Buffer Free, Acid Stable Low Dose Volume Rotavirus Vaccine

Abstract

A buffer free, acid stable, low dose volume rotavirus vaccine is disclosed. The vaccine is available in dose volume of less than 1 ml per dose for oral administration and it is without any buffer. The vaccine also does not require pre or post administration of any antacid at the time of oral administration of the vaccine to the subject to neutralize the stomach acid. The vaccine exemplifies nominal drop in vaccine titer at pH 2-4 for a time span of 30 minutes. The vaccine is stable at −20° C. for at least 60 months.

Claims

1. A liquid rotavirus vaccine composition comprising: a live attenuated rotavirus of strain 116E; trehalose at a concentration of about 0.5%; and lactalbumin hydrolysate at a concentration of about 0.05%; wherein the composition contains no buffer or antacid, and wherein the composition has a dose volume of about 0.5 ml.

2. The composition of claim 1, wherein the rotavirus has a titre of about 10.sup.6 FFU/0.5 ml.

3. The composition of claim 1, which can be maintained at from about 2° C. to about 8° C. for up to 36 months.

4. The composition of claim 1, which can be maintained at about 25° C. for 1-6 months.

5. The composition of claim 1, which can be maintained at about 37° C. for 1-3 months.

6. A liquid rotavirus vaccine composition comprising: a live attenuated rotavirus of strain 116E; sucrose at a concentration of about 30%; lactose or trehalose at a concentration of about 0.5%; and lactalbumin hydrolysate at a concentration of about 0.5%; wherein the composition contains no buffer or antacid, and wherein the composition has a dose volume of about 0.5 ml.

7. The composition of claim 6, wherein the rotavirus has a titre of about 10.sup.6 FFU/0.5 ml.

8. The composition of claim 6, which can be maintained at from about 2° C. to about 8° C. for up to 24 months.

9. The composition of claim 6, which can be maintained at about 25° C. for 1-4 months.

10. The composition of claim 6, which can be maintained at about 37° C. for 1-3 weeks.

11. A liquid rotavirus vaccine composition comprising: a live attenuated rotavirus of strain 116E; sucrose at a concentration of about 30%; maltose at a concentration of about 5.0%; and lactalbumin hydrolysate at a concentration of about 1.0%; wherein the composition contains no buffer or antacid, and wherein the composition has a dose volume of about 0.5 ml.

12. The composition of claim 11, wherein the rotavirus has a titre of about 10.sup.6 FFU/0.5 ml.

13. The composition of claim 11, which can be maintained at from about 2° C. to about 8° C. for up to 24 months.

14. The composition of claim 11, which can be maintained at about 25° C. for 1-4 months.

15. The composition of claim 11, which can be maintained at about 37° C. for 1-3 weeks.

16. A liquid rotavirus vaccine composition comprising: a live attenuated rotavirus of strain 116E; sucrose at a concentration of about 40%; lactose or trehalose at a concentration of about 0.5%; maltose at a concentration of about 0.5%; lactalbumin hydrolysate at a concentration of about 0.5%; and recombinant serum albumin at a concentration of about 0.35%; wherein the composition contains no buffer or antacid, and wherein the composition has a dose volume of about 0.5 ml.

17. The composition of claim 16, wherein the rotavirus has a titre of about 10.sup.6 FFU/0.5 ml.

18. The composition of claim 16, which can be maintained at from about 2° C. to about 8° C. for up to 36 months.

19. The composition of claim 16, which can be maintained at about 25° C. for 1-8 months.

20. The composition of claim 16, which can be maintained at about 37° C. for 1-6 weeks.

Description

DETAILED DESCRIPTION OF THE INVENTION

Embodiment 1: Buffered Rotavirus Formulations and Gastric Acidity

[0038] It has been mentioned above in the background of the invention that, usage of buffers is a common practice rotavirus vaccine formulation strategies that has being undertaken by various rotavirus vaccine manufacturers till date, and hence the Rota vaccine comprising the 116E strain at −20° C. formulation (SPG and DMEM) has been tested with citrate-bicarbonate buffer, and normal saline conditions initially.

[0039] Bharat Biotech International Limited obtained the human rotavirus strains 116E from National Institute of Health under a Material Transfer Agreement with National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, USA. The complete genomic sequence of rotavirus strains 116E is already reported in literature.

