 1% ) ectoin, 1% proline adjusted to pH 7.4 with sodium hydroxide (formulation F4). The concentration of the parasites is 1 X 107 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with a plateau of 1h after reaching each 15°C increase. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4°C. Two batches of lyophilized composition, also called a "lozenge" or "cake" were produced on separate days. They contained between 5.7 and 7.0% residual water.

Example 15: Reconstitution of the lyophilized composition of Example 14 after storage for 7 to 8 days at 4°C, measurement of viability and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine.

[00407] Prior to use, freeze-dried samples from 2 batches produced in the example above were brought back to atmospheric pressure by gently opening glass vials after 7 to 8 days of storage at 4°C. Dry cakes were resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of the resuspended tachyzoites, determined by flow cytometry, was between 11.2% and 72.6%.

[00408] An in vivo study was performed to evaluate the effectiveness of 2 batches of lyophilized tachyzoites as a vaccine against toxoplasmosis. For each batch, 3 to 6 mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites and a control mouse received the freeze-dried formulation bio-parasite free only (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased from 314% to 337% for the mice inoculated with the freeze-dried tachyzoites compared to the placebo group. Following a challenge with the RH strain of T. gondii on day 60, the mouse survival rate ranged from 83% to 100%) on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis.

Example 16: Reconstitution of the lyophilized composition of Example 14 after storage for 60 days at 4°C, measurement of viability and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine

[00409] A batch of freeze-dried samples produced in the above example was tested after 60 days of storage at 4 °C. The glass vials were brought back to atmospheric pressure by gently opening the glass vials after 60 days of storage . Residual water (water content) was 5.8%. Dry cakes were resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of resuspended tachyzoites, determined by flow cytometry, was 4.2%.

[00410] An in vivo study was performed to evaluate the effectiveness of this batch of lyophilized tachyzoites as a vaccine against toxoplasmosis. Six mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 freeze-dried tachyzoites and 2 control mice received the freeze-dried formulation bio-parasite free only (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased in 390% of the mice inoculated with the freeze-dried tachyzoites compared to the placebo group. Following a challenge with the T. gondii RH strain on day 60, the mouse survival rate was 83% on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis after 60 days of storage at 4 °C.

Example 17: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution formulation of Formulation F5.

[00411] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (ie Dulbecco's Modified Eagle Medium) supplemented with 10% DMSO (ie dimethyl sulfoxide) (formulation F5). The concentration of the parasites is 1 X 107 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4°C. A batch of freeze-dried composition, also called a "chip" or "cake", was produced.

Example 18: Reconstitution of the lyophilized composition of Example 17 after storage for 7 days at 4°C, measurement of viability and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine.

[00412] Before use, the lyophilized batch sample produced in the example above was brought back to atmospheric pressure by gently opening glass vials after 7 days of storage at 4°C. The dry cake was resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of the resuspended tachyzoites, determined by flow cytometry, was 57.6%.

[00413] An in vivo study was performed to evaluate the effectiveness of the batch of lyophilized tachyzoites as a vaccine against toxoplasmosis. Three mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites and a control mouse received the freeze-dried formulation bio-parasite free only (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased by 94% for the mice inoculated with the freeze-dried tachyzoites compared to the placebo group. Following a challenge with the T. gondii RH strain on day 60, the mouse survival rate was 67% on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis.

Example 19: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution formulation of Formulation F6.

[00414] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1,500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (ie Dulbecco's Modified Eagle Medium) supplemented with 0.1 M trehalose and 1 mg/ml EGCG (epigallocatechin gallate) (formulation F6). The concentration of the parasites is 1.10 7 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4°C. A batch of freeze-dried composition, also called a "chip" or "cake", was produced and contained 10.4% residual water.

Example 20: Reconstitution of the lyophilized composition of Example 19 after storage for 7 days at 4°C, viability measurement and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine.

[00415] Before use, the lyophilized batch sample produced in the example above was brought back to atmospheric pressure by gently opening glass vials after 7 days of storage at 4 °C. Dry cake was resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of resuspended tachyzoites, determined by flow cytometry, was 30.8%.

[00416] An in vivo study was performed to evaluate the effectiveness of batch freeze-dried tachyzoites as a vaccine against toxoplasmosis. Three mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites and a control mouse received the freeze-dried formulation bio-parasite free only (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased in 342% of the mice inoculated with the freeze-dried tachyzoites compared to the placebo group. Following a challenge with the T. gondii RH strain on day 60, the mouse survival rate was 67% on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis.

Example 21: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution formulation of Formulation F7

[00417] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation composed of PBS (Phosphate Buffered Saline Solution ie) supplemented with 0.1 M trehalose, 0.1 M sucrose, 2.5% autoclaved inulin, 0. 1M GSH (glutathione), 1% ectoin, 1% proline adjusted to pH 7.4 with sodium hydroxide (formulation F7). The concentration of the parasites is 1 X 107 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4°C. A batch of freeze-dried composition, also called a "pellet" or "cake" was produced and contained 4.0% residual water.

Example 22: Reconstitution of the lyophilized composition of Example 21 after storage for 7 days at 4°C, measurement of viability and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine

[00418] Before use, the lyophilized batch sample produced in the example above was brought back to atmospheric pressure by gently opening glass vials after 7 days of storage at 4 °C. Dry cake was resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of the resuspended tachyzoites, determined by flow cytometry, was 64.6%.

[00419] An in vivo study was performed to evaluate the effectiveness of the batch of lyophilized tachyzoites as a vaccine against toxoplasmosis. Three mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites and a control mouse received the freeze-dried formulation bio-parasite free only (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased in 337% of the mice inoculated with the freeze-dried tachyzoites compared to the placebo group. Following a challenge with the T. gondii RH strain on day 60, the mouse survival rate was 67% on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis.

