EA043650B1 - BUFFER-FREE, LOW-DOSE, ACID-FAST ROTAVIRUS VACCINE - Google Patents

Description

Field of technology

The present invention relates to the field of rotavirus vaccines. More specifically, the present invention relates to the field of rotavirus vaccines that do not contain a buffer or require the administration of any antacid before, during or after administration of such rotavirus vaccine. The present invention also relates to dose volume per unit of vaccine, in particular rotavirus vaccines.

State of the art

It is well known that rotaviruses are the leading cause of severe gastroenteritis in infants and older children worldwide. Even improvements in hygiene, water quality and sanitation, which can generally help control many of the bacteria and parasites that cause other types of diarrhea, do not sufficiently prevent the spread of rotavirus. Thus, vaccination is the only strategy that can significantly reduce the incidence of rotavirus infections in underdeveloped and developing countries around the world. Vaccination is more important in countries that lack the necessary economic support and infrastructure to control rotavirus infections in infected patients. To date, there have been at least four commercially available rotavirus vaccines worldwide, the first being Rotashield®, marketed by Wyeth Holdings, in 1999. This vaccine was a live oral tetravalent rotavirus vaccine produced using a vaccine based on 1 rhesus rotavirus, 3 reassorted rhesus and human viruses, licensed in the United States. However, the vaccine was withdrawn from the market after 14 months due to its association with adverse reactions such as intussusception.

The Rotashield® vaccine contained a composition comprising sucrose, monosodium glutamate, monopotassium phosphate, dipotassium phosphate, fetal bovine serum and neomycin sulfate in amphotericin medium. The corresponding patent publication relating to rotavirus vaccine compositions WO 2000006196 A2 discloses that said vaccine compositions may contain pharmaceutically acceptable excipients necessary to provide conditions approximating physiological conditions, such as pH adjusting and buffering agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanol aminooleate, citrate bicarbonate or the like. When the composition is administered orally, it may also be necessary to provide the individual with a buffer solution to partially neutralize gastric acid and protect the reassorted rotavirus upon entry into the intestine. Buffering solutions suitable for this application include sodium bicarbonate, citrate bicarbonate or the like.

The RotaTeq® vaccine is a pentavalent human-bovine (WC3) reassorted live attenuated oral vaccine manufactured by Merck & Co. containing the bovine rotavirus strain WC3, P7[5]G6, containing 5 reassorted variants, of which 4 reassorted variants with the VP7 gene from serotype G1, G2, G3 or G4 and 1 reassorted variant with the VP4 gene P1A[8] from the parental strain of human rotavirus with the remaining genes from the parental strain of bovine rotavirus WC3. The vaccine formulation (relevant Merck patent publications WO 1998/13065, WO 2001/08495 and WO 2002/011540) includes certain stabilizers in tissue culture media along with the presence of buffers in said formulation itself, said buffer being citrate sodium and sodium phosphate. The carboxylate according to the claims of these applications is used and acts as a buffer to ensure the resistance of the rotavirus antigen to the acidic environment of the stomach when the viral antigen is introduced into the stomach of the child.

Further, the Rotarix® vaccine is a monovalent vaccine containing a passaged strain of human rotavirus RIX4414, derived from strain 89-12. Rotarix® vaccine (manufactured by GlaxoSmithKline Biologicals (GSK)) is a live vaccine that contains an attenuated monovalent strain of human rotavirus G1P[8]. The vaccine is available in lyophilized form mixed with stabilizers for reconstitution using a calcium carbonate buffer diluent prior to administration of the vaccine to subjects.

Rotarix was prepared by reconstituting the lyophilized active ingredient (strain RIX4414 + excipients) using 1.0 ml of GSK CaCO ₃ buffer contained in a pre-filled syringe. After introducing said buffer into the vial containing the lyophilized vaccine, said vial was shaken well to resuspend said vaccine. The entire volume of the resuspended product was then drawn into the same syringe, the needle (or transfer device) of said syringe was removed, and said product was rapidly administered as a single oral dose. As an alternative, reconstitution of commercially available lyophilized vaccine would follow the same procedures using an oral applicator filled with buffer and a transfer adapter instead of a syringe and needle. Externally, the reconstituted vaccine should appear milky white. The indicated vaccine should be visually inspected for particulate matter and/or discoloration and should not be administered under any of these conditions.

- 1 043650 specified conditions. Rotarix should be administered orally. The child should be in a semi-recumbent position and the entire contents of the oral applicator containing the reconstituted vaccine should be administered onto the inner cheek. Rotarix should not be given by injection. Therefore, it is understood that reconstitution using a buffer diluent involves many steps, ranging from ensuring that reconstitution has been carried out properly until the time of administration of said vaccine. All of these factors undoubtedly cause decreased compliance with vaccination, which means partial protection against rotavirus infection rather than complete protection.

The corresponding GSK patent applications WO 2001/012797, WO 2005/02133, WO 2006/087205 relate to rotavirus vaccine formulations that contain a serially passaged live attenuated P43 rotavirus strain with specific mutations, also supplemented with an antacid recovery buffer. The G protein (VP4 and VP7) of the specified rotavirus strain is capable of inducing an immune response to at least 4 other rotavirus strains of serotypes G1 and at least one of G2-G14.

There are also other patented compositions of rotavirus vaccine with stabilizers and buffers from Bharat Biotech International Limited with the Indian “asymptomatic” strain 116E isolated from a child’s body. In the case of these compositions, a separate administration of an antacid buffer to the recipient of the vaccine is required to increase the pH of the stomach before the administration of the said vaccine, and the specified antacid buffer is a citrate-phosphate buffer necessary to neutralize the acidity of the stomach during the administration of the rotavirus vaccine antigen. Patent publication WO 2013160913 proposes rotavirus vaccine compositions containing rotavirus antigens, stabilizers and some new buffer combinations. Buffers according to this invention are premixed with rotavirus vaccine compositions to neutralize the highly acidic pH of the stomach without the need for a separate administration of an antacid before administering the vaccine.

Thus, it is clear that various rotavirus vaccines have been developed using different strains, both human viruses and reassorted animal viruses. There are several important differences in the characteristics of these strains, the most common of which are listed below.

Table 1. Differences between monovalent human vaccines and vaccines using reassorted animal viruses

Rotavirus vaccines with human strains Animal reassorted rotavirus vaccines Natural infection provides protection against subsequent rotavirus disease. Animal rotaviruses are naturally attenuated strains, which reduce replication in humans. Human viruses have a high ability to replicate in an infected organism and, therefore, Rotavirus vaccines with reassorted strains animal viruses are very demanding thus are attenuated by repeated passage in tissue cultures of multiple infected hosts. Since these are human strains, a vaccine titer of 10 ^L 4 FFU/0.5 ml is sufficient to induce the necessary immunogenicity against rotavirus infections in humans. high titres of the vaccine to be able to induce required immune response. Broad Spectrum Immunity acquired through various immune effector mechanisms. A wait is required for the neutralizing antibody to be present in the intestinal lumen. The resulting immunity is not as high as with human strain vaccines. Heterotypic protection is achieved through a broad immune response. This means that it provides cross protection against different strains. Reassorted designs vaccines must contain common human rotavirus antigens.

