Composition applicable as rotavirus vaccine and method for producing it

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are presented a composition and a method for producing it. The characterised composition contains: an effective amount of a viral antigen, which represents a live attenuated rotavirus pre-processed in 0.1% human serum albumin, and a pharmaceutically acceptable buffer. A method for producing a composition involves growing Vero cell culture pre-cultured in the presence of 5% foetal calf serum and 0.1% human serum albumin, infecting the above Vero cell culture with the live attenuated rotavirus, propagating the virus in the cell culture and adding a pharmaceutically acceptable buffer to the above virus.

EFFECT: presented inventions can be used to prevent rotavirus infection and/or rotavirus gastroenteritis.

11 cl, 17 dwg, 4 tbl, 2 ex

 

The technical field TO WHICH the INVENTION RELATES

The present invention relates to vaccine compositions containing the rotavirus, which can give a higher titer value and show property of increased stability. The composition may be in liquid or lyophilized form and has an extended shelf life, while maintaining its therapeutic efficacy. The invention also relates to methods of producing such viruses and methods for producing such compositions. In addition, the invention relates to prophylactic and therapeutic methods for their use.

The LEVEL of TECHNOLOGY

There are a number of vaccines against human viruses that are currently used. Such vaccines include vaccines against hepatitis A virus, hepatitis B virus, influenza virus, Japanese encephalitis virus B, measles virus, mumps virus, rubella virus (MMR), polio virus, rabies virus, variola virus, varicella zoster virus and varicella-zoster virus, yellow fever virus. In addition to increasing the number of vaccine products there are different compositions or preparations used and developed for this vaccine. The successful use of live virus vaccines depends not only on proper selection and delivery of the virus, but also on the maintenance of a sufficient titer or �aktivnosti, necessary for immune response. Inherent in living viruses lability is a particular challenge in the preparation of compositions associated with the stabilization and preservation of the viability of the vaccine during production, storage and administration. There are a number of compositions known in the field, to receive rotavirus vaccines, but they have shortcomings associated with one or more problems relating to storage stability.

Rotavirus is a genus of viruses that contain double-stranded RNA, in the family Reoviridae, and is transmitted by the fecal-oral route. It infects cells that line the small intestine and produces an enterotoxin, which induces gastroenteritis, leading to severe diarrhoea and sometimes death as a result of dehydration. Rotavirus infection is the most significant cause associated with diarrhea of death in newborns and young children. Each year rotavirus gastroenteritis results in the death 310000-590000 infants and young children worldwide.

All of rotavirus vaccine, developed so far, based on live strains of rotaviruses, which were isolated from humans or animals and subjected to reasontly in vitro, adapted to the cultures of cells and then cooked in the form of compositions for the pen�sentatives of delivery. And monovalent and polyvalent based on animal virus strains showed efficiency as candidates for vaccines.

The strain of human rotavirus 116E, natural reassortant man-bull and natural attenuated strain is a strain G9 man, in which nature was embedded one gene VP4 bull (VP = viral protein) homologous gene site P[11]. Strain I321, also known as G10P [11], mainly bull contains genes and gene has only two plot of human origin, VP5 and VP7. The two said strain of rotavirus vaccine were obtained separately in the form of pilot batches of liquid formulations of monovalent oral rotavirus vaccine for clinical trials conducted in India.

Bharat Biotech International Ltd. (BBIL) received strains of human rotaviruses, 116E and I321, from the National Institute of health (NIH) under a contract on transfer of materials from the National Institute of Allergy and infectious diseases (NIAID), NIH, Bethesda, USA. Original strains 116E (G9[P11]) and I321 (G10P[11]) have adapted to grow in cell culture passaging in primary kidney cells of the African green monkey (AGMK), then in cell substrate and then MA104 in serially pereselenyi AGMK cells (SPAGMK). MA104 cell substrates and SPAGMK not approved by the National regulatory authority (NRA) to obtain commercial vaccines. P�this preferred adaptation 116E and I321 and other strains of rotavirus vaccines to be approved, certified, licensed and fully characterized cellular substrate similar to the substrate on the basis of Vero cells and/or diploid cells, MRC-5.

Prior art known to the inventors includes the document WO 02/11540 A1, which describes the composition of rotavirus vaccines, which contain buffering agents suitable for oral administration of rotavirus vaccines. The compositions described in WO 02/11540 A1, also contain compounds for the stabilization of compositions of vaccines that prevent loss of activity. More specifically, to compositions described in WO 02/11540 requires a sugar, a phosphate and at least one carboxylate, at least one human serum albumin or an amino acid selected from glutamate, glutamine and arginine. However, the achieved stability is highly variable, especially at temperatures above 20°C, resulting in significant losses of activity of the compounds described in WO 02/11540 A1.

In WO 99/62500 (next 500), WO 2005/058356 ('356) A2 and WO 2001/012797 ('197) describes the use of stabilizers vaccines to obtain vaccine formulations and lyophilized vaccines, storage stable virus compositions, method of separation of variants of rotaviruses and live attenuated rotavirus liquid vaccine. In '500 described lyophilized vaccine against measles-epidemic�ski mumps-rubella, obtained with the use of a stabilizer consisting of hydrolyzed gelatin, sorbitol, phosphate, sodium chloride, sucrose, bicarbonate, glucose, human serum albumin and citrate. In the invention, the bet on the dual presence of increased amount of disaccharide and a polyhydric alcohol at a pH of from 6.0 to 7.0 to ensure thermal stability. Despite the need for a certain amount of ingredients that makes the invention expensive, not possible to achieve stability at ambient temperature. This in turn adds to the special requirements of infrastructure for storage of vaccines, which makes the invention even more costly. However, the majority of such compositions provide a limited storage stability and are therefore unprofitable.

In document PCT/IN07/00190, entitled "Composition, useful as vaccines are described stable vaccine. The invention is centered around the joint action of the first protein is a human serum albumin, the second protein that is at least partially hydrolysed, and combinations of the three different sugars. Furthermore, the invention is also based on the inclusion of trypsin in the culture medium during adaptation of the virus. Claimed the vaccine is stable for weeks at 37°C, within six months at 25°C and for one year at 2°C to 8°C.

From the above description, it is evident that despite its successes in the field of preparation of vaccine formulations, there remains a clear need for profitable live viral vaccine with increased stability and shelf life.

The present invention satisfies such a need by providing a live or live attenuated virus, which has improved properties and increased stability, either in the form of a combined mass of three separate charges of the virus from the same party, either in liquid or lyophilized composition. Stability with respect to the virus (such as rotavirus or rotavirus vaccine) in the present description should be understood as the titer of the virus at this time point, starting from the time of collection from cultured cells, during the stage of obtaining the total unpacked weight is prepared in the form of a vaccine composition. The authors of this invention after prolonged research has been able to develop a composition according to the present invention are useful as vaccines, which has increased stability in the form of the resulting total mass in the form of the prepared composition, in particular at ambient temperature.

The increased stability in a statistically meaningful metrics can be� achieved in the case of the use of the virus, who had contact with or been subjected to human serum albumin during the stage of growth and reproduction of the virus in cell culture. For the purposes of the present invention believe that the virus is being contacted with or exposed to human serum albumin, when the virus infected the host cell to multiply in the environment for cultivation/environment for cell growth with the addition of human serum albumin. Virus or population of viruses that thus were exposed to human serum albumin, referred to as "pre-processed" by a virus. A virus that has not been subjected to human serum albumin, referred to in the present description, "the normal virus." Pre-treated virus, or at the stage the crowd, either in the form of the composition, i.e. vaccines/prepared in the form of the composition of the vaccine has a higher stability (in statistically significant terms) than a normal virus.

The present invention further shows that the stability of the virus, either pre-treated virus, either a conventional virus, the composition can also be further enhanced or, at least, can be maintained, i.e., the stability can be saved or at least can be slowed gradually� achieving zero or zero storage stability in the case of in practice systems (i) and (ii): According to the system (i), to achieve higher or more for a long period of stability, the virus is prepared in the form of a composition containing a non-viral protein or a hydrolysate of such a protein or vegetable protein or similar protein, such as human serum albumin. An example of the hydrolysate may be, without limitation lactalbumin hydrolysate, yeast hydrolysate, peptone, gelatin hydrolysate and a hydrolysate of egg protein. Vegetable proteins include without limitation corn protein, wheat protein, protein of Bengal gram, protein of common bean, protein lentils, protein Lima beans, protein Turkish beans, soy protein, protein split peas. Human serum albumin has a natural or recombinant origin. The virus is prepared in the form of a composition with non-viral protein or a hydrolysate of such a protein simply by adding a composition used to obtain vaccine non-viral protein or a hydrolysate of such a protein. Imply that such a system (i) is a one-component system. According to the system of (ii) the virus is subjected to contacting with a non-viral protein or a hydrolysate of such a protein, as in the one-component system, disaccharides and 1-2 in the result are added to the composition used to produce vaccines, protein or protein hydrolyzate and 1-2 disaccharides. Imply that such a system (ii) is a two - and�and a three-component system, depending on whether, in the composition containing the virus, one disaccharide (two component system) or a combination of two different disaccharides (ternary system). In the practical use of the system (ii) the levels of stability observed in the one-component system, in addition increased.

Thus, in one aspect the present invention provides a composition containing pre-treated virus or a normal virus, which has a higher and/or more long-term stability.

The novelty of the invention consists in adding to the culture medium of human serum albumin during the multiplication of the virus to get viral antigen and vaccine composition with higher titer, increased shelf life and thermal stability even without the addition of stabilizers. Shelf life can be further enhanced with the addition of stabilizers described above. This yields a therapeutically improved vaccines with the introduction of simple, cost-favorable cost-effective way. In addition to technical improvements, the invention also ACID satisfies the criterion of economic significance.

The PURPOSE of the INVENTION

The main purpose of the present invention is a composition useful as rotavirus vaccine, have�her increased shelf life, while eliminating the disadvantages of the relevant prior art.

Another object is to obtain formulations of vaccines that contain live attenuated rotaviruses, able to give a higher value titer, to show the properties of high stability at ambient temperatures.

The composition may be in liquid or lyophilized form and has an extended shelf life, while maintaining its therapeutic efficacy/activity.

The invention also relates to methods of producing such viruses and methods for producing such compositions.

The invention, furthermore, relates to prophylactic and therapeutic methods of deterrence rotavirus infections by the introduction of vaccine formulations to subjects suffering from such infections.

BRIEF description of the DRAWINGS

Viral titer is indicated in the figures, corresponds to the number of blakebrough units (the FIGHT) in 0.5 ml collected or final mass of rotavirus 116E and prepared in the form of a vaccine composition. In the various figures presented in this publication, an indication of "biological method 1" means that the starting material used is a common virus, and an indication of "biological method 2" means that the source material used is pre-treated virus. Unless otherwise indicated, Yes�nye in the figures represent the stability of pre-treated virus. Fig.1-12H shows data for liquid formulations (Yid. = liquid), and Fig.13A-17C shows data for lyophilized formulations (lyophilized. = freeze-dried). Numeric indicators expressed as percentages, mean values by weight for the composition (compositions). For example, it should be understood that the statement "80% sucrose" means 80% of the mass. sucrose in the composition (wt./vol.). The RMS error for all time points ranged from ±0.40 to ±0,45.

Fig.1 shows the average titer obtained with the charges in the case of the biological method 1 (normal virus) and biological method 2 (pre-treated virus) in the five experiments.

Fig.2 shows the stability data collected for viruses, conventional virus (biological method 1) and pre-treated virus (biological method 2), in each case in the absence (Fig. 2A) or in the presence (Fig. 2B) stabilizers, namely, 5% LAH, 80% sucrose and 0.5% trehalose, in the liquid composition at 37°C.

Fig.3 (Yid.) shown data on the stability of pre-treated virus in four different formulations at 2-8°C (3A), 25°C (3B) and 37°C (3C). In each case: series 1 refers to the composition with 2.5% lactalbumin hydrolysate; series 2 refers to the composition with 10% lactalbumin hydrolysate and 0.5% trehalose; series 3 refers to the composition with 20% lactalbumin hydrolysate; and series 4 relative�are consistent with the composition with the combination of 2.5% lactalbumin hydrolysate, 0.5% starch and 0.5% trehalose.