[0040] Three Rotavirus vaccine formulations with rotavirus 116E stored at −20° C. were taken in 3 groups (Group I, II and III) as detailed below. The rotavirus vaccine compositions in all the three groups below comprise of live attenuated Rotavirus antigen of the strain 116E dissolved in 10% SPG (sucrose, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate) and Dulbecco's Minimum Essential Medium. The said vaccine formulations were treated separately as detailed below:

Group I: 0.5 ml of Rotavirus 116 E formulation A at −20° C.
Group II: 2 ml (equivalent 4 human doses of 0.5 ml per human dose) of Rotavirus 116 E formulation A mixed with 5 ml of Normal Saline.
Group III: 0.570 ml of Rotavirus 116 E formulation A+mix of [1.42 ml Citrate-Bicarbonate buffer+8 ml of 34.8 mEq HCl].

[0041] As given above, Group I includes the rotavirus vaccine formulation at −20° C. wherein no buffer or saline was added to the vaccine formulation and the vaccine titer was measured for next 120 minutes. IN Group II, 2 ml of rotavirus 116E vaccine formulation (stored at −20° C.) was taken and added with 5 ml of normal saline, and vaccine titer was measured at a target titer of 10.sup.A6 FFU/ml up to 120 minutes. In Group III, 0.57 ml of rotavirus 116E vaccine formulation (stored at −20° C.) was taken and added with 1.42 ml of Citrate Bicarbonate Buffer and 8 ml of 34.8 mEq Hydrochloric acid.

TABLE-US-00002 TABLE 2 Rotavirus vaccine formulation (at storage temperature: −20° C.) vaccine titers in presence of buffers at 0 minute, 60 minutes and 120 minutes. 0 mins 60 mins 120 mins Group# in FFU/0.5 ml in FFU/ml in FFU/ml Remarks Group I 10.sup.6.35 10.sup.6.17 10.sup.6.32 Highly stable Group II 10.sup.5.76 10.sup.5.84 10.sup.5.69 Fairly stable Group III 10.sup.4.61 10.sup.5.30 10.sup.5.11 Fairly stable

[0042] Therefore, the above table establishes that both for exposing the virus to normal saline conditions or in presence of buffer solutions, the rotavirus antigen titer is stable, and therefore the vaccine is stable.

Embodiment 2: Immunoperoxidase Assay of Rotavirus Vaccine Formulations at Different pHs

[0043] Example 1: Immunoperoxidase Assay was Done after Subjecting the Rotavirus Vaccine formulations directly to the acidic environment by direct addition of the 34.8 mEq of Hydrochloric Acid (HCl) to the rotavirus vaccine formulation. The pH of the vaccine formulations were directly brought down from pH 7 to various lower pH values of pH 2, and pH 4 by direct addition of acid, and subsequently the vaccine antigen titer was measured at the given lower pH values of pH 2, and pH 4. Following this, the vaccine titer was measured for up to 1 hour at various lower pH of 2 and 4. Following observations were made. The reaction mixture was studied at various time points and various pH levels. Details are shown below in the table below. The said rotavirus vaccine formulations (at storage temperature −20° C.) contains live attenuated rotavirus antigen 116E, 10% SPG (sodium phosphate glutamate) dissolved in Dulbecco's Minimum Essential Medium.