Motives for creating an invention

Thus, rotavirus is known to be very sensitive to acidic environments (Estes, M., Graham, D., Smith, E. and Gerba, S. (1979). Rotavirus Stability and Inactivation. Journal of General Virology, 43(2) , pp. 403-409). It has also been reported that the infantile gastroenteritis virus becomes unstable and the outer shell and capsomeres of these viruses are destroyed when exposed to an environment having a pH of less than 3.0 (Palmer E., Martin, M. and Murphy, F. (1977). Morphology and Stability

- 2 043650 of Infantile Gastroenteritis Virus: Comparison with Reovirus and Bluetongue Virus. Journal of General Virology, 35(3), pp. 403-414). It has also been reported that many rotavirus strains were inactivated within a minute by exposure to pH 2 media, and similar results were obtained using human gastric juice pH 1.8 (Weiss, C. and Clark, H. (1985). Rapid Inactivation of Rotaviruses by Exposure to Acid Buffer or Acidic Gastric Juice. Journal of General Virology, 66(12), pp. 2725-2730). Human rotavirus serotype 1 has also been reported to be completely inactivated by pH 2.5 media (Meng, Z., Birch, S., Heath, R. and Gust, I. (1987). Physicochemical Stability and Inactivation of Human and Simian Rotaviruses. Applied and Environmental Microbiology, 53(4), pp. 727-730). It is known that rotavirus in general is very unstable under the influence of acidic environments and is quickly inactivated. Inactivation rates of three bovine rotaviruses and several primate-derived rotaviruses were determined when exposed to acidic buffers at pH 2.0, pH 3.0, or pH 4.0. Each rotavirus was inactivated very quickly at pH 2.0 (an acidity most similar to normal fasting gastric acidity), and infectivity half-lives were determined to be 1 minute or less. Each rotavirus was inactivated at a much lower rate at pH 3.0; inactivation at pH 4.0 was minimal. Some differences in acid resistance have been found between different rotavirus strains. Although these determinations were performed at room temperature (23°C), experiments at different temperatures indicated an even higher rate of virus inactivation by acid at normal body temperature (37°C). Studies of rotavirus exposed to natural human gastric juice at pH 1.8 or pH 2.1 found rates of viral inactivation similar to those observed using glycine buffer at the same pH. (Ref: Geigy Scientific Tables, volume 1, 1981, page 126).

Inactivation of rotavirus occurs at a pH of 2.0 to 3.0, but at pH 4.0 there is no or minimal inactivation. Since the stomach contains a high gastric acid environment, until now it has been imperative to add buffers to rotavirus vaccine formulations, buffers in rotavirus formulations help maintain a constant pH of said rotavirus vaccine formulations, thereby providing the antacid or buffering capacity of said formulations vaccines. The antacid capacity (also called acid neutralizing capacity) of rotavirus vaccines has been measured using Baby Rossett Rice Assays in GSK patents. The acid neutralizing capacity of a particular formulation is defined as the measured time the pH is maintained above 4.0 and assessed using the pediatric Rossetta-Rice assay. The Baby Rosset assay reportedly simulates the intragastric environment of the human stomach. A recent GSK patent US 8821891 B2 proposes a lower concentration of phosphates with an increased concentration of carboxylate, preferably adipate, ranging from 100 mM to 1 M, preferably from 400 to 700 mM, in a rotavirus vaccine dose volume of only 1.5 ml per dose for a person. Adipate acts as a buffer that can withstand the high acidity of the stomach (pH 1-2). The ability to withstand the acidic environment of the human stomach is measured using the Rossetta-Rice test for children, obtaining a value of 8 to 23 minutes, which is reported to allow and is sufficient to avoid inactivation of the rotavirus antigen in the vaccine after administration of the specified rotavirus vaccine to the child .

Thus, for effective administration of a rotavirus vaccine that is capable of inducing an immunogenic response sufficient to provide protection against rotavirus infections, at least 1.5 to 2 ml of rotavirus vaccine formulation or reconstituted vaccine is required per oral human dose. to effectively ensure the passage of rotavirus antigen through the highly acidic environment of the stomach. Thus, with the help of the above descriptions of the state of the art of rotavirus vaccines available so far, those skilled in the art will also be able to easily understand and recognize that rotavirus antigen is a highly acid labile antigen and an antacid is always required for administration of rotavirus vaccine. or a buffer component, as a result of which the required quantity (based on the dosage in volumes per unit of vaccine) of the administered vaccine increases.

It is also certain that rotavirus antigen is a highly unstable virus, and the situation where the live attenuated rotavirus vaccine antigen may be more unstable is taken into account; Thus, the specified rotavirus antigen is supplemented with appropriate special vaccine stabilizers to ensure stabilization of the vaccine compositions, which increases the dose of the specified vaccine per volume per unit of vaccine. In addition, the inclusion of buffers or antacids or the need to reconstitute the vaccine using a buffer-based vaccine diluent also increases the vaccine dose volume per unit of vaccine. Therefore, providing a rotavirus vaccine that does not contain a buffer/antacid will undoubtedly reduce the dose volume per vaccine unit. At the same time, it is imperative that such a lack of buffer in the rotavirus vaccine does not impair the effectiveness and stability of the rotavirus vaccine.

Since the recipient of rotavirus vaccine is always a child in the 6-week to 6-month age group, it is likely that a significant amount of the rotavirus vaccine formulation administered into the child's mouth will be spilled. This creates a risk of partial administration of the vaccine, resulting in

- 3 043650 whereby the effectiveness of the vaccination procedure decreases and protection against the disease does not occur, despite receiving the specified vaccine. Thus, it is always desirable that the dose volume per unit of rotavirus vaccine be reduced as much as possible without compromising the effectiveness of the vaccine formulation. Currently, the minimum dose volume for rotavirus vaccine known in the art is 1.5 to 2.5 ml. The vaccines contain vaccine stabilizers and vaccine buffers that increase the overall dose volume of said vaccines. Thus, reducing the dose volume per unit of rotavirus vaccine while maintaining the same ability to increase the necessary immunogenicity for the prevention of rotavirus infections in children will reduce the likelihood of spillage of the vaccine composition administered to the child. Thus, it would not be at all obvious to provide a non-buffered rotavirus vaccine formulation in small doses of no more than 1 ml, preferably as little as 0.8 ml or most preferably as little as 0.5 ml per dose for a person who is equally or , rather, is more effective than rotavirus vaccine(s) provided with buffers or antacids, having a large dose volume per vaccine unit.