Fig.4 (Yid.) shown data on the stability of rotavirus in the composition containing and not containing 5% lactalbumin hydrolysate + 80% sucrose + 0.5% trehalose, which was stored at 2-8°C (4A), 25°C (4B) and 37°C (4C).

Fig.5 (Yid.) shown data on the stability of rotavirus in four different formulations at 2-8°C (5A), 25°C (5B) and 37°C (5C). In each case: series 1 refers to the composition containing a combination of 20% lactalbumin hydrolysate and 0.5% trehalose; series 2 refers to a composition containing a combination of 10% lactalbumin hydrolysate, 1.0% of lactose; series 3 refers to the composition comprising a combination of 5% lactalbumin hydrolysate, 80% of sucrose; and series 4 refers to a composition containing a combination of 10% lactalbumin hydrolysate and 50% maltose.

Fig.6 (Yid.) shown data on the stability of rotavirus in four different formulations at 2-8°C (6A), 25°C (6B) and 37°C (6C). In each case: series 1 refers to the composition containing the combination of 0.5% lactalbumin hydrolysate, 10% soy protein and 1.0% trehalose; series 2 refers to the composition with a combination of 0.5% lactalbumin hydrolysate, 10% soy protein and 1.0% lactose; series 3 refers to the composition with the combination of 5% lactalbumin hydrolysate, 2.5% of soy protein and 80% sucrose; and series 4 refers to a composition with a combination of 5% lactalbumin hydrolysate, 2.5% of soy protein and 50% maltose.

Fig.7 (Yid.) shown data SVID�teletubisie high stability of rotavirus in four different formulations at 2-8°C (7A), 25°C (7B) and 37°C (7C). In each case: series 1 refers to the composition containing a combination of 10% lactalbumin hydrolysate, 10% sucrose and 1.0% trehalose; series 2 refers to a composition containing a combination of 10% lactalbumin hydrolysate, 5% maltose and 1.0% trehalose; series 3 refers to the composition containing a combination of 2.5% lactalbumin hydrolysate, 80% sucrose and 1% trehalose; and series 4 refers to a composition containing a combination of 2.5% lactalbumin hydrolysate, 50% maltose and 1% trehalose.

Fig.8 (Yid.) shown data on the stability of rotavirus in four different formulations at 2-8°C (8A), 25°C (8B) and 37°C (8C). In each case: series 1 refers to the composition with a combination of conventional virus, 5% lactalbumin hydrolysate, 80% sucrose and 0.5% trehalose; series 2 refers to a composition containing a combination of pre-treated virus, 5% lactalbumin hydrolysate, 80% sucrose and 0.5% trehalose; series 3 refers to the composition with a combination of conventional virus, 0.1% of recombinant human serum albumin (rHSA) and 80% sucrose and 0.5% trehalose; series 4 relates to the composition with a combination of pre-treated virus, 0.1% of rHSA, 80% sucrose and 0.5% trehalose; series 5 relates to the composition containing the combination of conventional virus, 80% sucrose and 0.5% trehalose; and series 6 relates to a composition containing a combination of pre-treated virus, 80% sucrose and 0.5% trigal�SHL.

Fig.9 (Yid.) shown data on the stability of rotavirus in five different formulations at 37°C.

Fig.10 (Yid.) shows the stability of rotavirus in low titre in five different formulations at 2-8°C (10A) and 37°C (10B).

Fig.11A (Yid.) shows stability data of formulations pretreated rotaviruses, containing 20% hydrolyzed peptone, at 2-8°C, 25°C and 37°C.

Fig.11B (Yid.) shows stability data of formulations pretreated rotaviruses, containing a combination of 20% hydrolyzed peptone, 1% trehalose and 0.02% fucose, at 2-8°C, 25°C and 37°C.

Fig.11C (Yid.) shows stability data of formulations pretreated rotaviruses, containing 20% egg protein hydrolysate, at 2-8°C, 25°C and 37°C.

Fig.11D (Yid.) shows stability data of formulations pretreated rotaviruses, containing a combination of 20% egg protein hydrolysate, 0.5% trehalose, 1% D-sorbitol and 0.5% mannose, at 2-8°C, 25°C and 37°C.

Fig.11E (Yid.) shows stability data of formulations pretreated rotaviruses, containing 20% lactalbumin hydrolysate, at 2-8°C, 25°C and 37°C.

Fig.11F (Yid.) shows stability data of formulations pretreated rotaviruses, containing a combination of 20% lactalbumin hydrolysate, 0.5% trehalose, at 2-8°C, 25°C and 37°C.

Fig. 11G (Yid.) shows information about a stub�scenic spots of pre-processed compositions of rotaviruses, containing 20% yeast hydrolysate, at 2-8°C, 25°C and 37°C.

Fig.11H (Yid.) shows stability data of formulations pretreated rotaviruses, containing a combination of 20% yeast hydrolysate, 5% maltose and 0.5% lactose at 2-8°C, 25°C and 37°C.

Fig.12A (Yid.) shown data on the stability of conventional formulations of rotaviruses, containing 20% hydrolyzed peptone, at 2-8°C, 25°C and 37°C.

Fig.12B (Yid.) shown data on the stability of conventional formulations of rotaviruses, containing a combination of 20% hydrolyzed peptone, 1% trehalose and 0.02% fucose, at 2-8°C, 25°C and 37°C.

Fig.12C (Yid.) shown data on the stability of conventional formulations of rotaviruses, containing 20% egg protein hydrolysate, at 2-8°C, 25°C and 37°C.

Fig.12D (Yid.) shown data on the stability of conventional formulations of rotaviruses, containing a combination of 20% egg protein hydrolysate, 0.5% trehalose, 1% D-sorbitol and 0.5% mannose, at 2-8°C, 25°C and 37°C.

Fig.12E (Yid.) shown data on the stability of conventional formulations of rotaviruses, containing 20% lactalbumin hydrolysate, at 2-8°C, 25°C and 37°C.

Fig.12F (Yid.) shown data on the stability of conventional formulations of rotaviruses, containing a combination of 20% lactalbumin hydrolysate, 0.5% trehalose, at 2-8°C, 25°C and 37°C.

Fig.12G (Yid.) shown data on the stability of conventional formulations of rotaviruses, containing 20% yeast hydrolysate, at 2-8°, 25°C and 37°C.

Fig.12H (Yid.) shown data on the stability of conventional formulations of rotaviruses, containing a combination of 20% yeast hydrolysate, 5% maltose and 0.5% lactose at 2-8°C, 25°C and 37°C.

Fig.13 (lyophilized.) shown data on the stability of rotavirus in four different lyophilized formulations at 2-8°C (13A), 25°C (13B) and 37°C (13C). In each case: series 1 refers to the composition with 0.5% human serum albumin and 12% sucrose; series 2 refers to the composition with 0.5% lactalbumin hydrolysate, 0.5% trehalose; series 3 refers to the composition from 0.5% of soy protein and 0.5% trehalose; and series 4 refers to a 0.25% of polyvinylpyrrolidone, 0.5% trehalose.

Fig.14 (lyophilized.) shown data on the stability of rotavirus in four different lyophilized formulations at 2-8°C (14A), 25°C (14B) and 37°C (14C). In each case: series 1 refers to the composition with 0.5% human serum albumin, 12% sucrose and 0.1% starch; series 2 refers to the composition with 0.5% lactalbumin hydrolysate, 0.5% trehalose and 0.1% starch; series 3 refers to the composition from 0.5% soy protein, 0.5% trehalose and 0.1% starch; and series 4 refers to a 0.25% of polyvinylpyrrolidone, 0.5% trehalose and 0.1% starch.

Fig.15 (lyophilized.) shown data on the stability of rotavirus in four different lyophilized formulations at 2-8°C (15A), 25°C (15B) and 37°C (15C). In each case: series 1 refers to the composition with 0.5% human serum albumin, 12% sucrose and 0.1% starch and 304 mm bicarbonate; series 2 relates to the composition with 0.5% lactalbumin hydrolysate, 0.5% trehalose, 0.1% starch and 304 mm bicarbonate; series 3 refers to the composition from 0.5% soy protein, 0.5% trehalose, 0.1% starch and 304 mm bicarbonate; series 4 refers to a 0.25% of polyvinylpyrrolidone, 0.5% trehalose, 0.1% starch and 304 mm bicarbonate.

Fig.16 (lyophilized.) shown data on the stability of rotavirus in four different lyophilized formulations at 2-8°C (16A), 25°C (16B) and 37°C (16C). In each case: series 1 refers to the composition with 0.5% human serum albumin, 12% sucrose and 0.1% Arabian gum; series 2 refers to the composition with 0.5% lactalbumin hydrolysate, 0.5% trehalose and 0.1% Arabian gum; series 3 refers to the composition from 0.5% soy protein, 0.5% trehalose and 0.1% Arabian gum; and series 4 refers to a 0.25% of polyvinylpyrrolidone, 0.5% trehalose and 0.1% Arabian gum.

Fig.17 (lyophilized.) shown data on the stability of rotavirus in four different lyophilized formulations at 2-8°C (17A), 25°C (17B) and 37°C (17C). In each case: series 1 refers to the composition with 0.5% lactalbumin hydrolysate, 0.25% of polyvinylpyrrolidone; series 2 refers to the composition with 0.5% lactalbumin hydrolysate and 0.1% Arabian gum; series 3 refers to the composition with 0.5% lactalbumin hydrolysate and 0.1% pyridoxine·HCl; and series 4 refers to a 0.5% lactalbumin hydrolysate and 0.1% starch.

Summary of the INVENTION

According to the present�ademu the present invention provides a composition, containing:

(a) viral antigen that is a live attenuated rotavirus, and

(b) a pharmaceutically acceptable buffer with physiological pH,

the stability of the composition in relation to the titer of the virus is increased, because the influence of the virus that multiplies in the presence of human serum albumin on the stability higher than that of the virus, propagated in the absence of human serum albumin.

According to one variant, the composition may further contain at least one of stabilizers containing non-viral protein, or at least partially hydrolyzed, the hydrolysate of such a protein, or a disaccharide, or a combination of 2 of disaccharides.

Non-viral protein or protein hydrolysate may be a protein or a hydrolysate, as lactalbumin hydrolysate, yeast hydrolysate, hydrolyzed gelatin, hydrolyzed egg protein, hydrolyzed peptone or vegetable protein selected from corn protein, wheat protein, proteins of Bengal gram, protein of common bean, protein lentils, protein Lima beans, squirrel Turkish beans, soy protein, protein split peas, or similar protein, an example of such a protein is human serum albumin, preferably lactalbumin hydrolysate or hydrolysed soy protein, more preferably, the hydrolysis�t lactalbumin.

According to other variants used by the disaccharide can be such as trehalose or a combination of 2 of disaccharides consisting of sucrose and trehalose.

Thus, the composition according to the present invention may contain (a) viral antigen that is a live attenuated rotavirus, (b) a pharmaceutically acceptable buffer with physiological pH and (c) non-viral protein or protein hydrolysate.

Composition, which is disclosed in the present description above, may contain

(i) viral antigen that is a live attenuated rotavirus, which is described in this publication above, with a titer in the range from 103up to 108,5The FIGHT/0.5 ml,

(ii) a pharmaceutically acceptable buffer, representing a phosphate-citrate buffer (310/100 mm) with a pH from 6.8 to 8.0, as a diluent/carrier,

(iii) a protein hydrolysate, which is a hydrolysate of lactalbumin, in the range of 20-30% of the mass./vol., and

(iv) the disaccharide representing trehalose of about 0.5% wt./about. or sucrose for approximately 80% of the mass./vol., another disaccharide, representing trehalose of about 0.5% wt./about.

The composition comprises a live attenuated rotavirus capable of giving an increased titer on average from a minimum of 0.8 log up to a maximum of 1.1 log per milliliter when stored in ambient conditions, compared to W�Vym attenuated by rotavirus, propagated in the absence of human serum albumin.

Moreover, the live attenuated rotavirus propagated in the presence of 0.1% recombinant human serum albumin.