TABLE-US-00003 TABLE 3 Immunoperoxidase Assay results at a target titer of 10.sup.A5.0 FFU/0.5 ml dose at 0 minutes and 60 minutes by direct addition of acid to rotavirus vaccine formulations (storage temperature: −20° C.). Titer in FFU/0.5 mL dose Corrected Corrected Titer with Titer with Actual Dilution Log Actual Dilution titer Factor loss titer at 0 Factor at 0 after 1 after 1 after 1 Sample details minutes minutes hour hour hour. Remarks 0.5 ml Rotavirus 10.sup.4.95 NA 10.sup.4.94 10.sup.4.94 — — 116E vaccine formulation at pH 7.4 0.5 ml Rotavirus 10.sup.3.58 10.sup.5.05 10.sup.3.58 10.sup.5.05 Nil No effect 116E vaccine of pH formulation + 34.8 mEq HCl at pH 4.0 0.5 ml Rotavirus 10.sup.3.54 10.sup.5.01 10.sup.2.93 10.sup.4.40 0.55 Marginal 116E vaccine effect of formulation + 34.8 mEq pH HCl at pH 2.0 Note: Calculated reduction in Infectivity Titer value due to dilution = 10.sup.1.47 FFU. Therefore, actual vaccine titer after dilution is 10.sup.2.93 at pH 2. All the samples were titrated in triplicate by Immunoperoxidase Assay. The oral rotavirus vaccine (target titer 10.sup.5.0 FFU/0.5 mL) was stable at pH 4.0. Target Titer means, the minimum concentration of the rotavirus 116E antigen in the vaccine vial intended to be attained in the given vial. The original titer at neutral pH of the vaccine is 10.sup.4.95 FFU/0.5 ml at 0 minutes. The vaccine titer at pH 2 at 0 minutes is 10.sup.3.54 FFU/0.5 ml and the titer dropped to 10.sup.2.93 FFU/0.5 ml within 1 hour. So, after adding the dilution factor value of 1.47 due to addition of the acid, with the actual titer value (10.sup.2.93 FFU/0.5 ml), the corrected vaccine titer at pH 2 after 1 hour from addition of the acid is 10.sup.4.40 as compared to the original vaccine titer of 10.sup.4.95 at pH 7 at 0 minutes. Therefore, the vaccine experienced a total log loss in vaccine titer of 0.55 only at pH 2.0 within a period of 1 hour. This establishes that, even at lower pH value of 2, that is to say in a specific acidic environment as it appears in the human stomach, the rotavirus vaccine formulation is capable to sustain its vaccine titer at a value of 10.sup.4.40 FFU/0.5 ml. At such vaccine titer, the vaccine is capable to raise sufficient immune response against subsequent rotavirus infections. In a clinical trial carried out with Rota 116E vaccine at a titre of 10.sup.4.0, the vaccine yielded a 4 fold sero-conversion around 62%, which is quite encouraging (Nita Bhandari et al, J Infect Dis 2009, 200, 421-9).

[0044] Example 2: The same experiment has been repeated with a different vaccine (at storage temperature −20° C.), wherein the target titer is 10.sup.A6.0 FFU/0.5 ml (dose) Calculated titer values along with dilution factor are provided in the following table at various pH values at 0 minutes and after 1 hour. The said rotavirus vaccine formulations (at storage temperature −20° C.) contains live attenuated rotavirus antigen 116E, 10% SPG dissolved in DMEM.

TABLE-US-00004 TABLE 4 Immunoperoxidase Assay results at a target titer of 10.sup.A6.0 FFU/0.5 ml dose at 0 minutes and 60 minutes by direct addition of acid to rotavirus vaccine formulations (storage temperature: −20° C.). Corrected Titer ‘Corrected Titer with Dilution with Dilution Factor after 1 Log loss Vaccine Sample Factor at 0’ Hr in hour in after 1 Details pH FFU/0.5 ml FFU/0.5 ml hour Remarks 0.5 ml Rotavirus 7.2 10.sup.5.35 10.sup.5.30 0.05 No effect 116E vaccine (no loss) of pH formulation 0.5 ml Rotavirus 4.0 10.sup.5.33 10.sup.5.09 0.24 No effect 116E vaccine (marginal of pH formulation + 34.8 mEq loss) HCl 0.5 ml Rotavirus 2.0 10.sup.4.89 10.sup.4.21 0.68 Marginal 116E vaccine (marginal effect of formulation + 34.8 mEq loss) pH HCl

[0045] Conclusion: There was only loss in 0.24 log loss in vaccine titer in vaccine samples at pH 4.0, which is considered to be no effect on pH in practical circumstances kept for 1 hour. At pH 2.0, there was a 0.46 Log titer loss in the titer at 0 hour and a loss of 0.68 log 10 FFU at 1 hour. This is said to be having very marginal effect on pH on the vaccine titer values at such an acidic lower pH value of 2.

[0046] According to the above table, we see that vaccine titer of 10.sup.A4.89 FFU/0.5 ml at pH 2 is retained till a titer value of 10.sup.A4.21 FFU/0.5 ml; a log titer of 4.89-4.21 is good enough for replication of the rotavirus in the gut of an infant stomach at an age of 6 weeks to 2 years. The stomach acidity of an infant is also pH 2, representing a simulated gastric environment. The vaccine is still able to survive having a titer of 4.89 Log 10 FFU/0.5 ml, which is enough to cross the stomach and then get established into the intestinal villi and for further replication of rotavirus, responsible for generating immune response in the infant, thereby conferring protection and prevention of rotaviral gastroenteritis. Therefore, the vaccine was found to be stable at acidic pH of 2 for at least 1 hour duration without having any considerable loss in vaccine titer at all.