The prior art discloses rotavirus vaccine formulations, all of which must be stabilized in liquid formulations at refrigeration temperatures in the range of 5±3°C or in the form of lyophilized powders, which require reconstitution of the vaccine before administration of said vaccine using a suitable diluent. It has already been discussed that reconstitution of the vaccine in the case of lyophilized powder requires certain skills and care. In addition, separate manufacturing requirements for diluents on top of those for the vaccine double the cost of vaccine production as well as transportation costs. Lack of skill, care and adequate storage infrastructure contributes to the supply problem of lyophilized rotavirus vaccine. For liquid vaccines at 5±3°C the shelf life is limited to only 2 years. There is also a decrease in titer associated with vaccines provided at 2-8°C during storage and transport, which is undesirable. But it was found that in the case of rotavirus vaccine formulations at -20°C there was no loss at all during transportation and storage. The likelihood of vaccine contamination is also reduced to a much greater extent for vaccines at -20°C. In addition, a vaccine supply facility at -20°C has already been established in almost 120 countries to supply polio vaccine. Thus, existing vaccine storage and vaccine supply similar to that of polio vaccine would greatly contribute to reducing the burden of rotavirus disease worldwide. It would be desirable to develop alternative strategies that would further improve the stability of the rotavirus vaccine so that the vaccine would be available in the most remote places on the globe. Thus, this patent application discloses new rotavirus vaccine compositions, and said rotavirus vaccine compositions are stable for at least 5 years, are completely new and are completely undisclosed in the prior art.

Objective of the present invention

The main object of the present invention is to provide a buffer-free and acid-stable rotavirus vaccine.

Another main object of the present invention is to provide a rotavirus vaccine with a small dose volume of not more than 1 ml or even as little as 0.8 ml, preferably 0.5 ml.

Another object of the present invention is to provide a stable rotavirus vaccine composition with a vaccine volume of only 0.5 ml per dose.

Another object of the present invention is to reduce the spillage of rotavirus vaccine and minimize vaccine-related losses by the recipient of said vaccine and thereby increase compliance with vaccination.

Another object of the present invention is to provide a rotavirus vaccine composition that does not require the use of any specific dilution or reconstitution methods before or during administration of the vaccine.

Another object of the present invention is to provide a rotavirus vaccine that is stable for at least 5 years at -20°C.

Another object of the present invention is to provide a low dose volume rotavirus vaccine that is stable for 2 years at 5±3°C, 6 months at 25°C and 1 week at 37°C.

It is yet another object of the present invention to provide a low dose volume, unbuffered, rotavirus vaccine composition of no more than 1 ml that is stable in the stomach at pH 2 to 4 for at least 20 minutes.

Another object of the present invention is to provide a non-buffered rotavirus vaccine composition with a small dose volume of no more than 1 ml, which is stable at an acidic pH of 2-4 without any addition of buffers or acid stabilizing agents.

Another object of the present invention is to provide a low-dose, buffer-free, acid-fast rotavirus vaccine composition that does not require administration of

- 4 043650 any antacid or buffer before, or after, or during the administration of the specified vaccine to the subject.

Alternatively, it is also an object of the present invention to provide a rotavirus vaccine with a small dose volume of 0.5 ml, along with the presence of a buffer, which is equally effective against rotavirus infections in humans.

Another object of the present invention is to provide a low-dose, unbuffered rotavirus vaccine that is capable of inducing an immune response similar to or greater than that induced by rotavirus vaccines formulated with antacids or buffers.

Brief description of the invention

According to one embodiment of the present invention, a vaccine composition is provided wherein the rotavirus vaccine titer reduction is no more than 0.55 FFU/0.5 ml at a pH of 2-4 over a period of at least one hour.

In another embodiment, the present invention provides a vaccine composition wherein the rotavirus vaccine titer reduction is no more than 0.68 FFU/per 0.5 ml at pH 2-4 over a period of at least 20 minutes.

In another embodiment of the present invention, various rotavirus vaccine compositions are provided in dose volumes ranging from 1 ml to 0.5 ml, and these vaccine compositions do not contain any buffers or antacids and are characterized by a slight decrease in vaccine titer at pH 2 and pH over a period of time of 30 minutes.

Another embodiment of the present invention provides acid neutralizing properties of low dose volume rotavirus vaccine compositions of 0.5 to 1 ml per vaccine unit compared to buffered rotavirus vaccine compositions as determined by Rossetta-Rice pediatric assays.

Another embodiment of the present invention provides a non-buffered rotavirus vaccine with a dose volume of only 0.5 ml, which is capable of inducing a similar or stronger immune response based on four times the seroconversion after vaccination compared to the immune response according to the seroconversion induced using the composition vaccines in the presence of buffers or when antacids are administered.

In another embodiment, the present invention provides a method for the prevention of rotavirus diarrhea in humans, comprising the steps of administering an effective amount of a stabilized rotavirus vaccine composition containing rotavirus strain 116E, a combination of sugars selected from sucrose, lactose and trehalose, lactalbumin hydrolyzate, recombinant human serum albumin, without the presence of any buffer composition in the specified composition of the rotavirus vaccine.

In yet another embodiment, the present invention provides stable rotavirus vaccine compositions that are stable at -20°C for at least 5 years.

Another embodiment of the present invention provides stable rotavirus vaccine compositions at 2-8°C, 25°C and 37°C for 2 years, 6 months and 3 weeks, respectively.

Detailed Description of the Invention

Implementation 1: Buffer-derived rotavirus formulations and stomach acidity

In the above section describing the state of the art, it was mentioned that the use of buffers is a common strategy for the preparation of rotavirus vaccine, which has been used by various manufacturers of rotavirus vaccine so far, and therefore the composition of rotavirus vaccine (Rota vaccine) containing strain 116E at -20°C (SPG and Dulbecco's modified minimal essential medium (DMEM)) were tested using citrate bicarbonate buffer and saline conditions.

Bharat Biotech International Limited received human Rotasprus 116E strains from the National Institute of Health under a material transfer agreement with the National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, USA . The complete genomic sequence of rotavirus 116E strains has already been described in the literature.

Three rotavirus vaccine formulations containing rotavirus 116E, stored at -20°C, were classified into 3 groups (groups I, II and III), described in detail below. The rotavirus vaccine compositions in all three groups described below contained live attenuated rotavirus antigen strain 116E dissolved in 10% SPG (sucrose, potassium dihydrogen phosphate and potassium hydrogen phosphate) and Dulbecco's modified minimal growth medium. These vaccine formulations were processed separately from each other as detailed below.