According to another aspect of the invention also provides a method of receiving a live attenuated rotavirus according to claim 1, including:

(i) infection of host cells by rotavirus live attenuated;

(ii) growing the infected cells in the culture medium for cells, helping to maintain the growth of these cells, in a specified environment added human serum albumin, and collecting the specified rotavirus, and able to demonstrate improved stability.

In one particular aspect the present invention relates to compositions containing live and pre-treated virus or population of viruses) with a certain stability, and the stability of the virus is characterized by comparison with a conventional live virus or population of viruses, which multiply in the presence of human serum albumin (as in the case of the virus, called pre-processing), and in the case where the detected drop in titer on log 4, more than the difference between 4.5 and 7.5 FIGHT/0.5 ml, when both compositions with a live pre-treated with a virus or ordinary living Viru�Ohm kept at 37°C for four weeks after collection. The composition comprises a pharmaceutically acceptable buffer with or without added stabilizer, such as a protein hydrolysate, peptone, vegetable protein or disaccharide, in the composition. In another particular aspect the present invention relates to compositions containing live and pre-treated virus or population of viruses), is able to give titer, increased on average from a minimum of 0.8 log up to a maximum of 1.1 log per milliliter when stored in ambient conditions, compared to conventional live virus, and a pharmaceutically acceptable buffer, and pre-treated virus is able to give such a title without adding the composition of any stabilizer, such as a hydrolysate of non-viral protein, peptone, vegetable protein and disaccharide.

In each of the above aspects in cases where it is anticipated an additional stabilizer, the preferred additional stabilizer is a hydrolysate of lactalbumin. Disaccharide (e.g., trehalose) or a combination of different disaccharides (e.g., sucrose and trehalose) are the preferred stabilizer composition containing hydrolysate of lactalbumin. In one embodiment, lactalbumin hydrolysate in the composition has a concentration of about 5% wt./vol., sucrose - about 80% wt./vol., and trehalose - about 0.5% wt/vol. The composition may contain a recombinant human serum albumin (e.g., 0.1% wt./about.) as another additional stabilizer.

In one embodiment, the virus is a living rotavirus, such as live attenuated rotavirus. Preferably a live virus is a live virus of human rights, such as human rotavirus. In a particularly preferred embodiment, the human rotavirus is rotavirus strain 116E or I321. The composition according to the present invention is a vaccine. In one embodiment, the composition according to the present invention may contain live attenuated rotavirus with a titer in the range from 103up to 108,5The FIGHT/0.5 ml. of Live rotavirus is pre-treated with rotavirus.

In another General aspect, a method of receiving a live attenuated pre-treated rotavirus. The method includes the stage of infection of host cells by rotavirus live attenuated, growing the infected cells in the medium for culturing cells with the addition of human serum albumin, which allows the growth of cells, and collecting the pre-treated rotavirus. Assembled pre-treated rotavirus exhibits improved stability as compared to untreated prior�individual or an ordinary virus.

In yet another General aspect, the present invention also relates to a method of adaptation of the virus to a suitable cell substrate, such as Vero cells, serially pereselenie in a suitable environment, each passage is carried out in the medium in the absence or in the presence of human serum albumin derived from human, or recombinant human serum albumin.

DETAILED description of the INVENTION

The present invention relates to compositions and methods related to rotavirus live attenuated viral. Live attenuated rotaviruses exhibit the property of increased stability and is applicable for the prevention of rotavirus infection and/or rotavirus gastroenteritis in children.

In particular, the present invention relates to various approaches and systems for receiving rotavirus compositions which exhibit enhanced stability at this point of time and the stability, support for a certain period of time during storage. One approach is to use pre-treated virus in the starting material in the compositions according to the invention. Another approach is the use of various stabilizers to obtain increased stability when used virus is pretreated with a virus./p>

As defined above, a virus or population of viruses collected from cell cultures propagated in medium containing human serum albumin, referred to as "pre-treated" with a virus or population of viruses. Conversely, a virus or population of viruses collected from cell cultures in medium containing human serum albumin, referred to as "normal" with a virus or population of viruses. Live attenuated rotavirus sometimes in the present description is called a viral antigen or an antigen of a vaccine.

As described above, pre-treated virus exhibiting the properties of high stability compared with conventional virus. Each of the viruses, and pre-treated virus and a normal virus, show stability, more long-term supported during storage with the addition of one or more stabilizers, compared to the composition without additives. It should be understood that stabilizers are used to maintain stability, regardless of whether or not a virus, used as source material for composition after harvesting, pre-processed, in a broad sense includes three different system components.

One-component system contains non-viral protein or hydrolyzed protein such as part of the composition. Non-viral protein rigidroles protein serves as a stabilizer. Two-component system contains disaccharide in addition to non-viral protein or hydrolysate this protein. In a two-component system and a disaccharide, and a protein or its hydrolysate serve as stabilizers. A three-component system such two-component system, but contains additional disaccharide than the disaccharide in a two-component system.

In located on the simultaneous consideration of the request of the authors of the present invention No. 842/CHE/2006, the authors described a composition containing a viral antigen; a first protein selected from human serum albumin or recombinant human albumin, and a second protein which is at least partially hydrolysed selected from the lactalbumin hydrolysate, yeast hydrolysate, peptone and hydrolyzed egg protein, and preferably a combination of three different disaccharides, used the virus does not multiply in the presence of HSA. Liquid composition exhibits stable for 3-4 weeks at 37°C, six months at 25°C and one year at 2°C-8°C, whereas the freeze-dried composition exhibits stability for more than 50 weeks at 2°C-8°C, 25°C, 37°C.

The liquid composition according to the present invention is stable for 6 weeks at 37°C, for 6 months at 25°C and 24 months at 2-8°C.

Freeze-dried composition according to the present�the invention is stable for 16 weeks at 37°C, within 6 months at 25°C and 24 months at 2-8°C.

The composition can be a liquid composition or a lyophilized (dry form). The present invention relates to live attenuated rotaviruses and their compositions exhibiting improved and enhanced stability when stored at 2-8°C or at ambient conditions for a long period of time. The environmental conditions may be prevailing in a given place and normal atmospheric conditions (e.g. 25°C), but not in excess of about 37°C. the Compositions according to the present invention is able to maintain its capacity for immunization at the time of acquisition and during the period of time required for the shelf life of a commercial vaccine (i.e., the compositions are stable).

Thus, the composition according to the present invention exhibits stability over a longer period due to virus replication in the presence of human serum albumin, compared to adding it from the outside.

In one example, the composition according to the present invention comprises a viral antigen (pre-treated or normal virus), non-viral protein or protein that differs from a viral antigen. It should be understood that the term "non-viral protein" means any protein: lactalbumin�n, hydrolyzed yeast protein, gelatin, egg protein or vegetable protein, which is a corn protein, wheat protein, protein of Bengal gram, protein of common bean, protein lentils, protein Lima beans, protein Turkish beans, soy protein, protein split peas, and human serum albumin, a natural or recombinant origin. Preferably the protein is at least partially hydrolysed. In other words, hydrolysates of such proteins or peptone can be used in the compositions according to the present invention.

It is assumed that the phrase "a protein that is at least partially hydrolysed", used in the present description, the meaning refers to a variant, when the hydrolyzable protein was at least partially destroyed at the corresponding amino acid building blocks. Therefore, this phrase also means the inclusion of options, when the protein no longer exists in the form of complete molecules, and exists only in the form of a set of fragments. In addition, this phrase means the inclusion of options when protein is completely hydrolysed. It is assumed also that the phrase "protein hydrolysate" includes all these options, and such a phrase may include a fully hydrolyzed protein, i.e. a protein that is destroyed to the corresponding amino acids, or protein, partially destroyed, �AK is the set of peptides and amino acids.

Thus, the protein or at least partially hydrolyzed variant may represent a lactalbumin hydrolysate, yeast hydrolysate, peptone, gelatin hydrolysate, and egg protein hydrolysate or protein from plant source such as corn, wheat, Bengal gram, kidney beans, lentils, Lima beans, Turkish beans, soybeans, split peas, or homologous to the human protein, such as human serum albumin, which is derived from the human body or of recombinant origin. Such proteins and protein hydrolysates can be easily obtained by the skilled in the art, e.g. by acid hydrolysis, or can be obtained from a commercial source. In the present description it is shown that at low concentrations of rotavirus, especially when the concentration (titer) of rotavirus in the composition is 103The BATTLE in 0.5 ml, non-viral protein such as lactalbumin hydrolysate or soy protein, contributes to improved and higher stability compared to non-viral protein - serum human albumin or bovine serum albumin. Hydrolyzed lactalbumin is known to the person skilled in the art and commercially available. I believe that the hydrolysate of lactalbumin provides a perfect homogenization of the mixture with the protein of rotavirus, ka� in liquid form, and in dried form, and retains the molecules of the viral protein, even when the viral protein is present in low concentrations. Instead of or in addition to the hydrolysate of lactalbumin is also possible to use other additives such as yeast hydrolysate, peptone, gelatin hydrolysate and a hydrolysate of egg protein. The composition further comprises a disaccharide or a combination of the two disaccharides and pharmaceutically acceptable buffer. The disaccharide may be any of the following disaccharides: sucrose, lactose, maltose, trehalose, cellobiose, gentiobiose, melibiose, turanose and fucose. A three-component system contains a combination of two different types of disaccharides in addition to non-viral protein or a hydrolysate of such a protein. Such proteins or protein hydrolysates and disaccharides are referred to in this paragraph, referred to in the present description "stabilizers".

Stabilizers can be added to excipient, diluent or carrier (e.g., pharmaceutically acceptable buffer), which is commonly used in the pharmaceutical compositions of the virus. Such excipients or carriers are well known in this field. In particular, in a suitable diluent or pharmaceutically acceptable buffer one or more of the above stabilizers. In a preferred embodiment to contain the rotavirus sample first, add LAH, then �agarose. If you add a second disaccharide, the next in order the preferred disaccharide is trehalose. Composition without stabilizers is essentially a solution of the carrier or pharmaceutically acceptable buffer. In addition to these stabilizers, the composition may contain colorants, flavors, sweeteners, adsorbents and/or activators of fluidity. Preferably such compositions are buffered at the appropriate pH, generally from 6 to 8, preferably from 6.8 to 8.0. For example, in one embodiment, the composition according to the present invention can be prepared in a modified Dulbecco environment Needle.

In one aspect, the present invention relates to a composition comprising a population of live attenuated and pretreated rotaviruses. Pre-treated rotavirus has a certain level of stability in pharmaceutically acceptable excipient without any additional stabilizers in the composition. Population stability pretreated rotaviruses better compared to the population of living conventional attenuated rotaviruses, which are not subjected to such a pretreatment. As for the indicators of stability, the population of conventional rotaviruses has a falling titer in log 4, whereas pre-treated VIR�with has a drop in titer, significantly less than the fall in log 4, for the storage of both rotavirus at 37°C for four weeks after collection.

In another aspect, the present invention relates to compositions containing live attenuated pre-treated rotavirus, which is able to give titer, increased from a minimum of 0.8 log up to a maximum of 1.1 log per milliliter compared with conventional live attenuated rotavirus when stored in ambient conditions without any additional stabilizers in the composition.

In any case, the stability of pre-treated virus may be enhanced if the composition is added at least one stabilizer, such as lactalbumin hydrolysate, yeast hydrolysate, gelatin hydrolysate, and egg protein hydrolysate, peptone, vegetable protein and human serum albumin. The increased stability can be further enhanced by adding to the composition of the disaccharide or a combination of different disaccharides. Thus, in the preferred embodiment, the composition comprises a viral antigen, which is a pre-treated rotavirus protein than the viral antigen, at least partially hydrolyzed (such as, for example, lactalbumin, yeast protein, peptone, gelatin and egg protein, maize protein, WHI�OK wheat, of Bengal gram, French bean, lentil, Lima beans, Turkish beans, soybeans, split peas, or human serum albumin isolated from human body, or recombinant human serum albumin), and a disaccharide, or a combination of the two disaccharides. Composition with conventional virus is also preferable in the case where the composition contains a stabilizer, as in this composition can be achieved better stability of conventional virus compared with conventional virus in the composition without such stabilizers.