Example 23: Lyophilization of Toxo KO tachyzoites suspended in the aqueous solution formulation of the F1 formulation, after a first treatment of distilled water with 2.5% trehalose

[00420] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in a medium composed of DMEM (i.e., Dulbecco's Modified Eagle Medium) supplemented with 0.1 M trehalose, 0.1 M sucrose, 2.5% autoclaved inulin, 0. 1M GSH (glutathione), 1% ectoine, 1% proline adjusted to pH 7.4 with sodium hydroxide. After 30 minutes at 37°C, the suspension was centrifuged for 10 minutes at 1,500g. The supernatant was removed and the pellet resuspended in distilled water, 2.5% trehalose was added. After 1h at 37°C, the suspension was centrifuged for 10 min at 1,500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, inulin, 0.1M sucrose, 2.5% autoclaved, 0. 1M GSH (glutathione), 1% ectoine, 1% proline adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1 X 107 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4°C. A batch of freeze-dried composition, also called a "pellet" or "cake" was produced and contained 4.8% residual water.

Example 24: Reconstitution of the lyophilized composition of Example 23 after storage for 7 days at 4°C, measurement of viability and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine

[00421] Before use, the lyophilized batch sample produced in the example above was brought back to atmospheric pressure by gently opening glass vials after 7 days of storage at 4°C. The dry cake was resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of the resuspended tachyzoites, determined by flow cytometry, was 55.0%.

[00422] An in vivo study was performed to evaluate the effectiveness of batch freeze-dried tachyzoites as a vaccine against toxoplasmosis. Three mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites and a control mouse received the freeze-dried formulation bio-parasite free only (placebo). The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased in 226% of the mice inoculated with the freeze-dried tachyzoites compared to the placebo group. Following a challenge with the T. gondii RH strain on day 60, the mouse survival rate was 33% on day 90, the stage at which all mice receiving the placebo died after the challenge. These results confirm the ability of lyophilized tachyzoites to effectively vaccinate and protect mice against toxoplasmosis.

Example 25: Lyophilization of Toxo KO tachyzoites suspended in the formulation of the aqueous solution of formulation F5 by a second lyophilization process (Contra example-1)

[00423] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (ie, Dulbecco's Modified Eagle Medium) supplemented with 10% DMSO (ie, dimethyl sulfoxide). The concentration of the parasites is 2.5 X 107 / ml. 0.2 ml of such a parasite suspension was dispensed into 2 ml glass vials, which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 4 hours at a reduced temperature from 20°C to -55°C according to a kinetics of -1°C per minute. At the end of the freezing period, the lyophilization chamber was rendered inert with nitrogen gas before initiating drying at -40°C for 4h. The freeze dryer shelf temperature was then increased from -40°C to +5°C at a rate of 0.0625°C per minute with a plateau after 4h reaching each 15°C increase. The pressure was maintained below 50 Pa throughout the drying process. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C. The freeze-dried composition is also called a "lozenge" or "cake".

Example 26: Reconstitution of the lyophilized composition of Example 25 after storage for 7 days at 4°C, measurement of viability and in vivo study of the efficacy of the rehydrated lyophilized composition as a vaccine (Contra example-1)

[00424] Before use, the lyophilized batch sample produced in the example above was brought back to atmospheric pressure by gently opening glass vials after 7 days of storage at 4°C. Dry cake was resuspended in 0.5 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of resuspended tachyzoites, determined by flow cytometry, was 30.0%.

[00425] An in vivo study was performed to evaluate the effectiveness of batch freeze-dried tachyzoites as a vaccine against toxoplasmosis. Five mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites. The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased by 83% on day 30 compared to day 0. Following a challenge with the RH strain of T. gondii on day 60, all mice died before day 90. These results show that viable parasites can lead to an ineffective vaccine and suggest that freeze-dried tachyzoites must be viable and infectious to protect mice against toxoplasmosis.

Example 27: Lyophilization of Toxo KO suspended in the aqueous solution formulation of the F2 formulation by a second lyophilization process (Counter example-2)

[00426] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1,500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (ie, Dulbecco's Modified Eagle Medium) supplemented with 0.1 M trehalose and 5% ascorbic acid. The concentration of the parasites is 2.5 x 107 / ml. 0.2 ml of such a parasite suspension was dispensed into 2 ml glass vials, which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 4 hours at a reduced temperature from 20°C to -55°C according to a kinetics of -1°C per minute. At the end of the freezing period, the lyophilization chamber was rendered inert with nitrogen gas before initiating drying at -40°C for 4h. The freeze dryer shelf temperature was then increased from -40°C to +5°C at a rate of 0.0625°C per minute with a 4H hold upon reaching each 15°C rise. The pressure was maintained below 50 Pa throughout the drying process. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C. The freeze-dried composition is also called a "lozenge" or "cake".

Example 28: Reconstitution of the lyophilized composition of Example 27 after storage for 7 days at 4°C, measurement of viability and in vivo study of the efficacy of the lyophilized rehydrated composition as a vaccine (Contra example-2)

[00427] Before use, the lyophilized batch sample produced in the above example was brought back to atmospheric pressure by gently opening glass vials after 7 days of storage at 4°C. Dry cake was resuspended in 0.5 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of the resuspended tachyzoites, determined by flow cytometry, was 68.0%.

[00428] An in vivo study was performed to evaluate the effectiveness of batch freeze-dried tachyzoites as a vaccine against toxoplasmosis. Two mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites. The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased by 219% on day 30 compared to day 0. Following a challenge with the RH strain of T. gondii on day 60, all mice died before day 90. These results show that viable parasites can lead to an ineffective vaccine and suggest that freeze-dried tachyzoites must be viable and infectious to protect mice against toxoplasmosis.