Group I: 0.5 ml of composition A with rotavirus 116E at -20°C.

Group II: 2 ml (equivalent to 4 human doses of 0.5 ml per human dose) of Rotavirus 116E formulation A mixed with 5 ml saline.

Group III: 0.570 ml of composition A with rotavirus 116E + mixture [1.42 ml of citrate-bicarbonate buffer + 8 ml of 34.8 mEq HCl].

- 5 043650

As stated above, Group I contained a rotavirus vaccine formulation at -20°C, with no buffer or saline added to the vaccine formulation, and the vaccine titer was measured over the next 120 minutes. For Group II, 2 ml of rotavirus 116E vaccine formulation (stored at -20°C) was provided and 5 ml of saline was added and the vaccine titer was measured at a target titer of 10 ^L 6 FFU/ml for a maximum of 120 min. For group III, 0.57 ml of rotavirus 116E vaccine formulation (stored at -20°C) was provided and 1.42 ml of citrate bicarbonate buffer and 8 ml of 34.8 mEq hydrochloric acid were added.

Table 2. Vaccine titers for rotavirus vaccine formulations (at storage temperature -20°C) in the presence of buffers after 0, 60 and 120 min

Group number 0 min in FFU/0.5 ml 60 min in FFU/ml 120 min in FFU/ml Notes Group I 10 ⁶ ' ³⁵ 10 ⁶ ' ¹⁷ 10 ⁶ ' ³² Very stable Group II 10 ⁵ ' ⁷⁶ 10534 105.69 Quite stable Group III 10 ^{4 '61} 10 ⁵ ' ³⁰ 10 ⁵ ' ¹¹ Quite stable

Thus, the above table confirms that when the virus is exposed to saline conditions or in the presence of buffer solutions, the rotavirus antigen titer is stable, and therefore the said vaccine is stable.

Implementation option 2: Immunoperoxidase analysis of rotavirus vaccine compositions at different pH values.

Example 1: An immunoperoxidase assay was performed after rotavirus vaccine formulations were exposed to direct acidic conditions by directly adding 34.8 mEq of hydrochloric acid (HCl) to the rotavirus vaccine formulation. The pH of the vaccine formulations was reduced directly from pH 7 to various lower pH values of pH 2 and pH 4 by direct addition of acid, and then the antigen titer of or to the vaccine was measured at these lower pH values of pH 2 and pH 4. Thereafter, the titer vaccines were measured for no more than 1 hour at various lower pH values of 2 and 4. The following observations were made. The reaction mixture was studied at different times and at different pH levels. Details are presented in the table below. 3. The indicated rotavirus vaccine compositions (at a storage temperature of -20°C) contain live attenuated rotavirus antigen 116E, 10% SPG (monosodium phosphate glutamate), dissolved in Dulbecco's modified minimal nutrient medium.

Table 3. Results of immunoperoxidase analysis with a target titer of 10L5.0 FFU/0.5 ml dose 0 and 60 min after direct addition of acid to rotavirus vaccine formulations (storage temperature: -20°C)

Details about the sample Titer in FFU/0.5 ml dose Notes Actual titer at 0 minute Corrected titer with dilution factor at 0 minute Actual titer after 1 hour Adjusted titer with dilution factor after 1 hour Log decrease after 1 hour 0.5 ml of vaccine composition with rotavirus OM 116E at pH 7.4 104.95 n/a 1 ₀ 4.94 1 ₀ 4.94 - - 0.5 ml of vaccine composition with rotavirus OM 116E + 34.8 mEq HC1 at pH 4.0 10 ³ ' ⁵⁸ 10 ^{5 '05} 10 ³ ' ⁵⁸ 10 ^{5 '05} Nil No influence of pH 0.5 ml of vaccine composition with rotavirus OM 116E + 34.8 mEq HC1 at pH 2.0 10 ³ ' ⁵⁴ 10 ^{5 '01} 10 ^{2 '93} Yu ⁴ ' ⁴⁰ 0.55 Negligible influence of pH

Note: Calculated reduction in infectivity titer due to dilution =

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10 ^1.47 FFU. Thus, the actual titer of the vaccine after dilution is 10 ^2.93 at pH 2. All samples were titrated in triplicate using an immunoperoxidase assay. Oral rotavirus vaccine (target titer 10 ^5.0 FFU/0.5 ml) was stable at pH 4.0. Target titer refers to the minimum concentration of rotavirus 116E antigen in a vaccine vial that must be achieved in the specified vial. The initial titer at a neutral pH of the vaccine is 10 ^4.95 FFU/0.5 ml at 0 minutes. The vaccine titer at pH 2 at 0 minutes is 10 ^3.54 FFU/0.5 ml, and its value drops to 10 ^2.93 FFU/0.5 ml within 1 hour. Thus, after adding the dilution factor value 1.47 as a result of adding acid to the actual titer value (10 ^2.93 FFU/0.5 ml), the adjusted vaccine titer at pH 2 1 hour after adding acid is 10 ^4.40 , which is comparable to the initial vaccine titer 10 ^{4, 95} at pH 7 at 0 minute. Thus, the vaccine had a log total reduction in vaccine titer of only 0.55 at pH 2.0 for 1 hour. This confirms that even at a lower pH of 2, that is, in the particular acidic environment contained in the human stomach, the composition of the rotavirus vaccine is capable of maintaining the vaccine titer at a value of 10 ^4.40 FFU/0.5 ml. With such a vaccine titer, it is capable of inducing a sufficient immune response to subsequent rotavirus infections. In a clinical trial using the Rota 116E vaccine with a titer of 10 ^4.0 , the vaccine provided a 4-fold seroconversion rate of approximately 62%, which is a very encouraging result. (Nita Bhandari et al, J Infect Dis 2009, 200, 421-9).

Example 2.

The same experiment was repeated with a different vaccine (at storage temperature -20°C), and the target titer was 10 ^L 6.0 FFU/0.5 ml (dose). The calculated titer values together with the dilution factor are shown in the table below at various pH values at 0 minutes and after 1 hour. The indicated rotavirus vaccine formulations (at a storage temperature of -20°C) contain live attenuated rotavirus antigen 116E, 10% SPG, dissolved in DMEM.