Proteins, either individually or in combination of two proteins that may be present in the range of 0.01% (wt./about.) up to 80% (wt./vol.), preferably in the range from 0.05% to 50%. Preferably, the protein used as the stabilizer, is any of the following proteins: hydrolyzed lactalbumin, human serum albumin, and soy protein. Hydrolyzed lactalbumin may be present in the composition in a concentration of from about 0.01% to 70%, preferably from 0.1% to 30%. Soy protein may be present in the composition in a concentration of from about 0.01% to 70%, preferably from 0.05% to 20%. Human serum albumin preferably has a recombinant origin, and may be present in the composition in a concentration of from about 0.01% to 20%, preferably from 0.1% to 0.5%.

Preferably, the disaccharide that is used as a stabilizer may be any of disaccharides: trehalose, lactose and sucrose. The preferred concentration of trehalose is from about 0.01% to 70%, most preferably from 0.5% to 20%. The combination of two different disaccharides can be a combination of any two of disaccharides: sucrose, lactose, maltose, trehalose, cellobiose, gentiobiose, melibiose and turanose. For example, a combination of the two disaccharides can be any of combinations of sucrose and maltose, sucrose and lactose, sucrose and trehalose, maltose and trehalose or trehalose and lactose. A preferred combination is a combination of disaccharides sucrose and trehalose. Preferably the sucrose is present in a concentration of about 1-10% and from 70% (wt./about.) to 85% (wt./vol.), more preferably in a concentration of from 5% to 10% and from 80% to 85% (wt./vol.), and the trehalose is present in a concentration of about 0.01% (wt./about.) to 50.0% (wt./vol.), preferably from 0.5% to 20% (wt./vol.). The concentration of the combination of disaccharides or one disaccharide may be approximately 1-10%, or about 20% -85% (wt./vol.). In one embodiment, the concentration of disaccharides can have the following values: sucrose of about 1-10% and from 70% to 85% (wt./vol.), preferably from 5% to 10% or from 80% to 85% (wt./vol.), lactose from about 0.1% to 20.0% (wt./vol.), preferred�flax from 0.5% to 10% (wt./vol.), maltose from about 0.1% (wt./about.) up to 50% (wt./vol.), preferably from 5% to 50% (wt./vol.), trehalose approximately from 0.01% (wt./about.) to 70.0% (wt./vol.), preferably from 0.5% to 20.0% (wt./vol.).

Preferably the composition is buffered using phosphate-citrate buffer. Preferably a phosphate-citrate buffer has a concentration of approximately 310 mm phosphate and about 100 mm citrate. Pharmaceutically acceptable buffer may have a pH value in the range from 6.8 to 8.0. The buffer can be any of the buffers containing phosphates, carbonates, citrate, Tris, HEPES, and combinations thereof. The phosphate may have a concentration in the range from 10 mm to 1000 mm, preferably 50 mm to 310 mm. The carbonate may have a concentration in the range from 10 mm to 1000 mm, preferably 50 mm to 300 mm. Citrate may have a concentration in the range from 10 mm to 400 mm, preferably from 50 mm to 100 mm. Tris may have a concentration in the range from 0.1 mm to 1000 mm, preferably from 5 mm to 20 mm. HEPES may have a concentration in the range from 0.1 mm to 1000 mm, preferably 10 mm to 20 mm.

In some embodiments, as the stabilizer can also be used starch. Examples of starch are grains, wheat, corn and rice starch. The starch may be soluble, insoluble, partially or fully hydrolyzed starch. Starch can prise�estvovati in a concentration of from about 0.01% to 10%, preferably from 0.1% to 3.0%.

The composition may contain at least one diluent, such as an environment for tissue culture, saline, phosphate buffered saline or water. The preferred diluent is a modified Dulbecco Wednesday Needle (DMEM). The composition may further contain at least one chemical substance, such as ascorbic acid, acacia gum, Senegal gum, polyvinylpyrrolidine, pyridoxine•HCl (vitamin B6), and the concentration can be from about 0.1% to 20%, preferably from 0.25% to 5 wt%. composition. The composition preferably is a liquid composition with a live attenuated rotavirus. A preferred liquid composition contains pre-processed or regular rotavirus and stabilizers, lactalbumin hydrolysate (LAH) in the range of 20-30% of the mass./about. and trehalose of about 0.5% wt./about. Another preferred liquid composition contains pre-processed or regular rotavirus and stabilizers, lactalbumin hydrolysate in the composition in a concentration of about 5% wt./vol., sucrose about 80% wt./about. and trehalose of about 0.5% wt./about. The compositions according to the present invention can be used as a vaccine for vaccination against viral infections and associated with virus diseases. Rotavirus stammie (G9P[11]) and I321(G10P[11]) are natural reassortants man-bull, natural attenuated and give a considerable level of immunity to newborns and young children. Although it is preferable rotavirus person, other rotaviruses, which can be prepared in the form of a composition according to the present invention are bovine rotavirus, rotavirus reassortant swine and human rotaviruses-bull, rotavirus lambs, rotavirus sheep. It is required that suitable compositions and formulations described in this publication, maintained the stability of rotavirus in low titer, i.e. supported 103considering the fact that, as we know, the stability at low initial titer values that are supported during storage, is challenging. The rotavirus vaccine, which has an increased and/or more long-term stability, can be used for prophylaxis of viral infections, preferably of rotavirus infection and/or rotavirus gastroenteritis in children worldwide. Preferably the treatment or prevention include the introduction of three oral doses of an effective amount of the composition to the neonate aged 8-20 weeks in the temporary point dose 1.

In the following table (table 1) shows a comparison of the compositions of conventional rotavirus, i.e., rotavirus, propagated in the absence of human serum albumin 1-8), and rotavirus propagated in the presence of human serum albumin (1A-8A), and clearly shows that the compositions containing the virus, propagated in the presence of human serum albumin, have a higher stability.

Table 1
No. sampleThe fall in the log at 37°CTemporary pointThe fall in the log at 25°CTemporary point
154 weeks1,1924 weeks
1A3,7312 weeks0,9124 weeks
244 weeks1,0924 weeks
2A3,6320 weeks0,7624 weeks
34,45 3 weeks3,4816 weeks
3A3,312 weeks2,2124 weeks
44,434 weeks1,920 weeks
4A3,7212 weeksTo 1.6124 weeks
54,964 weeks1,6624 weeks
5A3,398 weeks1,0824 weeks
64,778 weeks0,8624 weeks
6A3,3212 weeks0,3924 weeks
75,394 weeks 1,9824 weeks
7A2,4412 weeks0,2824 weeks
84,464 weeks1,9624 weeks
8A3,120 weeks0,3424 weeks

The present study also relates to a method of adaptation of rotavirus, for example, natural reassortants man-bull, natural attenuated rotavirus strains 116E (G9P[11]) and I321 (G10P[11]), to suitable cells, such as Vero cells. In one embodiment, the adaptation is in serial passages, 2-20 passages, preferably 2-5 passages. Preferably, each passage occurs during a time period in the range of 24 hours and usually up to 6 days and a maximum period of 10 days. Preferably the virus is a human rotavirus. The method includes the use of an optimized dose of trypsin (0.1 μg/ml to 30 μg/ml) and/or calcium chloride (100 μg/ml to 1000 μg/ml) to activate the virus and the environment for the maintenance of the virus in which a high titer of 10 4up to 108The FIGHT/ml) collected virus obtained from 48 hours to six days. Also provides for the application of adapted strains to obtain a stable liquid compositions monovalent vaccines based on live rotavirus. In addition, the present invention discloses how to obtain a conventional and pre-treated rotaviruses. In addition, the present invention relates to the use of viral antigen, a protein, a combination of one or two different disaccharides for the production of compositions according to the present invention for the treatment or prevention associated with virus diseases, preferably associated with rotavirus disease.

WORKING EXAMPLES

The following working examples are presented to demonstrate preferred embodiments of the invention, but of course, they should not in any way be considered as limiting the scope of the present invention. The examples below are carried out using conventional methods that are well known and routine to specialists in this field, except when described otherwise. In addition, professionals in this field should be clear that the methods disclosed in the examples represent ways that, as discovered by the inventor, good work�up in the practice of the invention, and therefore, we can assume that they constitute preferred embodiments of the methods for its practical implementation. However, professionals in this field in light of the descriptions should be clear that can be implemented numerous changes of specific options, which are described and still obtain a like or similar result without departing from the essence and without leaving the scope of the invention.

Example 1. Obtaining populations of normal and pre-treated rotavirus in the form of total mass and indicators of stability

Bharat Biotech International Ltd. (BBIL) received strains of human rotaviruses, 116E and I321, from the National Institute of health (NIH) under a contract on transfer of materials from the National Institute of Allergy and infectious diseases (NIAID), NIH, Bethesda, USA. Original strains 116E (G9[P11]) and I321 (G10P[11]) have adapted to grow in cell culture passaging in primary kidney cells of the African green monkey (AGMK), then in cell substrate and then MA104 in serially pereselenyi AGMK cells (SPAGMK). MA104 cell substrates and SPAGMK not approved by the National regulatory authority (NRA) to obtain commercial vaccines. Therefore, the two vaccine strains of human rotaviruses (116E and I321) adapted to Vero cells, were grown separately in such cells, obtaining a population of viruses in the form of �BSA mass, and separately prepared in the form of pilot batches of liquid formulations of monovalent live attenuated oral rotavirus vaccine for clinical trials in humans. The strain of human rotavirus 116E, natural reassortant man-bull and natural attenuated strain is a strain G9 man, in which nature was embedded one gene VP4 bull (VP = viral protein) homologous gene site P[11]. Strain I321 called G10P [11], mainly bull contains genes and gene has only two plot of human origin, VP5 and VP7. Specific examples in this publication are described with reference to live and attenuated strain 116E.

In General, used the following method: For the cultivation of rotaviruses used working banks of Vero cells. Vero cells were propagated in modified Dulbecco environment Needle (DMEM) (Sigma®, MO, USA) with the addition of 5-10% fetal bovine serum. In the case of rotavirus requires cleavage by trypsin of one of the two major proteins of the outer shell VP4 in the presence of calcium chloride, so that they can infect Vero cells in vitro. Strains of rotavirus were received in the system of seed banks: viruses main Bank and Bank working viruses. Vero cells in medium without serum was infected with a selected strain and separate charges were made every 48 hours in the Techa�their time period, comprising 144 hours. Three separately collected material were pooled, yielding a total mass, and added a stabilizer containing sucrose, phosphate, glutamate. Such combined total collected mass of material stored at -70°C or 2-8°C. Used two methods of biological production process for obtaining a population of live attenuated viruses in the form of the total mass.

Ordinary viruses:A working cell Bank of Vero cells, which were stored in liquid nitrogen, used for the recovery and cultivation of monolayers of Vero cells for the production process. Two cryovials from a working cell Bank was carefully thawed, taking out tanks for storage in liquid nitrogen, and the cells were transferred to two sterile culture vial polystyrene T-150 to revitalize and added DMEM containing 5% fetal calf serum. Flasks for cultivation were incubated at 37°C for twenty-four hours. After the incubation period the medium from the cultures was drained and added to DMEM containing 5% fetal calf serum to stimulate the formation of fused monolayers.

Flasks were viewed under the microscope in relation to morphology and ability to multiply in your environment. Then the cells were propagated in two passages by getting several containers with cages for Ifni�of debugger the 116E rotavirus or I321.

Rotavirus 116E or I321 chose from Bank working virus system of seed banks, activated by trypsin and inoculable to infect cells. The computation of the multiplicity of infection was carried out in accordance with the population of cells. Cells were infected and maintained at 37°C for one hour for adsorption. After a period of adsorption in cell culture was added to DMEM without serum. Infected cell culture was maintained at 35°C, and separate charges were made every 48 hours during the time of 144 hours. After each individual collection in cell culture was added to DMEM without serum. The cell culture was stopped after the third separate collection. Filtered materials are separate charges collected in sterile containers and stored at 2-8°C. Materials are separate charges were combined, receiving a combined total weight, and stored at 2-8°C. had taken the sample to check the contents of the virus, the sterility of the material of each individual collection and combined total weight. The combined total mass stored at -70°C or 2-8°C in the stabilizer SPG (sucrose of 7.46%, potassium dihydrogen phosphate 0,0515%, potassium hydrophosphate of 0.128% and glutamate 0,101%). Also selected aliquots of samples of material specific fees without stabilizers to obtain data on stability at different pace�off-highway vehicles used.