Embodiment 3: In Vitro Experiment to Demonstrate that Rotavirus 116E Vaccine does not Require Antacid

[0047] Apart from the conclusions from the Clinical trial results that follow later in the patent specification, it was also proved in vitro simulating similar conditions of stomach acidity administering the vaccine directly without buffer.

[0048] Example 3.1: 1 mL of vaccine (formulation 3A) containing rotavirus 116E, combination of sucrose (50%), lactose (0.5%) and trehalose (0.5%), lactalbumin hydrolysate (1%), human serum albumin (0.4%), was taken and added with 1.25 mL of 0.1 N HCl and mixed well. Sample was taken and tested for virus content at various pH levels at different time intervals. The results are shown in the table 5.1 below.

TABLE-US-00005 TABLE 5.1 Immunoperoxidase assay of 1 ml vaccine with rotavirus 116E (formulation 3A) Virus content S. No Sample details (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

[0049] Example 3.2: 0.8 mL of vaccine containing rotavirus 116E, combination of sucrose (40%), and trehalose (0.5%), lactalbumin hydrolysate (20%), polyvinyl pyrrolidine (0.5%) (formulation 3B) was taken and added with 1.25 mL of 0.1 N HCl and mixed well. Sample was taken and tested for virus content at various pH levels at different time intervals. The results are shown in the table 5.2 below.

TABLE-US-00006 TABLE 5.2 Immunoperoxidase assay of 0.8 ml vaccine with rotavirus 116E (formulation 3B) Virus content Virus content S. No Sample details (FFU/mL) (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

[0050] Example 3.3: 0.5 mL of Rotavirus 116E vaccine containing combination of sucrose (60%), and trehalose (0.5%), soy protein (20%), pyridoxine-HCL (0.1%), polyvinyl pyrrolidine (0.25%) (formulation 3C) was taken and added with 1.25 mL of 0.1 N HCl and mixed well. Sample was taken and tested for virus content at various pH levels at different time intervals. The results are shown in the table 5.3 below.

TABLE-US-00007 TABLE 5.3 Immunoperoxidase assay of 0.8 ml vaccine with rotavirus 116E (formulation 3C) Virus content S. No Sample details (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

[0051] Example 3.4: 0.5 mL of Rotavirus 116E vaccine containing 10% SPG dissolved in DMEM (formulation 3D) was taken and added with 1.25 mL of 0.1 N HCl and mixed well. Sample was taken and tested for virus content at various pH levels at different time intervals. The results are shown in the table 5.4 below.

TABLE-US-00008 TABLE 5.4 Immunoperoxidase assay of 0.8 ml vaccine with rotavirus 116E (formulation 3D) Virus content S. No Sample details (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

[0052] Example 3.5: 0.5 mL of Rotavirus 116E vaccine containing sucrose (40%), trehalose (0.5%), lactose (5%), rHSA (0.5%). LAH (1%) and mixed buffer (ammonium acetate+ammonium bicarbonate+diammonium orthophosphate) (formulation 3D) was taken and added with 1.25 mL of 0.1 N HCl and mixed well. Sample was taken and tested for virus content at various pH levels at different time intervals. The results are shown in the table 5.5 below.

TABLE-US-00009 TABLE 5.5 Immunoperoxidase assay of 0.8 ml vaccine with rotavirus 116E (formulation 3D) Virus content S. No Sample details (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

[0053] Conclusions: From the above tables it is evident that at pH 2.0 for 30 minutes also any Rotavirus 116E vaccine formulations are able to withstand in absence of any buffer or any antacid.

Embodiment 4: Baby Rossette Rice Analysis

[0054] BRR assay is the validated procedure for acid neutralization capacity of rotavirus vaccine formulations. So selected vaccine formulations containing Citrate Phosphate Buffer and Citrate Bicarbonate Buffer are evaluated for Acid Neutralization Capacity using this assay. Invitro experiments were conducted under simulated conditions of infantile stomach using Baby Rosette Rice Assay (BRR). Various formulations with various combinations of buffering agents were selected for the acid neutralization experiments and the results are given in the table below at various pH values wherein the conditions were simulated as infant stomach. The vaccine formulations were diluted with water for injection up to 10 ml, then added 4 ml of 0.1 N HCl, then 0.5 ml of 0.1 N HCl added per minute until pH reaches 4.0.ANC is defined as the time in minutes taken to maintain the pH above 4.0.