Table 4. Results of immunoperoxidase analysis with a target titer of 10L6.0 FFU/0.5 ml dose 0 and 60 min after direct addition of acid to rotavirus vaccine formulations (storage temperature: -20°C)

Vaccine sample information pH Corrected titer with dilution factor at 0'h in FFU/0.5 ml Adjusted titer with dilution factor after 1 hour in FFU/0.5 ml Log decline after 1 hour Notes 0.5 ml of vaccine composition with rotavirus 116E 7.2 10 ⁵ ' ³⁵ 10 ⁵ ' ³⁰ 0.05 (no reduction) No influence of pH 0.5 ml of vaccine composition with rotavirus 116E + 34.8 mEq HC1 4.0 10 ⁵ ' ³³ 10 ^{5 '09} 0.24 (slight decrease) No influence of pH 0.5 ml of vaccine composition with rotavirus 116E + 34.8 mEq HC1 2.0 10 ^{4 '89} 10 ^{4 '21} 0.68 (slight decrease) Minor influence of pH

Conclusion: At pH 4.0 in the vaccine samples there was a decrease in vaccine titer, the log of which was only 0.24, which can be considered the absence of influence of pH under practical conditions maintained for 1 hour. At pH 2.0 there was a decrease in titer, the log of which was 0.46 at 0 hours, and the log10 of which in FRC was 0.68 at 1 hour. This suggests that pH has very little effect on vaccine titer values at a lower acidic pH value such as pH 2.

From the table above, it is clear that the vaccine titer of 10A4.89 FFU/0.5 ml at pH 2 decreases to a titer value of 10A4.21 FFU/0.5 ml; a log titer in the range of 4.89-4.21 is quite sufficient for rotavirus replication in the gastrointestinal tract of a child aged 6 weeks to 2 years. The acidity of the child's stomach is also pH 2, which reflects the model of the intragastric environment. The vaccine is still able to maintain its effect at a titer whose Log 10 in FFU/0.5 ml is 4.89, which is sufficient for passage through the stomach and subsequent penetration into the intestinal villi and for further replication of the rotavirus responsible for inducing the immune response in a child, which provides protection against rotavirus gastroenteritis and its prevention. Thus, the vaccine was found to be stable at an acidic pH of 2 for at least 1 hour without any significant decrease in vaccine titer.

Implementation 3: In vitro experiment to demonstrate that for a vaccine containing

- 7 043650 rotavirus 116E, no antacid required:

In addition to the conclusions from the clinical study, which are reported later in this description, the administration of the vaccine directly without buffer was also tested under similar conditions of exposure to the acidic environment of the stomach in vitro.

Example 3.1.

Provided 1 ml of vaccine (composition 3A) containing rotavirus 116E, a combination of sucrose (50%), lactose (0.5%) and trehalose (0.5%), lactalbumin hydrolyzate (1%), human serum albumin (0.4 %), added 1.25 ml of 0.1 N. HCl and mixed well. A sample was collected and tested for virus content at various pH levels at various time intervals. The results are presented in table. 5.1 below.

Table 5.1. Immunoperoxidase analysis of 1 ml of vaccine with rotavirus 116E (composition 3A)

Sample no. Sample Details Virus content (FFU/ml) 1 pH 2.0 - 0 minutes 5.72 2 pH 2.0 - 10 minutes 4.78 3 pH 2.0 - 30 minutes 3.84 4 pH 3.0 - 0 minutes 5.96 5 pH 3.0 - 10 minutes 5.96 6 pH 3.0 - 30 minutes 5.91 7 Control 6.10

Example 3.2.

To 0.8 ml of a vaccine containing rotavirus 116E, a combination of sucrose (40%) and trehalose (0.5%), lactalbumin hydrolyzate (20%), polyvinylpyrrolidine (0.5%) (composition 3B), 1.25 ml was added 0.1n. HCl and mixed well. A sample was taken and the virus content was determined at various pH levels at various time intervals. The results are presented in table. 5.2 below.

Table 5.2. Immunoperoxidase analysis of 0.8 ml of vaccine with rotavirus 116E (composition 3B)

Sample no. Sample Details Virus content (FFU/ml) Virus content (FFU/0.8 ml) 1 pH 2.0-0 minutes 4.71 4.62 2 pH 2.0 - 10 minutes 4.69 4.60 3 pH 2.0 - 30 minutes 4.41 4.32 4 pH 3.0 - 0 minutes 5.97 5.88 5 pH 3.0 - 10 minutes 5.94 5.85 6 pH 3.0-30 minutes 5.93 5.84 7 Control 6.10

Example 3.3.

To 0.5 ml of rotavirus 116E vaccine containing a combination of sucrose (60%) and trehalose (0.5%), soy protein (20%), pyridoxine-HCl (0.1%), polyvinylpyrrolidine (0.25%) (composition 3C), added 1.25 ml of 0.1 N. HCl and mixed well. A sample was collected and tested for virus content at various pH levels at various time intervals. The results are presented in table. 5.3 below.

Table 5.3. Immunoperoxidase analysis of 0.8 ml of vaccine with rotavirus 116E (composition 3C)

Sample no. Sample Details Virus content (FFU/0.5 ml) 1 pH 2.0 - 0 minutes 4.29 2 pH 2.0 - 10 minutes 4.21 3 pH 2.0 - 30 minutes 3.39 4 pH 3.0 - 0 minutes 5.20 5 pH 3.0 - 10 minutes 5.17 6 pH 3.0 - 30 minutes 5.08 7 Control 5.80

Example 3.4.

To 0.5 ml of rotavirus 116E vaccine containing 10% SPG dissolved in DMEM (composition 3D), 1.25 ml of 0.1 N was added. HCl and mixed well. A sample was collected and tested for virus content at various pH levels at various time intervals. The results are presented in table. 5.4 below.

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Table 5.4. Immunoperoxidase analysis of 0.8 ml vaccine with rotavirus 116E (3D composition)

Sample no. Sample Details Virus content (FFU/0.5 ml) 1 pH 2.0 - 0 minutes 4.78 2 pH 2.0 - 10 minutes 4.48 3 pH 2.0-30 minutes 4.29 4 pH 3.0 - 0 minutes 5.22 5 pH 3.0 - 10 minutes 5.17 6 pH 3.0 - 30 minutes 4.70 7 Control 5.80

Example 3.5.

To 0.5 ml of rotavirus 116E vaccine containing sucrose (40%), trehalose (0.5%), lactose (5%), recombinant human serum albumin (rHSA) (0.5%), lactalbumin hydrolyzate (LAH) (1%) and mixed buffer (ammonium acetate + ammonium bicarbonate + ammonium hydrogen orthophosphate) (composition 3D), 1.25 ml of 0.1 N was added. HCl and mixed well. A sample was collected and tested for virus content at various pH levels at various time intervals. The results are presented in table. 5.5 below.

Table 5.5. Immunoperoxidase analysis of 0.8 ml vaccine with rotavirus 116E (3D composition)

Sample no. Sample Details Virus content (FFU/0.5 ml) 1 pH 2.0-0 minutes 4.77 2 pH 2.0 - 10 minutes 4.65 3 pH 2.0 - 30 minutes 4.39 4 pH 3.0 - 0 minutes 5.21 5 pH 3.0 - 10 minutes 5.19 6 pH 3.0 - 30 minutes 4.87 7 Control 5.80

Conclusions: From the above tables it is obvious that at pH 2.0 for 30 minutes, any vaccine compositions containing rotavirus 116E are also capable of being stable in the absence of any buffer or any antacid.