Example 29: Lyophilization of Toxo KO tachyzoites suspended in the aqueous solution formulation of formulation F6 by a second lyophilization process (Counter example-3)

[00429] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (ie Dulbecco's Modified Eagle Medium) supplemented with 0.1 M trehalose and 1 mg/ml EGCG (epigallocatechin gallate). The concentration of the parasites is 2.5 X 107 / ml. 0.2 ml of such a parasite suspension was dispensed into 2 ml glass vials, which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 4 hours at a reduced temperature from 20°C to -55°C according to a kinetics of -1°C per minute. At the end of the freezing period, the lyophilization chamber was rendered inert with nitrogen gas before initiating drying at -40°C for 4h. The freeze dryer shelf temperature was then increased from -40°C to +5°C at a rate of 0.0625°C per minute with a 4H hold upon reaching each 15°C rise. The pressure was maintained below 50 Pa throughout the drying process. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C. The freeze-dried composition is also called a "lozenge" or "cake".

Example 30: Reconstitution of the lyophilized composition of Example 29 after storage for 7 days at 4 °C, measurement of viability and in vivo study of the efficacy of the lyophilized rehydrated composition as a vaccine (Contra example-3)

[00430] Before use, the lyophilized batch sample produced in the example above was brought back to atmospheric pressure by gently opening glass vials after 7 days of storage at 4°C. Dry cake was resuspended in 0.5 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of the resuspended tachyzoites, determined by flow cytometry, was 77.7%.

[00431] An in vivo study was performed to evaluate the effectiveness of batch freeze-dried tachyzoites as a vaccine against toxoplasmosis. Five mice were inoculated by intraperitoneal injection on day 0 with a dose of 4.10 6 lyophilized tachyzoites. The humoral immune response of mice was quantified on day 30 by titration of specific IgG antibodies against T. gondii in serum. The IgG titer was increased by 186% on day 30 compared to day 0. Following a challenge with the RH strain of T. gondii on day 60, all mice died before day 90. These results show that viable parasites can lead to an ineffective vaccine and suggest that freeze-dried tachyzoites must be viable and infectious to protect mice against toxoplasmosis.

Example 31: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution formulation of Formulation F8

[00432] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1 M trehalose, 0.1 M sucrose, 2.5% autoclaved inulin, 0.1M GSH (glutathione), 1% ectoine, 1% proline, 0.1% Pluronic F68 adjusted to pH 7.4 with sodium hydroxide (formulation F8). The concentration of the parasites is 1 X 107 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

Example 32: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution formulation of Formulation F9

[00433] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, inulin, 0.1M sucrose, 2.5% autoclaved, 0. 1M GSH (glutathione), 1% ectoin, 1% proline, 1% polyvinylpyrrolidone (PVP-40) adjusted to pH 7.4 with sodium hydroxide (formulation F9). The concentration of the parasites is 1.10 7 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

Example 33: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution formulation of Formulation F10

[00434] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, inulin, 0.1M sucrose, 2.5% autoclaved, 0. 1M GSH (glutathione), 1% ectoine, 1% proline, 1% polyethylene glycol (PEG-300), adjusted to pH 7.4 with sodium hydroxide (formulation F10). The concentration of the parasites is 1.10 7 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

Example 34: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution formulation of Fll formulation

[00435] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, inulin, 0.1M sucrose, 2.5% autoclaved, 0. 1M GSH (glutathione), 1% ectoin, 1% proline, 2% sodium alginate adjusted to pH 7.4 with sodium hydroxide (1 Fl formulation). The concentration of the parasites is 1.10 7 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure between 30 and maintained at 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and the freeze dryer shelf temperature was increased from -55 °C to +5 °C at a rate of 0.0625 °C per minute, with a plateau lh upon reaching each 15 °C rise. Secondary drying was completed through keeping samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

Example 35: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution formulation of Formulation F12

[00436] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1,500g. The supernatant was removed and the pellet resuspended in an aqueous formulation composed of PBS (Phosphate Buffered Saline Solution ie) supplemented with 0.1 M trehalose, 0.1 M sucrose, 2.5% autoclaved inulin and 5% of gelatin (formulation F12). The concentration of the parasites is 1.10 7 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

Example 36: Lyophilization of Toxo KO tachyzoites suspended in an aqueous solution formulation of Formulation F13

[00437] A suspension of Toxo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation composed of PBS (Phosphate Buffered Saline Solution ie) supplemented with 30% trehalose, 5% gelatin and 0.02% Pluronic F68 (F13 formulation). The concentration of the parasites is 1.10 7 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

Example 37: Lyophilization of KO Neo tachyzoites suspended in an aqueous solution formulation of Formulation F8

[00438] A suspension of Neo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e., Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, inulin, 0.1M sucrose, 2.5% autoclaved, 0, 1M GSH (glutathione), 1% ectoine, 1% proline, 0.1% Pluronic F68 adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1 X 107 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

Example 38: Lyophilization of Neo tachyzoites KO suspended in an aqueous solution formulation of Fl formulation

[00439] A suspension of Neo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1,500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, inulin, 0.1M sucrose, 2.5% autoclaved, 0. 1M GSH (glutathione), 1% ectoin, 1% proline, adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1.10 7 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C. The freeze-dried composition, also called a "lozenge" or "cake" contained 5.71% residual water.

EXAMPLE 38 bis: Reconstitution of the lyophilized composition of Example 38 after storage for 7 days at 4°C and measuring viability of the rehydrated lyophilized composition

[00440] Before use, the lyophilized batch sample produced in the above example was brought back to atmospheric pressure by gently opening glass vials after 7 days of storage at 4°C. The dry cake was resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of resuspended tachyzoites, determined by flow cytometry, was 8.5%.

Example 39: Lyophilization of Neo tachyzoites KO suspended in an aqueous solution formulation of Formulation F3

[00441] A suspension of Neo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (ie Dulbecco's Modified Eagle Medium) supplemented with 0.1 M trehalose, 0.1 M sucrose and 5% inulin. The concentration of parasites is 1.10 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C. The freeze-dried composition, also called a "lozenge" or "cake" contained 5.95% residual water.