TABLE-US-00010 TABLE 6 Details of Vaccine Formulations: Vaccine Rotavirus antigen 116E target titer of 10{circumflex over ( )}6 FFU/0.5 ml; Formulation 10% SPG, and DMEM (quantity sufficient) 4A Vaccine Rotavirus antigen 116E target titer of 10{circumflex over ( )}6 FFU/0.5 ml; Formulation 10% SPG, and DMEM (quantity sufficient) added to 4B 2.5 ml of Citrate Bicarbonate Buffer Vaccine Rotavirus antigen 116E target titer of 10{circumflex over ( )}6 FFU/0.5 ml, Formulation combination of atleast 2 Sugars (sucrose 50% and trehalose 4C 0.5%) and Lactalbumin hydrolysate 0.5%. (Sample 8 below). Vaccine Rotavirus antigen 116E target titer of 10{circumflex over ( )}6 FFU/0.5 ml, Formulation combination of atleast 2 sugars (Sucrose-40%, Trehalose- 4D 0.5%), 0.5% lactalbumin hydrolysate, 0.35% human serum albumin and 1.1M Phosphate Buffer.

[0055] Following observations (Table 7) were made for Baby Rosset Rice assay performed with the vaccine formulations 4A to 4D listed above.

TABLE-US-00011 TABLE 7 Baby Rosset Rice analysis in comparison to rotavirus vaccine formulations with and without buffer. Initial Vaccine Vaccine Titers in Volume Titers FFU/0.5 pH after of 0.1N Baby Rosset in ml Vaccine Initial addition HCl Rice Assay FFU/0.5 After 1 Formulations pH of Acid added Values ml hour Vaccine 7.48 1.95  4.0 mL BRR of less 10{circumflex over ( )}4.83 10{circumflex over ( )}3.83 formulation 4A at- than 0 minutes 20° C. without buffer (immediate drop of 0.5 mL dose. in pH) Vaccine 8.35 4.22 10.0 mL BRR of 12 10{circumflex over ( )}6.17 10{circumflex over ( )}6.15 formulation 4B at- minutes 20° C. with 2.5 ml of citrate bicarbonate buffer of 0.5 mL dose. Vaccine 7.28 3.98  5.0 mL BRR of 2 10{circumflex over ( )}5.30 10{circumflex over ( )}5.02 formulation 4C at minutes 5 ± 3° C. without buffer of 0.5 mL dose. Vaccine 6.75 4.33 10.5 mL BRR of 10{circumflex over ( )}6.12 10{circumflex over ( )}6.11 formulation 4D at approximately 5 ± 3° C. with buffer 13 minutes of 1.5 mL or 2 mL dose.

[0056] Conclusion: Therefore from the above table we find that, even if for the vaccine formulation 4A above which shows immediate drop from pH 7.48 to pH 1.95, the vaccine titer is retained from 10.sup.A4.83 FFU/0.5 ml and 10.sup.A3.83 FFU/0.5 ml. Same way, the vaccine titer is also retained in case of Vaccine formulation 4 C between 10.sup.A5.30 to 10.sup.A5.02 FFU/0.5 ml where the pH drops from 7.28 to pH of 3.98 within 2 minutes. Therefore, even if the BRR value is only 2 minutes, the vaccine titer is fairly retained i.e. 10.sup.A5.02 FFU/0.5 ml. This much amount of vaccine titer is enough for generation of immune response and thereby conferring protection and prevention of rotaviral gastroenteritis in human infants.

Embodiment 4: Clinical Trial without Buffer for ROTAVAC® Comprising Rotavirus 116E Strain

Hypothesis and Study Rationale

[0057] Natural transmission of rotavirus is assumed to occur via faecal-oral route. Rotaviruses in general are acid labile and it is believed that acidic environment affects the viability of the virus.