Implementation 4: Children's Rossetta-Rice (BRR) analysis

The BRR assay is a validated technique for the acid neutralizing ability of rotavirus vaccine formulations. Thus, selected vaccine formulations containing citrate-phosphate buffer and citrate-bicarbonate buffer are evaluated for acid neutralizing ability using the specified assay. In vitro experiments were performed under simulated pediatric gastric conditions using the pediatric Rossetta-Rice (BRR) assay. Various formulations with different combinations of buffering agents were selected for neutralization experiments, and the results are presented in the table below at various pH values at which conditions were simulated to match the infant's gastric environment. The vaccine compositions were diluted with water for injection to 10 ml, then 4 ml of 0.1 N was added. HCl, after which 0.5 ml per minute of 0.1 N was added. HCl until pH reaches 4.0. Acid neutralizing capacity (ANC) is defined as the time in minutes required to maintain pH above 4.0.

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Table 6. Details of vaccine formulations

Composition of vaccine 4A Rotavirus antigen 116E with a target titer of 10 ^L 6 FFU/0.5 ml; 10% SPG and DMEM (sufficient amount) Composition of vaccine 4B Rotavirus antigen 116E with a target titer of 10 ^L 6 FFU/0.5 ml; 10% SPG and DMEM (sufficient amount) added to 2.5 ml citrate bicarbonate buffer Composition of the 4 C vaccine Rotavirus antigen 116E with a target titer of 10 ^L 6 FFU/0.5 ml, a combination of at least 2 sugars (50% sucrose and 0.5% trehalose) and 0.5% lactalbumin hydrolyzate. (Sample 8 below). Composition of the 4D vaccine Rotavirus antigen 116E with a target titer of 10 ^L 6 FFU/0.5 ml, a combination of at least 2 sugars (40% sucrose, 0.5% trehalose), 0.5% lactalbumin hydrolyzate, 0.35% human serum albumin and 1 .1 M phosphate buffer.

The following observations were made in the pediatric Rossetta-Rice analysis using vaccine formulations 4A-4D above (Table 7).

Table 7. Rossetta-Rice analysis for children for rotavirus vaccine formulations with buffer ____________ versus formulations without ____________

Vaccine compositions Initial pH pH after adding acid The volume of added 0.1 and. NS1 Rossetta-Rice analysis values for children Initial vaccine titers in FFU/0.5 ml Vaccine titers in FFU/0.5 ml after 1 hour Vaccine composition 4A at -20°C without buffer with a dose of 0.5 ml 7.48 1.95 4.0 ml BRR less than 0 minutes (instantaneous pH drop) 10 ^L 4.83 1O ^L 3.83 Vaccine composition 4B at -20°C with 2.5 ml citrate bicarbonate buffer with a dose of 0.5 ml 8.35 4.22 10.0 ml BRR 12 minutes 10 ^l 6.17 10 ^l 6.15 Composition of vaccine 4C at 5+3 °C without buffer with a dose of 0.5 ml 7.28 3.98 5.0 ml BRR 2 minutes 10 ^l 5.30 10 ^l 5.02 Composition of the 4D vaccine at 5+3°C with a buffer in a dose of 1.5 ml or 2 ml 6.75 4.33 10.5 ml BRR approximately 13 minutes 10 ^l 6.12 10 ^l 6.11

Conclusion: Thus, from the above table it is clear that even for the vaccine composition 4A indicated above, which demonstrates an instantaneous drop in pH from 7.48 to 1.95, the vaccine titer is kept in the range of 10 ^L 4.83 FFU/0, 5 ml up to 10 ^L 3.83 FFU/0.5 ml. Likewise, in the case of vaccine formulation 4C, the vaccine titer is maintained in the range from 10L5.30 to 10L5.02 FFU/0.5 ml when the pH value drops from 7.28 to 3.98 within 2 minutes. Thus, even if the BRR is only 2 min, the vaccine titer is maintained at an acceptable level, i.e. is 10L5.02 FFU/0.5 ml. This significant titer of the vaccine is sufficient to ensure the induction of an immune response and thereby provide protection against rotavirus gastroenteritis and its prevention in children.

Implementation option 4: Clinical trial of the ROTAVAC® vaccine containing rotavirus strain 116E, without adding a buffer.

Hypothesis and rationale for the study

Natural transmission of rotavirus is believed to occur through the fecal-oral route. Rotaviruses in general are acid labile, and an acidic environment is thought to affect the viability of the virus.

Given the acid-labile nature of rotaviruses, they are extremely successful in causing widespread infection of mammals, most of which have gastric pH values of approximately 2. One suggestion is that the child's stomach may be somewhat more amenable to rotavirus survival than the adult stomach, as as the pH level in a child's stomach is usually approximately 3.2 compared to the adult value of approximately 1.0. This could explain the fact that 60 to 90% of human cases of rotavirus disease occur in children under 3 years of age.

Study plan

To test this hypothesis, a large multicenter randomization study was conducted.

- 10 043650 bath controlled trial to evaluate and compare the immune response to the vaccine in subjects receiving ROTAVAC vaccine with citrate bicarbonate buffer with those receiving ROTAVAC without buffer.

Accordingly, the trial included the following three treatment groups:

Group I (received ROTAVAC® 5 minutes before injection of 2.5 ml of buffer),

Group II (received ROTAVAC® without buffer),

Group III (received ROTAVAC® mixed with 2.5 ml of buffer immediately before administration).

The investigator and safety officials (study coordinators) were blinded to treatment group assignments. Blinding was provided by an experienced nurse who administered the vaccine according to the designated treatment group. All study participants received 3 doses of the vaccine at an interval of 4 weeks. The immune response to the vaccine was checked 4 weeks after the third dose of the vaccine (day 84) and compared with baseline values (day 0). Immunogenicity was determined based on geometric mean titers (GMT) of serum anti-rotavirus IgA and seroconversion (fold increase in antibody levels from the period before vaccination to the period after vaccination) in the treatment groups.

Research results and conclusions

The immune response after vaccination was comparable between treatment groups, with no statistically significant difference in the anti-rotavirus IgA response. It is important to note that the seroconversion and GST values obtained in group II (ROTAVAC® administered without buffer) were similar to those obtained in the other two treatment groups in which ROTAVAC® was administered with buffer.

Anti-rotavirus IgA GMT after vaccination in the group where ROTAVAC was administered without buffer was 20.7 U/ml compared with 19.6 U/ml and 19.2 U/ml in the two groups that received antacid buffer 5 minutes before the vaccine and simultaneously in the form of a mixture with the vaccine, respectively (Table 8). Differences between groups were not statistically significant (p>0.05, Student's T-test).