Example 39 bis: Reconstitution of the lyophilized composition of Example 39 after storage for 7 days at 4 °C and measuring feasibility of the rehydrated lyophilized composition

[00442] Before use, the lyophilized batch sample produced in the example above was brought back to atmospheric pressure by gently opening glass vials after 7 days of storage at 4 °C. The dry cake was resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of resuspended tachyzoites, determined by flow cytometry, was 5.7%.

Example 40: Lyophilization of KO Neo tachyzoites suspended in an aqueous solution formulation of F3 formulation

[00443] A suspension of Neo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1,500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (ie Dulbecco's Modified Eagle Medium) supplemented with 0.1 M trehalose, 0.1 M sucrose and 5% autoclaved inulin. The concentration of the parasites is 1.10 7 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa for the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and the freeze dryer shelf temperature was increased from -55 °C to +5 °C at a rate of 0.0625 °C per minute with a plateau lh upon reaching each 15 °C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

Example 40 bis: Lyophilization of Neo tachyzoites KO suspended in an aqueous solution formulation of Formulation F2

[00444] A suspension of Neo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (ie Dulbecco's Modified Eagle Medium) supplemented with 0.1 M trehalose and 5% ascorbic acid adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1 X 107 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C. The freeze-dried composition, also called a "lozenge" or "cake" contained 9.08% residual water.

Example 40b: Reconstitution of the lyophilized composition of Example 40bis after storage for 7 days at 4°C and measurement of the viability of the rehydrated lyophilized composition

[00445] Prior to use, the lyophilized batch sample produced in the example above was brought back to atmospheric pressure by gently opening glass vials after 7 days of storage at 4°C. The dry cake was resuspended in 1 ml of DMEM (rehydration medium) introduced with a flow rate of 250 μl/second. The viability of resuspended tachyzoites, determined by flow cytometry, was 11.7%.

Example 41: Lyophilization of Neo tachyzoites KO suspended in an aqueous solution formulation of Formulation F14

[00446] A suspension of Neo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1 M trehalose, 0.1 M sucrose and 5% autoclaved inulin (formulation F14) . The concentration of the parasites is 1 X 107 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

EXAMPLE 41bis: Lyophilization of Neo tachyzoites KO suspended in an aqueous solution formulation of Formulation F4.

[00447] A suspension of Neo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.2M trehalose, 0.15M sucrose, 0.1M GSH (glutathione), 1% ectoin, 1% proline adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1.10 7 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55°C for 20h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and the freeze dryer shelf temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. of samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C. The lyophilized composition, also called a "lozenge" or "cake" contained 5.96% residual water.

Example 41b: Reconstitution of the lyophilized composition of Example 41bis after storage for 7 days at 4°C and measurement of the viability of the rehydrated lyophilized composition

[00448] Before use, the lyophilized batch sample produced in the example above was brought back to atmospheric pressure by gently opening glass vials after 7 days of storage at 4°C. The dry cake was resuspended in 1 ml of DMEM (rehydration medium) introduced at a flow rate of 250 μl/second. The viability of resuspension tachyzoites, determined by flow cytometry, was 10.0%.

Example 42: Lyophilization of Neo KO tachyzoites suspended in an aqueous solution formulation of Formulation F9.

[00449] A suspension of Neo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, inulin, 0.1M sucrose, 2.5% autoclaved, 0. 1M GSH (glutathione), 1% ectoine, 1% proline, 1% polyvinylpyrrolidone (PVP-40) adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1.10 7 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

Example 43: Lyophilization of Neo tachyzoites KO suspended in an aqueous solution formulation of Formulation F10.

[00450] A suspension of Neo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, inulin, 0.1M sucrose, 2.5% autoclaved, 0. 1M GSH (glutathione), 1% ectoine, 1% proline, 1% polyethylene glycol (PEG-300), adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1.10 7 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

Example 44: Lyophilization of Neo tachyzoites KO suspended in an aqueous solution formulation of Formulation F12.

[00451] A suspension of Neo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1, 500g. The supernatant was removed and the pellet resuspended in an aqueous formulation composed of PBS (Phosphate Buffered Saline Solution ie) supplemented with 0.1 M trehalose, 0.1 M sucrose, 2.5% autoclaved inulin, 5% of gelatin. The concentration of the parasites is 1 X 107 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

Example 45: Lyophilization of Neo tachyzoites KO suspended in an aqueous solution formulation of Formulation F13.

[00452] A suspension of Neo KO tachyzoites in a culture medium, freshly egressed from host cells, was centrifuged for 10 minutes at 1500g. The supernatant was removed and the pellet resuspended in an aqueous formulation composed of PBS (Phosphate Buffered Saline Solution ie) supplemented with 30% trehalose, 5% gelatin and 0.02% Pluronic F68. The concentration of the parasites is 1 X 107 / ml. After an LH equilibration period, 1 ml of such a parasite suspension was dispensed into 10 ml glass vials which were placed on the shelf of a lyophilizer. Samples were kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying was carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure was maintained below 2 Pa and on the lyophilizer shelf the temperature was increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. holding samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials were mechanically sealed. The vacuum was then gently broken and the samples stored at 4 °C.

Example 46: Lyophilization of Neospora caniemum tachyzoites suspended in an aqueous solution formulation of Formulation Fl.

[00453] A suspension of a live attenuated strain of Neospora caniem a tachyzoite in a culture medium, freshly egressed from host cells, is centrifuged for 10 min at 1, 500g. The supernatant is removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e., Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, 0.1M sucrose, 2.5% autoclaved inulin, 0 1M GSH (glutathione), 1% ectoine, 1% proline, adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1.10 7 / ml. After a period of LH equilibration, 1 ml of such a suspension is dispensed parasite into 10 ml glass vials which are placed on the shelf of a lyophilizer. Samples are kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying is carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure is maintained below 2 Pa and on the lyophilizer shelf the temperature is increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. Keeping samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials are mechanically sealed. The vacuum is then gently broken and the samples stored at 4 °C.