Getting pre-treated viruses:A working cell Bank of Vero cells, which were stored in liquid nitrogen, used for the recovery and cultivation of monolayers of Vero cells for the production process. Two cryovials from a working cell Bank was carefully thawed, taking out tanks for storage in liquid nitrogen, and the cells were transferred to two sterile culture vial polystyrene T-150 to revitalize and added DMEM containing 5% fetal calf serum and 0.1% human serum albumin. Flasks for cultivation were incubated at 37°C for twenty-four hours. After the incubation period the medium from the culture bottles was decanted and added to DMEM containing 5% fetal calf serum and 0.1% human serum albumin to stimulate the formation of fused monolayers.

Recruited a second group of cells each of which contained 0,1%, 0,2%, 0,3%, 0,5% or 1% human serum albumin (derived from the human body) along with 5% fetal calf serum in DMEM. Also recruited a second group of cells each of which contained 0,1%, 0,2%, 0,3%, 0,5% or 1% human serum albumin (recombinant nature) along with 5% fetal calf serum in DMEM.

Cryoprobes with rotavirus 116E or I321 chose from Bank working virus systems� seed banks and preparing inoculate, to infect a cell culture Vero. Determining the multiplicity of infection was carried out in accordance with the population of cells. Culture cells twice washed with phosphate-saline buffer with a pH from 7.4 to 7.6. Culture of cells infected and maintained at 37°C for one hour for adsorption of the virus. After a period of adsorption in cell culture was added DMEM with 0.1% human serum albumin. In cell culture was added human serum albumin obtained from the human body, or recombinant human serum albumin. Infected cell culture was maintained at 37°C and carried out multiple fees every 48 hours, and culture added to the medium containing the corresponding human serum albumin (derived from human and recombinant human serum albumin). Collections were carried out in a sterile container. The collected materials were stored at 2-8°C. Infected cultures were maintained at 37°C for reproduction of viruses, and cells were maintained until the third gathering, and the cultivation was stopped after the third collect. Materials three harvests from each group were pooled, receiving a combined total weight, and stored at 2-8°C. had taken the sample to check the contents of the virus and the sterility of each material combined total mass of individual sat�the moat. The combined total mass stored at -70°C or 2-8°C in the stabilizer SPG (sucrose of 7.46%, potassium dihydrogen phosphate 0,0515%, potassium hydrophosphate of 0.128% and glutamate 0,101%). Selected aliquots of material specific fees without stabilizers to obtain data on stability at different temperatures.

Fig.1 shows data on the average titer obtained for conventional pre-treated virus and the virus in the five experiments. All five experiments were carried out using the same parameters to demonstrate that the presence of HSA in the culture medium during reproduction of rotavirus on the substrate of Vero cells resulted in a higher titer than without HSA. Carried out three separate gathering on the 2nd, 4th and 6th day after infection and the material were pooled; the titer was an average titer for 3 charges at 2°-8°C. Pre-treated virus gave a higher yield of the title. The minimum difference of the average titer was 0.8 log and a maximum of 1.1 log per milliliter.

Example 2. The composition of normal and pre-treated rotavirus in liquid and lyophilized forms, and the effect of each composition on the parameters of stability

The combined material in the form of the total mass, which was stored at 2-8°C or -70°C, was prepared in the form of a finite total mass on the basis of the target titer of 103up to 108, The FIGHT/0.5 ml, and the resulting material filled the tank in the form of a vaccine. Based on the titer of the combined total weight took a calculated amount of aggregate material and added to a pre-determined volume of the final total weight, containing stabilizers, antibiotics and buffers. The final total weight of filled vials. In the compositions used different stabilizers in different combinations and concentrations, such as lactalbumin hydrolysate (LAH), trehalose, sucrose, starch, lactose, maltose, soy protein, rHSA (not including residual rHSA, which could be transferred from the pre-treated virus collected as a result of the implementation of the method of producing pretreated virus). Aliquots of 0.5 ml of a composition containing the virus, aseptically transferred into vials with a volume of 2.0 ml and stored at 2-8°C, 25°C and 37°C. the stability Parameters were tested at regular intervals to show the stability of conventional pre-treated virus and virus after preparation of compositions with various stabilizers (see table 2).

The preparation of liquid formulations 1-25:Various compositions were prepared under aseptic conditions by calculating the amount of stabilizers, buffers and the amount of viral antigen and the target titer. Samples from each formulation were carried out aseptically and IU�or separately, indicating the sample number, the date and purpose of sample storage at specific temperatures. The vials with samples were stored at 2°-8°C, 25°C and 37°C. the number of samples were coded, and they are periodically tested in respect of credits in accordance with the survey plan stability. The results in the figures from Fig.1 according to Fig.12.

Obtaining lyophilized formulations 26-45:Various compositions were prepared under aseptic conditions by calculating the amount of stabilizers, buffers and the amount of viral antigen and the target titer. Prepared finite mass of material is aseptically filled vials for lyophilization and subjected to freeze-drying process during the period from 42 hours to 48 hours. The freeze-drying process usually has three parts: pre-cooling, primary drying and secondary drying. The freeze-drying cycle was established to primarily support cryohydrate point of various stabilizers used in different combinations. After completion of the freeze drying vials properly sealed closed in a vacuum. Samples from each formulation were carried out aseptically and labeled separately, indicating the sample number, the date and purpose of sample storage at specific temperatures. The vials with samples were stored at 2°-8°C, 25°C and 37°C. Samples�sci periodically tested in respect of credits in accordance with the survey plan stability. Liofilizirovanny the contents of the vials pererestorani, using water for injection.

0,0 0,0 0,0 0,0
Table 2
no prep-rata (Fig.)Stabilizer
LAHSucroseTrehaloseStarchLactoseMaltoseSoyaHSAPVPBicarbonate, mmHarav. gumPyr·HCl
1
(2B)
5,080,00,50,00,00,00,00,00,00,00,00,0
2
(3)
2,50,00,0 0,00,00,00,00,00,00,00,00,0
3
(3)
10,00,00,50,00,00,00,00,00,00,00,00,0
4
(3)
20,00,00,00,00,00,00,00,00,00,00,00,0
5
(3)
2,50,00,50,50,00,00,00,00,00,00,0
6
(4)
5,0800,50,00,00,00,00,00,00,00,00,0
7
(5)
20,00,00,50,00,00,00,00,00,00,00,00,0
8
(5)
10,00,00,00,01,00,00,00,00,00,00,00,0
9
(5)
5,0 800,00,00,00,00,00,00,00,00,00,0
10
(5)
10,00,00,00,00,0500,00,00,00,00,00,0
11
(6)
0,50,01,00,00,00,010,00,00,00,00,00,0
12
(6)
0,50,00,00,01,00,010,00,00,00,00,0
13
(6)
5,080,00,00,00,00,02,50,00,00,00,00,0
14
(6)
5,00,00,00,00,050,02,50,00,00,00,00,0
15
(7)
10,0101,00,00,00,00,00,00,00,00,00,0
(7)10,00,01,00,00,05,00,00,00,00,00,00,0
17
(7)
2,5801,00,00,00,00,00,00,00,00,00,0
18
(7)
2,50,01,00,00,050,00,00,00,00,00,00,0
19
(8)
5,080,00,50,00,0 0,00,00,00,00,00,00,0
20
(8)
5,080,00,50,00,00,00,00,00,00,00,00,0
21
(8)
0,0800,50,00,00,00,00,10,00,00,00,0
22
(8)
0,0800,50,00,00,00,00,10,00,00,0
23
(8)
0,0800,50,00,00,00,00,00,00,00,00,0
24
(8)
0,0800,50,00,00,00,00,00,00,00,00,0
25
(9 and 10)
0,00,00,00,00,00,00,00,00,00,00,00,0
26
(13)
0,012,00,0 0,00,00,00,00,50,00,00,00,0
27
(13)
0,50,00,50,00,00,00,00,00,00,00,00,0
28
(13)
0,00,00,50,00,00,00,50,00,00,00,00,0
29
(13)
0,00,00,50,00,00,00,00,00,250,00,0
30
(14)
0,012,00,10,00,00,00,50,00,00,00,0
31
(14)
0,50,00,50,10,00,00,00,00,00,00,00,0
32
(14)
0,00,00,50,10,00,00,50,00,00,00,00,0
33
(14)
0,0 0,00,50,10,00,00,00,00,250,00,00,0
34
(15)
0,012,00,00,10,00,00,00,50,03040,00,0
35
(15)
0,50,00,50,10,00,00,00,00,03040,00,0
36
(15)
0,00,00,50,10,00,00,5 0,00,03040,00,0
37
(15)
0,00,00,50,10,00,00,00,00,253040,00,0
38
(16)
0,0120,00,00,00,00,00,50,00,00,10,0
39
(16)
0,50,00,50,00,00,00,00,00,00,00,10,0
40
(16)
0,00,00,50,00,00,00,50,00,00,00,10,0
41
(16)
0,00,00,50,00,00,00,00,00,250,00,10,0
42
(17)
0,50,00,00,00,00,00,00,00,250,00,00,0
43
(17)
0,50,00,00,0 0,00,00,00,00,00,00,10,0
44
(17)
0,50,00,00,00,00,00,00,00,00,00,00,1
45
(17)
0,50,00,00,10,00,00,00,00,00,00,00,0
*Abbreviations and explanations:
No. composition - the number of the composition;
PVP - polyvinylpyrrolidone;
Bicarbonate - 304 mm bicarbonate;
Harav. gum - acacia gum;
Pyr·HCl - pyridoxine·HCl.
The compositions 1-24 are liquid formulations with stabilizers.
In the compounds 19, 20, 23 and 24 �sportvan normal virus.
In the compositions 21 and 22 used pre-treated virus.
Composition 25 is a liquid composition containing only buffer without stabilizers.
The compositions 26-45 are lyophilized compositions.

Fig.2 shows the stability data of conventional pre-treated virus and virus in the absence (2A) or presence (2B) stabilizers: 5% LAH + 80% sucrose + 0.5% trehalose, in a liquid composition (composition 1) and kept at 37°C. At this temperature, observed that the stability of the virus gradually decreases, starting from 0 day up to the 16th week in the presence or without stabilizers. In the absence of stabilizers falling titer pre-treated virus is 3.2 log after 4 weeks at 37°C, whereas the fall of the normal titer of the virus is 4.0 log. On the basis of Fig.2B it can be noted that the presence of stabilizers slowed the drop in titer to levels observed in the absence of stabilizers. In addition, in studies in the presence or in the absence of stabilizers shown that the reduction of the titer is slightly slower in the case of preprocessed virus than common virus, which suggests that the HSA used in the production method, has made a significant contribution to stability.

Fig.3 shows data on the stability of rotavirus in four different formulations at 2-8°C (3A),25°C (3B) and 37°C (3C). In each case, the series 1 relates to the composition with 2.5% lactalbumin hydrolysate, series 2 refers to the composition with 10% lactalbumin hydrolysate and 0.5% of trehalose, series 3 relates to the composition with 20% lactalbumin hydrolysate, and series 4 refers to a composition with a combination of 2.5% lactalbumin hydrolysate, 0.5% starch and 0.5% trehalose. At 2-8°C was not falling titer in the series 3 up to twenty-four months, and observed a decline in titer by 0.09 log-0,49 log in series 1, 2 and 4 through 14 months - 24 months at 2-8°C. At 25°C in series 1-4 watched the fall of a titer in the range of 0.94 to log 2,69 log after 3 months and in the range of 1.19 to 4.19 log in 6 months, and in the range of 1.89 to 5,39 12 months. At 37°C in series 1-4 watched the fall of a titer in the range from 1.39 to 2.49 log in six weeks, and in series 2 and 3 observed a decline in titer from 3,09 2,99 to log in 10 weeks. In series 1 and 4, the titer was zero in ten weeks.