Table 8. Geometric mean titers and 95% CI for results (day 0 and day 84)

Inspection Group I(N = 290) Group II (N = 287) Group III (N = 286) SGT 95% CI SGT 95% CI SGT 95% CI 0 DAY 10.5 (9.4, 11.7) 10.8 (9.7, 11.9) 10.2 (9.2, 11.3) 84 DAY 19.6 (17.0, 22.7) 20.7 (17.9, 24.0) 19.2 (16.8, 22.1)

N = Number of subjects assessed in each group;

GMT = geometric mean;

95% CI (LI, HL) = confidence intervals (lower limit, upper limit)

Similarly, the fourfold seroconversion observed in the groups receiving ROTAVAC® without and with antacid buffer had similar values of 29.2, 24.5 and 25.1%, respectively (Table 9). In addition, the lower limit of the 95% confidence interval for the difference in seroconversion (2-fold) between treatment groups was more than -10%, indicating a noninferior magnitude of the immune response achieved with all three treatment regimens (Table 10).

Table 9. Seroconversion and 95% CI (2-, 3-, or 4-fold change in antibody titers) between baseline and 84 days after vaccination

Frequency of changes Group I (N = 290) Group II (N = 287) Group III (N = 286) % 95% CI % 95% CI % 95% CI 2 30.7 (25.7, 36.2) 35.2 (29.9, 40.9) 33.5 (28.3, 39.2) 3 27.6 (22,8,33,1) 32.4 (27,3,38,1) 31.8 (26.7, 37.4) 4 24.5 (19,8,29,7) 29.2 (24.3, 34.8) 25.1 (20.5, 30.5)

N = Number of subjects assessed in each group;

(95% CI) = confidence intervals (lower limit, upper limit)

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Table 10. Difference in % 2-fold seroconversion after vaccination in treatment groups

% 2-fold seroconversion after vaccination (day 84) Group I (N = 290) Group II (N = 287) Group III (N = 286) % 95% CI % 95% CI % 95% CI DAY 84 30.7 (25.7, 36.2) 35.2 (29,9,40,9) 33.5 (28.3, 39.2) Difference in % seroconversion with 95% CI (two-tailed χ-square test) Group % (95% CI difference) Group I versus group II 4.5 (-3.5, 12.5) Group II versus group III 1.6 (-6.5, 9.7) Group I versus group III 2.9 (-4.7, 10.5)

Reactogenicity and safety for solicited and unsolicited adverse events were comparable across the three groups, with no statistically significant difference.

ROTAVAC® vaccine was well tolerated in all three treatment groups that received the vaccine with or without an antacid buffer.

Implementation Option 5: New formulations containing rotavirus 116E

Stability data for liquid formulations of ORV 116E vaccine at 37°C, 25°C and 2-8°C

The following rotavirus vaccine formulations were tested at a dose volume of 0.5 ml, without buffer (samples 1-12, 14-16) and with a mixed buffer at a dose volume of 0.5 ml (sample 13) over an extended period of time, up to 5 years. Below are details of the various formulations along with their stability data at 37°C for up to 4 weeks, 25°C for at least 6 months and 5±3°C for up to 2-5 years. These studies confirm that these unbuffered rotavirus vaccine formulations at dose volumes of 0.5 ml are also inherently stable over extended periods of time at refrigerated temperatures for at least 2 years. Rotavirus vaccine formulations have also been found to be stable over a period of 5 years at refrigerated temperatures.

Sample 1 was prepared containing rotavirus 116E, 50% sucrose, 0.5% lactose, 0.5% human serum albumin (HSA) and 0.05% lactalbumin hydrolysate.

Sample 1 stability: ____________ _____ ____________________

Pace. 0 day 1M 2M zm 6M 9M 12M 24M 36M 60M 5±3°C 6.02 6.0 6.43 6.21 6.16 6.16 6.07 25°C 6.02 6.07 5.81 5.9 5.51 4.65 37°С 6.02 5.32 2.63

Sample 2 was prepared containing rotavirus 116E, 50% sucrose, 0.5% trehalose, 0.5% HSA and 0.5% lactalbumin hydrolysate.

Sample 2 stability:

Pace. 0 day 1M 2M ZM 6M 9M 12M 24M 36M 60M 5+3 °C 6.09 6.14 6.19 6.07 6.10 6.05 6.01 25°C 6.09 6.02 6.06 5.63 5.51 2.48 37°C 6.09 5.47 3.23

Sample 3 was prepared containing rotavirus 116E, 50% sucrose, 1.0% trehalose, 0.5% HSA and 0.5% lactalbumin hydrolysate.

Sample 3 stability:

Pace. 0 day 1M 2M ZM 6M 9M 12M 24M 36M 60M 5±3°C 6.22 6.24 6.26 6.14 6.20 6.13 6.04 25°С 6.22 6.18 6.15 5.80 5.34 2.03 37°C 6.22 5.35 3.22

Sample 4 was prepared, containing rotavirus 116E, 40% sucrose, 0.5% lactose and 1.0% lactalbumin hydrolysate.

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Sample 4 stability:

Sample 5 was prepared, containing rotavirus 116E, 40% sucrose, 5.0% maltose and 1.0% lactalbumin hydrolysate.

Sample 5 stability:

Sample 6 was prepared, containing rotavirus 116E, 40% sucrose, 0.5% trehalose and 1% lactalbumin hydrolyzate.

Sample 6 stability:

Pace. 0 day 1M 2M ZM 6M 9M 12M 15M 24M 60M 5+3 °C 6.19 6.15 6.10 6.03 6.22 6.13 25°C 6.19 5.87 5.43 5.12 3.25 37°C 6.19 4.02 2.52

Sample 7 was prepared containing rotavirus 116E, 40% sucrose, 1.0% trehalose and 1.0% lactalbumin hydrolysate.

Sample 7 stability:

Sample 8 was prepared containing rotavirus 116E, 50% sucrose, 0.5% trehalose and 0.5% lactalbumin hydrolysate.

Sample 8 stability:

Sample 9 was prepared, containing rotavirus 116E, 70% sucrose, 0.5% trehalose.

Sample stability 9:______________________________________________________________

Pace. 0 day 1M 2M ZM 6M 15M 18M 24M 36M 60M 5±3°C 6.38 6.31 6.23 6.44 6.35 6.26 6.18 25°C 6.38 6.41 6.35 6.23 5.57 37°С 6.38 5.07 4.01

Sample 10 was prepared containing rotavirus 116E, 50% sucrose, 0.5% lactose, 0.5% maltose, 0.5% HSA and 0.05% lactalbumin hydrolysate.