Example 47: Lyophilization of Neospora hughesi tachyzoites suspended in an aqueous solution formulation of formulation Fl.

[00454] A suspension of a live attenuated strain of Neospora tachyzoites hughesi in a culture medium, freshly egressed from host cells, is centrifuged for 10 min at l, 500g. The supernatant is removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, 0.1M inulin sucrose, 2.5% autoclaved, 0.1M GSH (glutathione), 1% ectoin, 1% proline, adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1.10 7 / ml. After a period of LH equilibration, 1 ml of such a suspension is dispensed parasite into 10 ml glass vials which are placed on the shelf of a lyophilizer. Samples are kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying is carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure is maintained below 2 Pa and on the lyophilizer shelf the temperature is increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. Keeping samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials are mechanically sealed. The vacuum is then gently broken and the samples stored at 4 °C.

Example 48: Lyophilization of Sarcocystis neurona bradyzoites suspended in an aqueous solution formulation of Formula Fl

[00455] A suspension of a live attenuated strain of Sarcocystis neurona bradyzoites in a culture medium, freshly egressed from host cells, is centrifuged for 10 min at 1,500g. The supernatant is removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, 0.1M inulin sucrose, 2.5% autoclaved, 0.1M GSH (glutathione), 1% ectoin, 1% proline, adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1.10 7 / ml. After a period of LH equilibration, 1 ml of such a suspension is dispensed parasite into 10 ml glass vials which are placed on the shelf of a lyophilizer. Samples are kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying is carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure is maintained below 2 Pa and on the lyophilizer shelf the temperature is increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. Keeping samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials are mechanically sealed. The vacuum is then gently broken and the samples stored at 4 °C.

Example 48 bis: Lyophilization of Sarcocystis neurona merozoites suspended in an aqueous solution formulation of formulation Fl.

[00456] A suspension of a live attenuated strain of Sarcocystis neurona merozoites in a culture medium, freshly harvested from host cells, is centrifuged for 10 min at 1,500g. The supernatant is removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, 0.1M inulin sucrose, 2.5% autoclaved, 0.1M GSH (glutathione), 1% ectoin, 1% proline, adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1.10 7 / ml. After a period of LH equilibration, 1 ml of such a suspension is dispensed parasite into 10 ml glass vials which are placed on the shelf of a lyophilizer. Samples are kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying is carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure is maintained below 2 Pa and the freeze dryer shelf temperature is increased from -55 °C to +5 °C at a rate of 0.0625 °C per minute, with a plateau lh upon reaching each 15 °C rise. Keeping samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials are mechanically sealed. The vacuum is then gently broken and the samples stored at 4 °C.

Example 49: Lyophilization of Leishmania donovani promastigotes suspended in an aqueous solution formulation of formulation Fl.

[00457] A suspension of a live attenuated strain of promastigotes forms of Leishmania donovani in a culture medium, is centrifuged for 10 min at l, 500g. The supernatant is removed and the pellet resuspended in aqueous media solution formulation composed of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1 M trehalose, 0.1 M sucrose, 2.5% autoclaved inulin, 0.1M GSH (glutathione), 1% ectoine, 1% proline, adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1.10 7 / ml. After a period of LH equilibration, 1 ml of such a suspension is dispensed parasite into 10 ml glass vials which are placed on the shelf of a lyophilizer. Samples are kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying is carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure is maintained below 2 Pa and on the lyophilizer shelf the temperature is increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. Keeping samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials are mechanically sealed. The vacuum is then gently broken and the samples stored at 4 °C.

Example 50: Lyophilization of Leishmania infantum promastigotes suspended in an aqueous solution formulation of Fl.

[00458] A suspension of a live attenuated strain of promastigotes forms of Leishmania infantum in a culture medium, is centrifuged for 10 min at l, 500g. The supernatant is removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1 M trehalose, 0.1 M sucrose, 2.5% autoclaved inulin , 0.1M GSH (glutathione), 1% ectoin, 1% proline, adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1 X 107 / ml. After a period of LH equilibration, 1 ml of such a suspension is dispensed parasite into 10 ml glass vials which are placed on the shelf of a lyophilizer. Samples are kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying is carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure is maintained below 2 Pa and on the lyophilizer shelf the temperature is increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. Keeping samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials are mechanically sealed. The vacuum is then gently broken and the samples stored at 4 °C.

Example 51: Lyophilization of Plasmodium falciparum sporozoites suspended in an aqueous solution formulation of Fl.

[00459] A suspension of a live attenuated strain of Plasmodium falciparum sporozoites in a culture medium, freshly harvested from host cells, is centrifuged for 10 minutes at 1,500g. The supernatant is removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e., Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, inulin, 0.1M sucrose, 2.5% autoclaved, 0 1M GSH (glutathione), 1% ectoine, 1% proline, adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1.10 7 / ml. After a period of LH equilibration, 1 ml of such a suspension is dispensed parasite into 10 ml glass vials which are placed on the shelf of a lyophilizer. Samples are kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying is carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure is maintained below 2 Pa and on the lyophilizer shelf the temperature is increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. Keeping samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials are mechanically sealed. The vacuum is then gently broken and the samples stored at 4 °C.

Example 51 bis: Lyophilization of Plasmodium falciparum merozoites suspended in an aqueous solution formulation of formulation Fl.

[00460] A suspension of a live attenuated strain of Plasmodium falciparum merozoites in a culture medium, freshly harvested from host cells, is centrifuged for 10 minutes at 1,500g. The supernatant is removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e., Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, inulin, 0.1M sucrose, 2.5% autoclaved, 0 1M GSH (glutathione), 1% ectoine, 1% proline, adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1 X 107 / ml. After a period of LH equilibration, 1 ml of such a suspension is dispensed parasite into 10 ml glass vials which are placed on the shelf of a lyophilizer. Samples are kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying is carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure is maintained below 2 Pa and on the lyophilizer shelf the temperature is increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. Keeping samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials are mechanically sealed. The vacuum is then gently broken and the samples stored at 4 °C.