Fig.4 shows data on the stability of rotavirus 116E in the composition in the presence or in the absence of stabilizers (5% lactalbumin hydrolysate + 80% sucrose + 0.5% trehalose), which was stored at 2-8°C (4A), 25°C (4B) and 37°C (4C). At 2-8°C in the composition of rotavirus without lactalbumin hydrolysate and two disaccharides was a gradual decrease of the titer 1,84 log up to 24 months. In the case of rotavirus with 5% lactalbumin hydrolysate and a mixture of 80% sucrose and 0.5 % trehalose were observed �Aden titer of up to 24 months. At 25°C in the composition of rotavirus without lactalbumin hydrolysate and combinations of the two disaccharides observed reduction in titer of 6.0 log up to 12 months. In the case of rotavirus with 5% lactalbumin hydrolysate and a mixture of 80% sucrose and 0.5% trehalose was observed gradually falling titer by 2.81 log up to 12 months. At 37°C in the composition of rotavirus without lactalbumin hydrolysate and combinations of the two disaccharides was observed a General decrease of the titer and the titer was zero in 6 weeks. In the case of rotavirus with 5% lactalbumin hydrolysate and a mixture of 80% sucrose and 0.5% trehalose was observed to gradually decline in the titer of 3.85 log up to 16 weeks.

Fig.5 shows data on the stability of rotavirus in four different formulations at 2-8°C (5A), 25°C (5B) and 37°C (5C). In each case, the series 1 relates to the composition with a combination of 20% lactalbumin hydrolysate and 0.5% of trehalose, series 2 refers to the composition with the combination of 10% lactalbumin hydrolysate, 1.0% of lactose, series 3 relates to the composition with the combination of 5% lactalbumin hydrolysate, 80% sucrose, and series 4 refers to a composition with a combination of 10% lactalbumin hydrolysate and 50% maltose. At 2-8°C, the titer did not decrease in episodes 1 and 3 up to 24 months, and in series 2 and 4 are not observed falling titer of up to nine months and observed a decline of 0.37-0,57 log in 24 months. At 25°C was not falling titer in all four series of up to three month� at 25°C and a drop of 1.47 log in 7 months in series 1 and 3. In series 2 and 4, the titer was reduced by 3,47 is 4.07 log in seven months. At 37°C in series 1-4 watched the fall of a titer in the range of 1.37 to 2.77 log in four weeks and in the range from 1.77 to 4.07 log in 6 weeks.

Fig.6 shows data on the stability of liquid formulations of rotavirus 116E at 2-8°C (6A), 25°C (6B) and 37°C (6C). In each case, the series 1 relates to the composition with a combination of 0.5% lactalbumin hydrolysate, 10% soy protein and 1.0% of trehalose, series 2 refers to the composition with a combination of 0.5% lactalbumin hydrolysate, 10% soy protein and 1.0% lactose, series 3 relates to the composition with the combination of 5% lactalbumin hydrolysate, 2.5% of soy protein and 80% sucrose, and series 4 refers to a composition with a combination of 5% lactalbumin hydrolysate, 2.5% of soy protein and 50% maltose. At 2-8°C was not falling titer in series 3 and 4 up to twenty four months, and in series 1 and 2 was observed a slight decrease of titer 0.17 and 0.27 log. At 25°C in series 1-4 watched the fall of a titer in the range from 1.07 to 2.17 log after 3 months and the drop in titer in the range from 2.7 to 4.47 log in eight months. At 37°C in series 1-4 watched the fall of a titer in the range from 1.16 5.07 log in four weeks and the drop in titer in the range from 2.1 to 6.17 log in series 2, 3 and 4, and a zero titer in series 1 after eight weeks.

Fig.7 presents the high stability of the liquid formulations of the 116E rotavirus at 2-8°C (7A), 25°C (7B) and 37°C (7C). In each case� series 1 relates to the composition with the combination of 10% lactalbumin hydrolysate, 10% sucrose and 1.0% of trehalose, series 2 refers to the composition with the combination of 10% lactalbumin hydrolysate, 5% maltose and 1.0% of trehalose, series 3 relates to the composition with a combination of 2.5% lactalbumin hydrolysate, 80% sucrose and 1% trehalose, and series 4 refers to a composition with a combination of 2.5% lactalbumin hydrolysate, 50% maltose and 1% trehalose. At 2-8°C was not falling titer in series 3 and 4 up to twenty four months, and in series 1 and 2 observed a decline in titer by 0.19 to 0.4 log 15 months. At 25°C in series 1-4 watched the fall of a titer in the range from 0.89 to 1.59 log in three months and a fall in the titer in the range from 2.89 to 4,49 log in seven months. At 37°C in series 1-4 watched the fall of a titer in the range of 0.69 to 3.19 log in four weeks and the drop in titer in the range from 1.39 to 4.89 log in six weeks.

Fig.8 shows the high stability of the liquid formulations of the 116E rotavirus at 2-8°C (8A), 25°C (8B) and 37°C (8C). In each case, the series 1 relates to the composition with a combination of common rotavirus, 5% lactalbumin hydrolysate, 80% sucrose and 0.5% trehalose, series 2 refers to the composition with a combination of pre-treated rotavirus, 5% lactalbumin hydrolysate, 80% sucrose and 0.5% trehalose, series 3 relates to the composition with the combination of antigen, obtained by biological method 1, with 0.1% HSA and 80% sucrose and 0.5% trehalose, and series 4 refers to a composition with a combination of antigen, �must register by biological method 2, Of 0.1% HSA, 80% sucrose and 0.5% trehalose, series 4 relates to the composition with the combination of antigen, obtained by biological method 2, with 0.1% HSA, 80% sucrose and 0.5% trehalose, series 5 relates to the composition with the combination of antigen, obtained by biological method 1, 80% sucrose and 0.5% trehalose, series 6 relates to the composition with the combination of antigen, obtained by biological method 2, 80% sucrose and 0.5% trehalose. At 2°-8°C was not falling titer in the series 1, 2 and 4 through 24 months, and the series 3, 5 and 6 saw no reduction up to 18 months and a fall of about 0.2 log was observed after 24 months. At 25°C in series 1, 2, 3 and 4 observed a decline in titer in the range of 0.64 to 1.44 log in six months and the drop in titer in the range of 2.64 to 3,05 log eleven months later. In series 5 and 6 watched the fall of a titer in the range from 2.44 to 2.82 in six months and the fall from 5,09 to 5,02 11 months. At 37°C in series 1, 2, 3 and 4 observed a decline in titer in the range of 1.51 to 4,83 log in six weeks, and a fall in titer in the range from 4.0 to 5,04 log after sixteen weeks. In series 5 and 6 watched the fall of a titer in the range of 2.92 to 3,54 log in six weeks, and the titer was zero through sixteen weeks.

Fig.9 shows data on the stability of rotavirus in five different formulations at 37°C. In the composition, wherein no stabilizer, watched the deep fall of the value of the titer and the titer mills�lsya zero in six weeks. The final total weight of the material, which was a pre-treated virus with 80% sucrose and 0.5% trehalose, has had a drop of 2.2 log after 4 weeks, 2,92 log in six weeks, on 6,02 log in 8 weeks, and the titer was zero in ten weeks. The final total weight 20% lactalbumin hydrolysate had a fall 1.39 log in four weeks, falling 2.09 log in six weeks, to 2.29 log after eight weeks and drop on 5,39 log after 16 weeks. The final total weight 20% lactalbumin hydrolysate and 0.5% trehalose had a falling titre 4,97 through 16 weeks. The final total mass of the combination of 5% lactalbumin hydrolysate, 80% sucrose and 0.5% trehalose had a gradual decline from the first week in its 16th week, falling by 1.1 log in 6 weeks, falling 2.42 log in 12 weeks and the fall of 3.85 log after 16 weeks. When the total mass of the prepared formulations with 80% sucrose and 0.5% trehalose, the stability of the vaccine at 37°C was observed for up to one week and slow the collapse down to four weeks and a sharp drop in six weeks. The total weight, prepared in the form of a composition with 20% lactalbumin hydrolysate, had improved stability and was able to persist up to four weeks, when the fall in titer was less than 1.5 log, and a gradual decrease in titer was observed up to 16 weeks. Not seen a sharp drop in titer� in the case of 20% lactalbumin hydrolysate and 0.5% trehalose through 16 weeks at 37°C compared with the vaccine, containing only 20% of lactalbumin. When the total mass of the prepared formulations with 5% lactalbumin hydrolysate, 80% sucrose and 0.5% trehalose, we observed a decline in the titer of less than 1.5 log after six weeks and fall 3.85 logs through 16 weeks.

Fig.10 shows the stability of rotavirus vaccine with a low titer (less than 104) in five different formulations at 2-8°C (10A) and 37°C (10B). At 2-8°C in the composition, wherein no stabilizer, watched the fall of the value of the titer and the titer was equal to 0.6 after 24 months. In the composition with 80% sucrose and 0.5% trehalose observed a decline of 1.8 log in 24 months with 20% lactalbumin hydrolysate in the composition with 20% lactalbumin hydrolysate and 0.5% trehalose observed a decline of 0.8 and 0.89 log log, and in the composition with 5% lactalbumin hydrolysate, 80% sucrose and 0.5% trehalose observed a decline in titer of 0.7 log 24 months. At 37°C in the composition, wherein no stabilizer, observed a strong decline in the value of the titer and the titer was equal to 0.8 in four weeks. The final total weight, which was prepared with 80% sucrose and 0.5 % trehalose, had a decline of 2.06 log after 4 weeks, whereas the total mass with 20% lactalbumin hydrolysate had a decline of 2.48 log in six weeks, the bulk prepared with 20% lactalbumin hydrolysate and 0.5% trehalose had a falling titer 2,79 8 weeks. The final total weight was prepared in the wee�e composition with a combination of 5% lactalbumin hydrolysate, 80% sucrose and 0.5% trehalose and observed a gradual decline from the first to the 10th week, falling by 1.2 log was observed after 4 weeks, falling by 1.7 log in 8 weeks and drop by 2.5 log after 10 weeks. Fig.10 demonstrates the fact that the hydrolysate of lactalbumin concentrations in some undoubtedly improves the stability of rotavirus vaccines with low titer at 37°C.

Fig.11A-11H shows the stability data of liquid compositions of the pretreated virus with different stabilizers. The same data are shown in table 3 below.

Fig.12A-12H shows data on the stability of liquid formulations of conventional virus with different stabilizers. The same data is presented below in table 4.