Sample 10 stability:

Pace. 0 day 1M 2M ZM 6M 15M 18M 24M 36M 60M 5+3 °C 6.25 6.15 6.03 6.34 6.08 6.12 6.11 6 25°C 6.25 6.14 6.27 6.12 5.88 37°C 6.25 5.02 4.35

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Sample 11 was prepared containing rotavirus 116E, 50% sucrose, 0.5% trehalose, 0.5% maltose, 0.5% HSA.

Sample 11 stability:

Pace. 0 day 1M 2M ZM 6M 9M 12M 24M 36M 60M 5±3°C 6.68 6.62 6.95 6.84 6.64 6.65 6.37 6.72 6.05 25°C 6.68 6.50 6.20 6.04 5.32 4.47 37°C 6.68 6.10 5.58 4.26

Sample 12 was prepared containing rotavirus 116E, 50% sucrose, 0.5% trehalose, 0.5% HSA and 0.05% lactalbumin hydrolysate.

Stability of sample 12: _______________________ _____

Pace. 0 day 1M 2M ZM 6M 9M 12M 24M 36M 60M 5±3°C 6.62 6.76 6.98 7.00 6.86 6.74 5.99 6.46 6.17 25°C 6.62 6.46 6.42 5.99 5.13 3.25 37°C 6.62 5.58 5.06

Sample 13 was prepared containing rotavirus 116E, sucrose (40%), trehalose (0.5%), lactose (5%), rHSA (0.5%), LAH (1%) and mixed buffer (ammonium acetate + ammonium bicarbonate + ammonium hydroorthophosphate).

Sample 13 stability:

Pace. 0 day 1M 2M ZM 6M 9M 12M 24M 36M 60M 5±3°C 6.61 6.59 6.58 6.60 6.53 6.54 6.18 6.01 5.83 25°C 6.61 6.46 6.44 5.79 5.25 4.15 37°C 6.61 5.56 5.01

Stability of sample 13 at -20°C:

Pace. 0 day 1M 2M ZM 6M 9M 12M 24M 36M 60M -20°С 6.61 6.59 6.59 6.56 6.54 6.52 6.21 6.11 6.13 6.09

Samples 14, 15 and 16 were prepared using rotavirus 116E, 10% SPG dissolved in DMEM. Samples 14, 15 and 16 were stored at -20°C and their stability was checked for 60 months. Vaccine formulations 13, 14 and 15 were found to be very stable at -20°C for a period of at least

0.5 ml are stable at -20°C for 5 years, at 2-8°C for 2 years, at 25°C for 6 months and at 37°C for 1 week. It has also been found that, regardless of the composition of the vaccine, said rotavirus vaccine at a dose volume of 0.5 to 1 ml is also acid-resistant, as presented in embodiment 3 at a pH in the range of 2-4. Thus, the applicants would like to mention that, regardless of the details of the vaccine compositions, any rotavirus vaccine containing live attenuated rotavirus 116E is able to withstand the highly acidic environment in the child's stomach and ensure the induction of the necessary immune response to rotavirus infections. The vaccine compositions presented in this patent application are provided only for the purpose of clarifying the breadth and scope of the present invention. It is not practical for the applicant to provide details of all possible vaccine formulations, although the applicant has decreed

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Claims (12)

There are numerous preparation strategies in Embodiment 3, Example 3 and Embodiment 5. Thus, all possible modifications apparent to one skilled in the art for the preparation strategy of a rotavirus vaccine containing rotavirus strain 116E are also included in the present invention. It should be understood and construed accordingly that vaccine compositions containing live attenuated rotavirus 116E, which are not specifically included in the present patent application, are also within the scope of the present invention. CLAIM 1. Rotavirus vaccine containing: a) rotavirus strain 116E; b) a combination of sugars selected from sucrose at a concentration of 40-70%, lactose at a concentration of 0.5% and trehalose at a concentration of 0.5-1%; c) lactalbumin hydrolyzate at a concentration of 0.05-1% and d) recombinant human serum albumin at a concentration of 0.4-0.5%, wherein said vaccine does not contain a buffer or antacid, wherein said vaccine is stable in the stomach at pH 2 to pH 4 without reducing the titer, and is able to withstand the acidic environment of the stomach in humans for at least 20 minutes and does not require the administration of any antacid or buffer before or after administration of the vaccine. 2. Rotavirus vaccine according to claim 1, containing rotavirus 116E, wherein the dose volume per unit of vaccine is no more than 1 ml. 3. Rotavirus vaccine according to claim 1, characterized in that the dose volume per unit of vaccine is 0.8 ml per unit of vaccine. 4. Rotavirus vaccine according to claim 1, characterized in that the dose volume per unit of vaccine is 0.5 ml per unit of vaccine. 5. Rotavirus vaccine according to claim 1, characterized in that said vaccine is stable at -20°C for at least 60 months. 6. Rotavirus vaccine according to claim 1, characterized in that said vaccine is stable at 2-8°C for at least 36 months, at 25°C for 6 months and at 37°C for at least 4 weeks 7. Rotavirus vaccine according to claim 1, characterized in that the induced immune response, determined by fourfold seroconversion after vaccination, is similar or stronger than the immune response, determined by seroconversion, achieved using the vaccine composition in the presence of buffers or with the administration of antacids. 8. Rotavirus vaccine according to claim 1, characterized in that said vaccine is a liquid oral vaccine composition. 9. A method of inducing an immune response to rotavirus in a subject, comprising oral administration of 0.5 ml of a stabilized rotavirus vaccine not containing any antacid or buffer, said vaccine containing rotavirus strain 116E; a combination of sugars selected from sucrose at a concentration of 40-70%, lactose at a concentration of 0.5% and trehalose at a concentration of 0.5-1%; lactalbumin hydrolyzate at a concentration of 0.05-1%; recombinant human serum albumin in a concentration of 0.4-0.5%, without the presence of any buffer or antacid in the specified rotavirus vaccine, and the specified vaccine is stable in the stomach at pH 2 to pH 4 without reducing the titer, able to withstand exposure to acidic gastric environment in a person for at least 20 minutes and does not require the administration of any antacid or buffer before or after administration of the vaccine to the subject. 10. The method according to claim 9, characterized in that said rotavirus vaccine remains stable for a period of at least 60 months at -20°C. 11. The method according to claim 9, characterized in that said rotavirus vaccine remains stable for a period of at least 3 years at 2-8°C, a period of at least 6 months at 25°C and for a period of at least 4 weeks at 37°C. 12. The method of claim 9, wherein said vaccine, when administered orally to a subject in the absence of an antacid or buffer, is capable of inducing a similar or stronger immune response, as measured by four times the seroconversion following vaccination, compared to the immune response, as measured by the seroconversion achieved by using the vaccine composition either in the presence of buffers or when administering antacids. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2