Example 52: Lyophilization of Plasmodium vivax sporozoites suspended in an aqueous formulation solution of Formulation Fl

[00461] A suspension of a live attenuated strain of Plasmodium vivax sporozoites in a culture medium, freshly harvested from host cells, is centrifuged for 10 min at 1500g. The supernatant is removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e., Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, inulin, 0.1M sucrose, 2.5% autoclaved, 0 1M GSH (glutathione), 1% ectoine, 1% proline, adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1.10 7 / ml. After a period of LH equilibration, 1 ml of such a suspension is dispensed parasite into 10 ml glass vials which are placed on the shelf of a lyophilizer. Samples are kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying is carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure is maintained below 2 Pa and the freeze dryer shelf temperature is increased from -55 °C to +5 °C at a rate of 0.0625 °C per minute, with a plateau lh upon reaching each 15 °C rise. Keeping samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials are mechanically sealed. The vacuum is then gently broken and the samples stored at 4 °C.

Example 52 bis: Lyophilization of Plasmodium vivax merozoites suspended in an aqueous solution formulation of Formula Fl

[00462] A suspension of a live attenuated strain of Plasmodium vivax merozoites in a culture medium, freshly harvested from host cells, is centrifuged for 10 min at 1500g. The supernatant is removed and the pellet resuspended in an aqueous formulation solution consisting of DMEM (i.e. Dulbecco's Modified Eagle Medium) supplemented with 0.1M trehalose, 0.1M inulin sucrose, 2.5% autoclaved, 0.1M GSH (glutathione), 1% ectoin, 1% proline, adjusted to pH 7.4 with sodium hydroxide. The concentration of the parasites is 1.10 7 / ml. After a period of LH equilibration, 1 ml of such a suspension is dispensed parasite into 10 ml glass vials which are placed on the shelf of a lyophilizer. Samples are kept freezing at atmospheric pressure for 23 hours at a reduced temperature from 20°C to -75°C according to a kinetics of -1°C per minute. Primary drying is carried out at -55 °C for 20 h with a pressure maintained between 30 and 80 Pa during the first 7 hours and then below 2 Pa. For secondary drying, the pressure is maintained below 2 Pa and on the lyophilizer shelf the temperature is increased from -55°C to +5°C at a rate of 0.0625°C per minute with an LH plateau upon reaching each 15°C rise. Keeping samples at +5 °C for 4 h at a pressure of less than 2 Pa. At the end of the lyophilization process, the glass vials are mechanically sealed. The vacuum is then gently broken and the samples are stored at 4 °C.

[00463] Having thus described in detail the preferred embodiments of the present invention, it is to be understood that the invention defined by the appended claims should not be limited by certain details set out in the above description of how many obvious variations thereof are possible without departing from the spirit or scope of it.

Claims (17)