12
weeks
Table 3
Liquid compositions of the 116E rotavirus
Detailed description of the composition
Hydrolyzed peptone - 20%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C5,99Of 5.755,484,854,053,722,98At 2.26
25°C5,996.18 of6,0166,08To 5.895,945,985,825,695.08 mm
2°C-8°C5,99Of 6.026,055,976,015,956,13Of 6.02
Detailed description of the composition
Hydrolyzed peptone - 20%
Trehalose - 1%
Fucose - 0,02%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C6,286,115,65The 5.455,284,97Is 4.574,053,322,65
25°C6,286,126,136,036,055,986,216,25,945,825,52
2°C-8°C6,286,15Of 6.02 6,176,156,12The 6.25USD 6.16
Detailed description of the composition
Hydrolyzed egg protein - 20%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C6,11The 5.56Of 5.68To 4.564,11Of 3.983,25With 2.816,11 The 5.56
25°C6,116,165,985,976,14Of 6.024,99The 4.253,953,9

2°C-8°C6,116.18 of6,16,036,046,086,126.18 of
Detailed description of the composition
Hydrolyzed egg protein - 20%
Trehalose - 0,5%
D-sorbitol and 1%
Mannose - 0,5%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C6,17Is 5.58Of 5.024,894,15Of 3.753,052,45
25°C6,176,126,036,055,976,045,845,344,97To 4.56
2°C-8°C6,176,236,0466,156,196,236,17
Detailed description of the composition
Hydrolyzed lactalbumin - 20%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C6,084,96With 2.811,781,12
25°C6,086,126,215,966,22To 5.896,015,925,434,984,42
2°C-8°C6,086.18 of6,366,116,065,996,126,23
Detailed description of the composition
Hydrolyzed lactalbumin - 20%
Trehalose - 0,5%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C6,12To 4.563,462,662,061,89Of 1.35
25°C6,126,216,156,066,136.18 ofThe 6.256,315,855,675,26

2°C-8°C6,126.32 per 6,216,266,116,156,016,07
Detailed description of the composition
Yeast hydrolysate - 20%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C6,226,013,35Of 1.760,83
25°C6,226,16,236,036,135,765,285,18Of 5.024,89A 4.24
2°C-8°C6,226,286,45USD 6.166,046,156,276.35 mm
Detailed description of the composition
Yeast hydrolysate - 20%
Maltose - 5%
Lacto�and - 0,5%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C5,986,033,45Of 2.58Of 1.52
25°C5,98 6,136.32 per6,226.18 of6,236,315,985,38Of 4.884,02
2°C-8°C5,986,036,156,286,136,226,116.18 of

Table 4
Detailed description of the composition
Hydrolyzed peptone - 20%
0
days
the titer
1
week
2
of the week
3
of the week/td>
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C6,135,934,753,211,13
25°C6,136.18 of6,236,316,086,16,155,985,55 5,284,94
2°C-8°C6,136,196,226,086,015,996,116,23
Detailed description of the composition
Hydrolyzed peptone - 20%
Trehalose - 1%
Fucose - 0,02%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C6,316,114,593,342,32
25°C6,316,286,136,016,055,956,216,38Of 5.755,525,22
2°C-8°C6,31 6.35 mm6,286,175,936,126,226,31
Detailed description of the composition
Hydrolyzed egg protein - 20%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°COf 6.02Of 4.542,67 1,57
25°COf 6.026,176,196,085,975,184,373,682,54

2°C-8°COf 6.026.18 of6,236,296,115,936,12For 6.24
Detailed description of the composition
Hydrolyzed egg protein - 20%
Trehalose - 0,5%
D-sorbitol and 1%
Mannose - 0,5%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°COf 6.02Is 5.58Or 3.282,761,59
25°COf 6.026,056,116,145,945,976,13Of 5.294,514,12
2°C-8°COf 6.026,236,126,286,336,156,016,17
Detailed description of the composition
Hydrolyzed lactalbumin - 20%
0
days
the titer
1
week
2
of the week
3
b> of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C6,084,96With 2.811,781,12
25°C6,086,126,215,966,22To 5.896,015,925,43 4,984,42
2°C-8°C6,086.18 of6,366,116,065,996,126,23
Detailed description of the composition
Hydrolyzed lactalbumin - 20%
Trehalose - 0,5%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
<> of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C6,12To 4.563,462,662,061,89Of 1.35
25°C6,126,216,156,066,136.18 ofThe 6.256,315,855,675,26

2°C-8°C6,12 6.32 per6,216,266,116,156,016,07
Detailed description of the composition
Yeast hydrolysate - 20%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C6,22 6,013,35Of 1.760,83
25°C6,226,16,236,036,135,765,285,18Of 5.024,89A 4.24
2°C-8°C6,226,286,45USD 6.166,046,156,276,3
Detailed description of the composition
Yeast hydrolysate - 20%
Maltose - 5%
Lactose - 0,5%
0
days
the titer
1
week
2
of the week
3
of the week
4
of the week
6
weeks
8
weeks
12
weeks
16
weeks
20
weeks
24
of the week
36
weeks
48
weeks
72
of the week
97
weeks
37°C5,986,033,45Of 2.58Of 1.52
25°C5,986,136.32 per6,226.18 of6,236,315,985,38Of 4.884,02
2°C-8°C5,986,036,156,286,136,226,116.18 of

Fig.13-17 shows data on the stability of lyophilized formulations of rotavirus 116E, No. compositions 26-45, in each case at 2°-8°C (A), 25°C (B) and 37°C (C).

This example demonstrates that some compositions are suitable for maintaining stability at 2°-8°C for prolonged periods. The example also show�Vaeth, some compositions are particularly suitable for storage at 25°C or even at 37°C.

All publications, patents and applications for patents referred to in the description are an indicator of the level of specialists in the field of technology to which the present invention relates. All publications, patents and applications for patents included in the present description to the same extent as in the case when specifically and separately indicated that each individual publication or application for the grant of the patent incorporated by reference. Although the above invention is described in detail with illustration and example for purposes of clearer understanding, it will be obvious that in practice can be implemented some changes and modifications in the scope of the attached claims.

1. Stable composition as a vaccine to prevent rotavirus infection, comprising:
(a) an effective amount of viral antigen, which is a pre-processed live attenuated rotavirus, and
(b) a pharmaceutically acceptable buffer with physiological pH value,
in this case, the pre-processed a live attenuated rotavirus rotavirus is who contacted or was subjected to a 0.1% serum albumin human rights during the stage of growth and reproduction of the virus in cul�ur Vero cells, where the culture of Vero cells were initially cultured in the presence of 5% fetal calf serum and 0.1% human serum albumin to pre-treatment of the virus.

2. A composition according to claim 1, further comprising at least one stabilizer comprising a hydrolysate of non-viral such partially hydrolyzed protein, or a combination of the two disaccharides.

3. A composition according to claim 2, in which the specified protein hydrolysate is a hydrolysate of lactalbumin.

4. A composition according to claim 2, in which the combination of the two disaccharides include sucrose and trehalose.

5. The composition of claim 2 containing
(a) viral antigen that is a live attenuated rotavirus
(b) a pharmaceutically acceptable buffer with physiological pH and
(c) non-viral protein or protein hydrolysate.

6. The composition of claim 2 containing
(a) viral antigen that is a live attenuated rotavirus, and
(b) a pharmaceutically acceptable buffer with physiological pH value,
(c) non-viral protein or protein hydrolysate, and
(d) a combination of at least two disaccharides.

7. A composition according to claim 6, containing
(a) viral antigen that is a live attenuated rotavirus specified in claim 1, with a titer in the range from 103up to 108,5The FIGHT/0.5 ml,
(b) a pharmaceutically acceptable buffer, represent�rd a phosphate-citrate buffer (310 mm phosphate and 100 mm citrate) with a pH from 6.8 to 8.0, as a diluent/carrier,
(c) hydrolysed protein, representing a lactalbumin hydrolysate, at a concentration of about 5% wt./about.,
(d) one disaccharide representing sucrose, at a concentration of about 80% wt./about. another disaccharide, representing trehalose, at a concentration of about 0.5% wt./about.

8. A composition according to claim 1, in which live attenuated rotavirus able to give titer, increased on average from a minimum of 0.8 log up to a maximum of 1.1 log per milliliter during storage at ambient conditions compared with the live attenuated rotavirus propagated in the absence of human serum albumin.

9. A composition according to any one of the preceding paragraphs, which represents a liquid composition.

10. A composition according to claim 9, wherein the liquid composition is stable for 6 weeks at 37°C for 6 months at 25°C and for 24 months at 2-8°C.

11. A method of obtaining a stable composition according to claim 1, including
(i) cultivation of a culture of Vero cells initially cultured in the presence of 5% fetal calf serum and 0.1% human serum albumin;
(ii) the risk of infection from these cell culture Vero (i) a live attenuated rotavirus in the culture of Vero cells (i), which is a sequential processing of live attenuated rotavirus by contacting the company�of irusa or the exercise of influence of 0.1% human serum albumin during the stage of growth and reproduction of the virus in cell culture for propagation of the virus;
(iii) adding to the specified virus pharmaceutically acceptable buffer with physiological pH.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions deal with infectious molecule of nucleic acid, coding infectious porcine Torque teNO viruses (PTTV), which contains at least one copy of genome sequence, selected from the group, consisting of sequences, corresponding to genotypes or subtypesPTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA, as well as to biologically functional plasmid or viral vector, containing such infectious nucleic genome sequence, and host-cell, containing such plasmid or vector. In addition claimed inventions include live, attenuated expressible with vector application and purified recombinant capsid subunit or killed viral vaccines for protection against PTTV infection, as well as methods of immunisation of pigs against PTTV viral infection by said vaccine introduction.

EFFECT: characterised inventions can be used to prevent infection, caused by porcine Torque teNo virus.

23 cl, 53 dwg, 5 tbl, 24 ex

FIELD: biotechnology.

SUBSTANCE: characterised strain was isolated from diseased pigs and produced by serial passages on sensitive hetero- and homologous cell cultures and deposited in the collection of the FSBI "Federal Animal healthcare centre" under the registration number of FMD virus strain A No.2187/Kuti/2013 (production). The presented strain is reproduced in monolayer cell culture of porcine kidney (PK), passaged cell cultures of kidney of Siberian mountain ibex (SMIK-30), VPK-21 and IB-RS-2. During 18÷24 hours of incubation the virus yield in the said cell cultures reaches the values of 6.0÷7.0 lg TCD50/cm3. At high multiplicity of infection (1÷10 TCD/cell) causes TCID50 after 5 hours, while maintaining the original characteristics when passaging in cell cultures for 5 passages.

EFFECT: invention can be used to monitor the antigenic and immunogenic activity and for producing biological products for diagnostics and specific prophylaxis of FMD of type A.

6 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology. The subject of the invention is a new strain of the Sendai virus Sen293nsk1 adapted to effective replication in the human cell culture HEK293. The produced strain of the Sendai virus possesses lower virulence for laboratory mice and higher ability to destroy human tumour cells. The strain is supposed to be used experimentally as a therapeutic oncolytic preparation for treating malignant diseases. The invention can be used in treating oncologic diseases.

EFFECT: invention enables providing higher clinical effectiveness in oncologic diseases by using the murine Sendai virus non-pathogenic for humans, possessing an increased oncolytic activity and intensifying anti-tumour immunity.

1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to such compositions and pharmaceutical compositions, which include poxviruses, and namely to those, which include extracellular enveloped viruses. Claimed invention also relates to such method, which is intended for production of poxviruses, as well as poxviruses, obtained in accordance with claimed invention. In addition, claimed invention also relates to application of claimed poxviruses and said composition for medication preparation.

EFFECT: obtaining pharmaceutical compositions, which include poxviruses.

11 cl, 3 dwg

FIELD: biotechnology.

SUBSTANCE: method of detection and differentiation of a genome of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates comprises: extraction of the DNA from the biological material, posing a multiplex polymerase chain reaction using synthesised primers complementary to regions of genes M130R and M151R of the myxoma virus of rabbit, and having the following nucleotide composition: MF 5'TGG-AGC-TTT-TCA-AGC-ATT 3', MR 5'ATA-TCT-CGG-CTC-TAG-GGC-GAG 3', MZ 5' [FAM]-AG-CGT-CGG-ACG-TCT-TCG-TT-[RTQ1] 3', VF 5'AGC-CCT-ATA-AAC-CCG-TAG-ACG-AAC 3', VR 5'CAA-GCT-TTT-TTT-TAT-CCT-CGT-CCG 3', VZ 5' [R6G]-TCG-ACG-GTT-TCG-TCC-GCC-TTC-TTG-[BHQ2] 3', DNA amplification of the virus and evaluation of the reaction. The present inventions may be used in veterinary virology for the detection and differentiation of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates.

EFFECT: increase in accuracy.

2 cl, 6 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, virology and immunology. Particularly, the present invention refers to a new avian astrovirus; to antibodies and their fragments targeted to the above new virus; to antigen preparations, proteins and DNA molecules of new avian astrovirus; to vaccines based on the above new virus or to its antigen preparations, protein or DNA; to methods for producing such vaccines and to diagnostic kits. The present invention can be used in veterinary science.

EFFECT: preparing the new avian astrovirus.

11 cl, 5 dwg, 4 tbl, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for preparing influenza A or B viruses in a cell culture, and a composition of the cell culture for preparing influenza A or B viruses.

EFFECT: invention provides the serum-free culture medium, avoids the need for the stage of cell culture medium replacement, prepares the influenza viruses with the high live virus recovery and can be used for the active immunisation of individuals and for producing the antibodies for various applications, including passive immunisation and diagnostic immunoassays.