1. Lyophilized composition containing freeze-dried protozoa of an intracellular nature characterized by the fact that said lyophilized composition being devoid of protozoan host cells containing a moisture content of less than 12% by weight and being capable of reconstitution to restore said protozoa to infectious and viable states, wherein said viable protozoa account for more than 1% of the freeze-dried protozoa, further wherein said lyophilized composition comprises elements of the culture medium of said protozoa and at least one cryoprotectant and at least , an osmoprotectant and/or at least one antioxidant and/or at least one other additive, wherein: i) said culture medium is chosen from DMEM, RPMI or PBS; ii) said cryoprotectant is chosen from DMSO, a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide or a mixture thereof, in particular sucrose, trehalose, glucose, inulin or a mixture thereof; iii) the osmoprotectant is ectoine; iv) the antioxidant is selected from GSH, EGCG, ascorbic acid or a mixture thereof; and v) the other additive is proline. 2. Lyophilized composition, according to claim 1, characterized by the fact that said freeze-dried protozoa are fragile state protozoa, in particular selected from the tachyzoite state, the bradyzoite state, the sporozoite state, the promastigote state, the amastigote state, epimastigote state, trypomastigote state or merozoite state. 3. Lyophilized composition, according to any one of claims 1 to 2, characterized by the fact that it is capable of reconstitution to restore the immunogenic activity and/or the prophylactic activity of said frozen-dried protozoan. 4. Lyophilized composition, according to any one of claims 1 to 3, characterized in that it comprises: - DMEM, RPMI or PBS components, - 0 μmol to about 300 μmol of sucrose, preferably 0 μmol, 100 μmol or 150 µmol, - 0 µmol to about 1000 µmol trehalose, preferably 0 µmol, 100 µmol, 200 µmol or 880 µmol, - 0 µmol to about 400 µmol inulin (fructose equivalent), preferably 0 µmol, 155 μmol or 309 μmol, - 0 μmol to about 400 μmol ascorbic acid, preferably 0 μmol or 284 μmol, - 0 μmol to about 10 μmol EGCG, preferably 0 μmol or 2 μmol, - 0 to about 200 μmol GSH, preferably 0 μmol or 100 μmol, - 0 μmol to about 150 μmol proline, preferably 0 μmol or 87 μmol, - 0 μmol to about 150 ectoine, preferably 0 μmol or 70 μmol, being said micromolar values given per 1 ml of the suspension to be lyophilized, provided that the amount of at least one of the components among sucrose, trehalose, inulin, ascorbic acid, EGCG, GSH, proline or ectoine is different from 0 μmol. 5. Lyophilized composition, according to any one of claims 1 to 4, characterized in that said freeze-dried protozoa are virulent protozoa, attenuated protozoa or avirulent protozoa, preferably, in which said freeze-dried protozoa belong to the phylum Apicomplexa, or wherein said freeze-dried protozoa are live attenuated strains and/or recombinant strains of Leishmania spp., or wherein said freeze-dried protozoa are live attenuated strains and/or recombinant strains of Plasmodium spp. 6. Lyophilized composition, according to claim 5, characterized by the fact that said freeze-dried protozoa belong to the Apicomplexa phylum, preferably to the Sarcocystidae phylum, preferably, in which said freeze-dried protozoa are live attenuated strains of Toxoplasma spp. or from Neospora spp. or recombinant live attenuated strains of Toxoplasma spp., Neospora spp., Sarcocystis spp. or a combination of said strains, more preferably, wherein said freeze-dried protozoa are recombinant live attenuated strains of Toxoplasma gondii, Neospora caninum, Neospora hughesi, Sarcocystis neurona or a combination of said strains. 7. Lyophilized composition, according to claim 5, characterized in that said freeze-dried protozoa are attenuated live strains and/or recombinant strains of Leishmania spp., preferably, in which said freeze-dried protozoa are attenuated live strains and/or recombinant strains of Leishmania donovani, Leishmania infantum or a combination of said strains. 8. Freeze-dried composition, according to claim 5, characterized in that said freeze-dried protozoa are live attenuated strains and/or recombinant strains of Plasmodium spp., preferably, wherein said freeze-dried protozoa are live attenuated strains and/or recombinant strains of Plasmodium falciparum. 9. Process for obtaining a lyophilized composition containing lyophilized protozoa of an intracellular nature, devoid of protozoan host cells, containing a moisture content of less than 12% by weight and being capable of reconstitution to restore said protozoa to viable and infectious states, in which said viable protozoa representing more than 1% of the lyophilized protozoa characterized by the fact that it comprises: a) an initial step of suspending the protozoa of an intracellular nature and devoid of protozoan host cells in an aqueous formulation solution comprising a culture medium of said protozoa and at least one cryoprotectant, further comprising at least one osmoprotectant and/or at least one antioxidant and/or at least one other additive to obtain a protozoan suspension in an aqueous formulation solution, wherein: i) the culture medium is chosen from DMEM, RPMI, or PBS; ii) the cryoprotectant is chosen from DMSO, a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide or a mixture thereof, preferably sucrose, trehalose, glucose, inulin or a mixture thereof; iii) the osmoprotectant is ectoine; iv) the antioxidant is selected from GSH, EGCG, ascorbic acid or a mixture thereof; and v) the other additive is proline, in which the process additionally comprises: b) a lyophilization step of a suspension of protozoa of an intracellular nature, devoid of protozoan host cells in an aqueous formulation solution, in which said lyophilization step of the said suspension comprises: - a primary drying step of a frozen suspension to obtain a dry primary composition, preferably, wherein said primary drying step is carried out at about -55° C, for about 20 h and/or said primary drying step is carried out at a pressure ranging from about 30 to about 80 Pa, for 0 to 7 h, and then at a pressure below 2 Pa for the rest of the time, and - a secondary drying step of the dried composition primary to obtain a freeze-dried composition with a moisture content of less than 12% by weight, preferably, wherein said secondary drying step is carried out at a pressure of less than 2 Pa and at an increasing temperature from -55 °C to + 5 °C at a rate comprised between about 0.01 °C/min to about 0.2 °C/min, and said secondary drying comprises a temperature step of 1 h at each increase of 15 °C, said temperature being kept at + 5 °C for 4 hours after reaching said temperature. 10. Process according to claim 9, characterized in that the lyophilization step of said suspension comprises a step of freezing the suspension to obtain a frozen suspension, preferably, in which said freezing step is carried out for 4 to 23 h, at atmospheric pressure, lowering the initial temperature of the protozoan suspension to a temperature comprised between about -40 °C and about -80 °C, preferably -75 °C; said initial temperature of the protozoan suspension being comprised between about 15°C and about 25°C, preferably about 20°C; and said temperature reduction being carried out at a rate comprised between about -0.1°C/min and about -10°C/min, preferably at a rate of about 1°C/min. 11. Process according to any one of claims 9 and 10, characterized in that the step of lyophilizing said suspension comprises: - a step of freezing the suspension to obtain a frozen suspension, preferably, in which said step of freezing is carried out for 4 to 23 h, at atmospheric pressure, lowering the initial temperature of the protozoan suspension to a temperature comprised between about -40°C and about -80°C, preferably -75°C; - wherein said initial temperature of the protozoan suspension is comprised between about 15°C and about 25°C, preferably about 20°C; and wherein additionally said temperature reduction is performed at a rate comprised between about -0.1°C/min and about -10°C/min, preferably at a rate of about 1°C/min, - a primary drying step of said frozen suspension to obtain a dry primary composition, preferably wherein said primary drying step is carried out at about -55 °C, for about 20 h and/or said primary drying step is carried out at a pressure ranging from about 30 to about 80 Pa, for 0 to 7 h, and then at a pressure below 2 Pa for the rest of the time, and - a secondary drying step of the primary dry composition to obtain a freeze-dried composition with a moisture content of less than 12% by weight, preferably, wherein said secondary drying step is carried out at a pressure of less than 2 Pa and at an increasing temperature from -55 °C to + 5 °C at a rate comprised between about 0.01°C/min to about 0.2°C/min, preferably at a rate of about 0.0625°C/min and/or said secondary drying comprises a temperature step of 1 h for each increase of 15 °C, said temperature being maintained at + 5 °C for 4 h when reaching said temperature. 12. Process according to claim 9, characterized in that said aqueous formulation solution comprises or consists of: - DMEM, RPMI or PBS, - 0 mM to about 3000 mM of DMSO, preferably 0 mM or 1280 mM, - 0 mM to about 300 mM sucrose, preferably o mM or 100 mM or 150 mM, - 0 mM to about 1000 mM trehalose, preferably 0 mM, 100 mM, 200 mM or 880 mM, - 0 mM mM to about 400 mM inulin (fructose equivalent), preferably 0 mM, 155 mM or 309 mM, - 0 mM to about 400 mM ascorbic acid, preferably 0 mM or 284 mM, - 0 mM to about 10 mM EGCG, preferably 0 mM or 2 mM, - 0 mM to about 200 mM GSH, preferably 0 mM or 100 mM, - 0 mM to about 150 mM proline, preferably 0 mM or 87 mM, - 0 mM to about 150 mM ectoine, preferably 0 mM or 70 mM, provided that the concentration of at least one of the components among DMSO, sucrose, trehalose, inulin, ascorbic acid, EGCG, GSH, proline or ectoine is different from 0 mM; preferably, said aqueous formulation solution comprises or consists of - DMEM, - 20 mM to about 300 mM trehalose, - 20 mM to about 300 mM sucrose, - 20 mM to about 400 mM autoclaved inulin (equivalent to fructose), - 20 mM to about 200 mM GSH, - 20 mM to about 150 mM ectoine, - 20 mM to about 150 mM proline, and is adjusted to pH 7.4 with sodium hydroxide, or said aqueous formulation solution comprises or consists of: - DMEM, - 20 mM to about 300 mM trehalose, - 20 mM to about 400 mM ascorbic acid, and is adjusted to pH 7.4 with sodium hydroxide , or said aqueous formulation solution comprises or consists of: - DMEM, - 20 mM to about 300 mM trehalose, - 20 mM to about 300 mM sucrose, - 20 mM to about 400 mM inulin (equivalent fructose), or said aqueous formulation solution comprises or consists of: - DMEM, - 20 mM to about 300 mM trehalose, - 20 mM to about 300 mM sucrose, - 20 mM to about 200 mM GSH, - 20 mM to about 150 mM ectoine, - 20 mM to about 150 mM proline, and is adjusted to pH 7.4 with sodium hydroxide, or said aqueous formulation solution comprises or consists of: - DMEM, - 200 mM to about 3000 mM DMSO, or said aqueous formulation solution comprises or consists of: - DMEM, - 20 mM to about 300 mM trehalose, - 0.1 mM to about 10 mM EGCG, or said aqueous formulation solution comprises or consists of: - PBS, - 20 mM to about 300 mM trehalose, - 20 mM to about 300 mM sucrose, - 20 mM to about 400 mM autoclaved inulin (fructose equivalent), - 20 mM to about 200 mM GSH, - 20 mM to about 150 mM ectoine, - 20 mM to about 150 mM proline, and is adjusted to pH 7.4 with sodium hydroxide, or said solution aqueous formulation comprising or consisting of: - DMEM, - 0.1 M trehalose, - 0.1 M sucrose, - 2.5% autoclaved inulin, - 0.1 M GSH, - 1% ectoin, - 1% proline, and is adjusted to pH 7.4 with sodium hydroxide, or said aqueous formulation solution comprises or consists of: - DMEM, - 0.1 M trehalose, - 5% ascorbic acid, and is adjusted to pH 7.4 with sodium hydroxide, or said aqueous formulation solution comprising or consisting of: - DMEM, - 0.1 M trehalose, - 0.1 M sucrose, - 5% inulin, or said aqueous formulation solution comprises or consists of: - DMEM, - 0.2 M trehalose, - 0.15 M sucrose, - 0.1 M GSH, - 1% ectoine, - 1% proline, and is adjusted to pH 7.4 with sodium hydroxide, or said aqueous formulation solution comprises or consists of: - DMEM, - 10% DMSO, or said aqueous formulation solution comprises or consists of: - DMEM, - 0, 1 M trehalose, - 1mg/ml EGCG, or said aqueous formulation solution comprising or consisting of: - PBS, - 0.1 M trehalose, - 0.1 M sucrose, - 2.5% inulin autoclaved, - 0.1 M GSH, - 1% ectoine, - 1% proline, and adjusted to pH 7.4 with sodium hydroxide. 13. Lyophilized composition containing lyophilized protozoa of an intracellular nature, devoid of protozoan host cells, having a moisture content of less than 12% by weight and being capable of reconstitution to restore said protozoa to viable and infectious states, said viable protozoa representing more of 1% of lyophilized protozoa characterized by the fact that it is obtained by the process as defined by any one of claims 9 to 12. 14. Rehydrated lyophilized composition characterized in that it comprises a lyophilized composition as defined in any one of claims 1 to 5 in a rehydration medium, wherein said lyophilized composition contains lyophilized protozoa of an intracellular nature, is devoid of protozoan host cells and contains a moisture content of less than 12% by weight, said rehydrated lyophilized composition being such that the protozoa contained therein are viable and infectious and said viable protozoa representing more than 1% of the lyophilized protozoa. 15. Therapeutic or vaccine composition characterized by the fact that it comprises the rehydrated lyophilized composition as defined by claim 14. 16. Process for the reconstitution of a lyophilized composition as defined by any one of claims 1 to 5, said lyophilized composition contains lyophilized protozoa of an intracellular nature, being devoid of protozoan host cells and containing a moisture content of less than 12% by weight characterized in that it comprises a step of adding a rehydrated medium to said lyophilized composition to obtain a lyophilized rehydrated composition, wherein said protozoa are viable and infectious, said viable protozoa representing more than 1% of the lyophilized protozoa; preferably, wherein said step of adding the rehydration medium is performed at a rate comprised between about 25 µl per second and about 1000 µl per second, preferably at a rate of about 250 µl per second. 17. Lyophilized composition, according to any one of claims 1 and 14, or a process, according to any one of claims 9 and 13, characterized in that said viable protozoa represent more than 1% of the dried-frozen protozoa after a storage of said freeze-dried composition for a period of time longer than 2 weeks at a temperature of -25°C to 25°C, preferably at a temperature of -25°C to 2°C, or at a temperature of 2°C to 8 oC, or at a temperature of 2 oC to 6 oC, or at a temperature of 8 oC to 25 oC.