22 cl, 24 dwg, 47 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for producing viral-like influenza virus particles (IVP) in a plant or a part thereof. The method involves the expression of a new influenza virus protein HA in plants and purification thereof. The invention also aims at IVPs containing the influenza virus protein HA and herbal lipids. The invention also refers to nucleic acids coding an improved influenza virus HA, as well as to vectors. The IVPs can be used in developing the influenza vaccines or for the treatment of existing vaccines.

EFFECT: presented group of inventions can be used in medicine.

18 cl, 44 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention relates to medical virology and deals with an influenza virus strain. The vaccine strain B/60/Massachusetts/2012/10 is a reassortant, obtained by crossing the "wild" virus B/Massachusetts/2/2012 with the cold-adapted temperature-sensitive virus B/USSR/60/69 -attenuation donor. The strain B/60/Massachusetts/2012/10 is deposited in the State Collection of Viruses FSBI D.I.Ivanovsky Scientific Research Institute of Virology Russian Ministry of Health under No 2740, actively reproduces in developing chicken embryos at the optimal temperature of 32°C, is characterised by temperature sensitivity and cold-adaptedness and safety for laboratory animals. Reassortant inherited genes, which code surface antigens of virus hemagglutinin (HA) and neuraminidase (NA), from the epidemic parent virus and remaining six genes, which code internal non-glycosylated proteins, from the attenuation donor.

EFFECT: claimed invention can be applied in practical healthcare for the prevention of influenza morbidity among adults and children.

4 tbl

FIELD: veterinary medicine.

SUBSTANCE: presented subline of cells A4C2/9k is highly sensitive to the ASF virus. The growth medium is used as medium Needle-MEM with 10% blood serum of swine. The inoculating concentration is 80-100 thousand cells/ml. The mitotic index to 2-3 days of cultivation is 25-35. The subline of cells is deposited at the Specialized collection of cell cultures of agricultural and game animals at the All-Russian research institute of experimental veterinary medicine under the number of 87.

EFFECT: invention enables to isolate the African swine fever virus without prior adaptation in reaction of hemadsorption and provides its accumulation in titre.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to veterinary science and biotechnology. Immunogenic compositions containing a canine influenza virus and a canine respiratory coronavirus, can contain Bordetella bronchiseptica, pertactin, canine para-influenza virus and serotype 2 canine adenovirus; they are effective for treating or preventing the complex of infectious respiratory diseases in dogs.

EFFECT: using the immunogenic compositions is accompanied by no immunologically adverse action between various viral and bacterial antigens.

20 cl, 3 dwg, 4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: composition containing an isolated native biologically active Sendai virus strain deposited in the American Type Culture Collection (ATCC) No. PTA-13024, and a pharmaceutically acceptable carrier, is administered into a patient. A group of inventions also refers to a pharmaceutical composition containing the isolated, genetically engineered, biologically active Sendai virus strain prepared on the basis of a viral material deposited in the American Type Culture Collection (ATCC) No. PTA-13024.

EFFECT: higher clinical effectiveness in treating oncological diseases ensured by the oncolytic action of Sendai virus and the viral stimulation of the antitumour immunity.

21 cl, 1 dwg, 8 ex

FIELD: veterinary medicine.

SUBSTANCE: proposed vaccine contains concentrated antigenic materials from the strain "Adeno III WBR-1-DEP" of the third serotype of subgroup I, reproduced in a passaged culture of cow embryo kidney cells, from strain "Adeno IV Weybridge CT2-DEP" of 4th serotype of subgroup II, reproduced in a passaged culture of calf kidney cells Taurus-1, from the strain "TKA-VIEV-B2-DEP" of herpesvirus of type I, reproduced in a passaged culture of cow embryo kidney cells, from the strain "SF-4-DEP" of virus of parainfluenza-3, reproduced in a passaged culture of cow embryon lung cells from the strain "VK-1-DEP" of viral diarrhoea virus - mucosal disease of cattle, reproduced in a passaged culture of cow embryo kidney cells. The concentrated and avirulent antigenic materials, formalin inactivated, are combined with an adjuvant in an effective ratio.

EFFECT: vaccine induces the high level of antibodies against the said infections and provides reliable protection of newborn calves, young stock of rearing and fattening period.

3 cl, 7 tbl, 9 ex

FIELD: veterinary medicine.

SUBSTANCE: proposed vaccine contains concentrated antigenic materials from the strain "Adeno III WBR-1-DEP" of the third serotype of subgroup I, reproduced in a passaged culture of cow embryo kidney cells, from strain "Adeno IV Weybridge CT2-DEP" of 4th serotype of subgroup II, reproduced in a passaged culture of calf kidney cells Taurus-1, from the strain "TKA-VIEV-B2-DEP" of herpesvirus of type I, reproduced in a passaged culture of cow embryo kidney cells, from the strain "SF-4-DEP" of virus of parainfluenza-3, reproduced in a passaged culture of cow embryon lung cells from the strain "VK-1-DEP" of viral diarrhoea virus - mucosal disease of cattle, reproduced in a passaged culture of cow embryo kidney cells. The concentrated and avirulent antigenic materials, formalin inactivated, are combined with an adjuvant in an effective ratio.

EFFECT: vaccine induces the high level of antibodies against the said infections and provides reliable protection of newborn calves, young stock of rearing and fattening period.

3 cl, 7 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: invention refers to veterinary virology and biotechnology, and concerns a bovine viral diarrhoea, rotavirus and coronavirus infections vaccine. The described vaccine contains an active substance and a target additive. As the active substance, the vaccine contains a mixture of an avirulent purified antigen material of the strain NADL-VNIIZZh-DEP of bovine viral diarrhoea of the family Flaviviridae, of the genus Pestivirus, an avirulent purified antigen material of the strain 101 VNIIZZh-DEP of bovine rotavirus of the family Reoviridae, of the genus Rotavirus, and an avirulent purified antigen material of the strain VNIIZZh-DEP of bovine coronavirus of the family Coronaviridae, of the genus Coronaivirus deposited in the Russian National Collection of Microorganism Strains used in veterinary science and animal industry. The strains are taken in the relation of 1:1:1 in the amounts to provide the protective immune activity of each antigen in an animal's body after the target preparation is introduced.

EFFECT: vaccine induces a high antibody concentration and provides the expressed and long-lasting immunity in the immunised animals .

31 cl, 9 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to veterinary microbiology and concerns a method for preparing a cattle parainfluenza-3 vaccine. The presented method involves preparing a virus-containing material of a cattle parainfluenza-3 virus strain, infecting a continuous cell culture with the virus-containing material, culturing the cattle parainfluenza-3 virus, collecting a virus-containing fluid, inactivating it and preparing a liquid end product with the virus-containing fluid inactivated in an oxidant solution prepared by electrolysis of 10.0-20.0% sodium chloride to achieve pH values 7.0-8.0, the oxidant concentration of 0.7-0.9% and an oxidantion-reduction potential of +1000±50 mV and an inactivation agent consumption of 4.5-5.0 cm3 per 0.8-1.0 l of the virus-containing fluid with the virus-containing fluid inactivation being one-staged in the content of available chlorine Cax=250-500 mg/l for 60-70 min and at temperature 37-38°C at pH 7.2-7.4.

EFFECT: presented solution allows obtaining a high-efficacy, high-quality product with the reduced concentration of the inactivation agent and shorter processing time.

2 cl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to veterinary virology and biotechnology. The vaccine contains an active substance and a target additive. As the active substance, the vaccine contains a mixture of an avirulent purified antigen material of the strain VGNKI-4 of PI-3 virus of the family Paramyxoviridae, the genus Parainfluenza, the subtype Parainfluenza virus 3, the collection of VGNKI No. VGNKI-4-DEP, of an avirulent purified antigen material of the strain VNIIZZh of cattle IRT virus of the family Herpesviridae, the genus Varicellavirus, of the collection of VGNKI VNIIZZh-DEP and of an avirulent purified antigen material of the strain NADL-VNIIZZh of cattle VD virus of the family Flaviviridae, of genus Pestivirus, of the collection FGU VGNKI NADL-VNIIZZh- DEP. The strains are taken in the relation of 1:1:1 in the amounts to provide the protective immune activity of each antigen in an animal's body after the target preparation is introduced.

EFFECT: vaccine induces the high antibody level in immunised animals; the immunity in the immunised animals is formed 10-15 days after the repeated introduction of the vaccine and preserved for at least 6 months; when the above antigens are combined, a synergetic effect is observed that enables reducing the quantity of antigens in the preparations with the preserved and continuous immunity.

27 cl, 9 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to veterinary virology and biotechnology. The vaccine contains an active substance and a target additive. As the active substance, the vaccine contains a mixture of an avirulent purified antigen material of the strain VGNKI-4 of PI-3 virus of the family Paramyxoviridae, the genus Parainfluenza, the subtype Parainfluenza virus 3, the collection of VGNKI No. VGNKI-4-DEP, of an avirulent purified antigen material of the strain VNIIZZh of cattle IRT virus of the family Herpesviridae, the genus Varicellavirus, of the collection of VGNKI VNIIZZh-DEP and of an avirulent purified antigen material of the strain NADL-VNIIZZh of cattle VD virus of the family Flaviviridae, of genus Pestivirus, of the collection FGU VGNKI NADL-VNIIZZh- DEP. The strains are taken in the relation of 1:1:1 in the amounts to provide the protective immune activity of each antigen in an animal's body after the target preparation is introduced.

EFFECT: vaccine induces the high antibody level in immunised animals; the immunity in the immunised animals is formed 10-15 days after the repeated introduction of the vaccine and preserved for at least 6 months; when the above antigens are combined, a synergetic effect is observed that enables reducing the quantity of antigens in the preparations with the preserved and continuous immunity.

27 cl, 9 tbl, 4 ex

FIELD: veterinary medicine.

SUBSTANCE: invention relates to immunoenzymometric test system for serologic diagnosis of reovirus infection in cattle and the monitoring of post-vaccination immunity level. The presented immunoenzymometric test system comprises a specific antigen of the strain "Reo 1 Lang-DEP" of reovirus of type I, inactivated by 0.08% solution of 1,2-aminoethylaziridine representing a specific protein in a concentration of 5-10 mkg/cm3 adsorbed on the surface of polystyrene cavities in carbonate-bicarbonate buffer with merthiolate in a final concentration of 0.1-0.2 mg/cm3, a control positive serum obtained to the antigen of reovirus of type I with the activity in IFA 1:3200-1:6400, control negative serum, anti-species conjugate, potassium dihydrogen phosphate, potassium phosphate dibasic, sodium chloride, a chromogen (ortho-phenylenediamine), hydrogen peroxide, stop reagent and panels for carrying out reaction of the immunoenzymatic assay.

EFFECT: test system enables to diagnose reovirus infection of cattle with high sensitivity.

2 dwg, 3 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary science, and concerns a Newcastle disease (ND), reovirus tenosynovitis (RVT) and avian metapneumoviral infection (aMPVI) associated inactivated emulsion vaccine containing an active substance and a target additive. As the target additive, the vaccine contains the oil adjuvant Montanide ISA-70 VG.

EFFECT: invention provides induction in vaccinated poultry of high antibody level to ND, RTV and aMPVI agents in 28 days after the application which appears to remain unchanged for 12 months and transovarially transmitted to offsprings.

7 cl, 7 ex, 7 dwg, 5 tbl

FIELD: veterinary virology, biotechnology.

SUBSTANCE: new industrial strain of rotavirus serotypes G8P7 in cattle is obtained by successive passages in 3-day old culture of MDBK cells. The strain is deposited in strains microorganism collection in VGNKI at registration number "TE87"-DEP. Virus of the strain № "TE87"-DEP multiplies good in cell cultures MDBK and Marc-145 and for 72-96 h it accumulates with the titers 4.5-7.5 lg TCD50/ml. The strain is stable and retains indices in successive passage in sensitive biological systems for 9-10 passages (observation period). The strain № "TE87"-DEP elicits the broad antigen spectrum of action and high biological activity and can be used for preparing effective diagnostic preparations.

EFFECT: valuable properties of strain.

3 dwg, 3 tbl, 5 ex

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