[0058] Given their acid-labile nature of rotaviruses, are so efficient in ubiquitously infecting mammals, most of which have gastric pH values around 2. One possibility is that the human infant stomach may be somewhat more permissive for survival of rotavirus than the adult stomach, as infant gastric pH levels tend to be approx. 3.2 compared with adults at approximately 1.0. This could account for the fact that 60 to 90% of reported human rotavirus disease occurs in children below the age of 3 years.

Study Design

[0059] In order to test this hypothesis a large multicenter randomized controlled trial was undertaken to evaluate and compare the vaccine immune response in subjects receiving ROTAVAC® with citrate bicarbonate buffer to those who receive ROTAVAC® without the buffer.

[0060] Accordingly, the study included the following three treatment groups: [0061] Group I (received ROTAVAC® with 5 minutes prior administration of 2.5 ml of buffer). [0062] Group II (received ROTAVAC® without buffer), [0063] Group III (received ROTAVAC® immediately mixed with 2.5 ml of buffer prior to administration).

[0064] The study investigator and safety assessors (study coordinators) were blinded to the treatment group assignment. Blinding was achieved by a study nurse administering the vaccine as per the allocated treatment group. All subjects received 3 doses of the vaccine given 4 weeks apart. Vaccine immune response was tested 4 weeks after the third vaccine dose (day 84) and compared to baseline (day 0). Immunogenicity was tested in terms of Geometric Mean Titres (GMTs) of serum anti rotavirus IgA and seroconversion (fold rise in antibody levels from pre-vaccination to post vaccination) in the treatment groups.

Study Results and Conclusions

[0065] Post vaccination immune response was comparable with no statistically significant difference in the anti-rotavirus IgA response between the treatment groups. Importantly, seroconversion and GMTs achieved in group II (ROTAVAC® administered without buffer) was similar to that in the other two treatment groups where ROTAVAC® was administered with buffer.

[0066] Post vaccination anti-rotavirus IgA GMTs in the group where ROTAVAC® was administered without buffer was 20.7 U/mL in comparison to 19.6 U/mL and 19.2 U/mL in the two groups which received antacid buffer 5 minutes prior to vaccine and simultaneously mixed with vaccine respectively (Table 8). The differences between the groups were not statistically significant (p>0.05, Student's T-test).

TABLE-US-00012 TABLE 8 Geometric Mean Titres & 95% CI for results (day 0 & day 84) Group I Group II Group III (N = 290) (N = 287) (N = 286) Visit GMT 95% CI GMT 95% CI GMT 95% CI DAY0 10.5 (9.4, 11.7) 10.8 (9.7, 11.9) 10.2 (9.2, 11.3) DAY84 19.6 (17.0, 22.7) 20.7 (17.9, 24.0) 19.2 (16.8, 22.1) N = Number of Subjects evaluated in each group; GMT = Geometric Mean; 95% CI (LL, UL) = Confidence Intervals (Lower Limit, Upper Limit)

[0067] Similarly, the four-fold seroconversion observed in the groups that received ROTAVAC® without antacid buffer and with buffer was similar at 29.2%, 24.5% and 25.1% respectively (Table 9). Further, the lower limit of the 95% confidence interval of the difference of seroconversion (2-fold) between the treatment groups was >-10%, indicating non-inferiority in the immune response achieved with all three treatment regimens (table 10).

TABLE-US-00013 TABLE 9 Seroconversion and 95% CI (2, 3 or 4 fold change in antibody titres) between baseline and day 84 post-vaccination Group I Group II Group III Fold (N = 290) (N = 287) (N = 286) Change % 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 evaluated in each group; (95% CI) = Confidence Intervals (Lower Limit, Upper Limit)

TABLE-US-00014 TABLE 10 Difference in % 2-fold seroconversion at post vaccination in the treatment groups % 2-fold Seroconversion post 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 2-Sided, Chi-Square Test) Group % (95% CI of difference) Group I Vs Group II 4.5 (−3.5, 12.5) Group II Vs Group III 1.6 (−6.5, 9.7) Group I Vs Group III 2.9 (−4.7, 10.5)

[0068] The reactogenicity and safety with respect to 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 the antacid buffer.

Embodiment 5: New Rotavirus 116E Formulations

[0069] Stability Data of ORV 116E Liquid Formulations at 37° C., 25° C. and 2-8° C.

[0070] The rotavirus vaccine formulations of a dose volume of 0.5 ml without buffer (samples 1 to 12, 14 to 16), and with mixed buffer at 0.5 ml dose volume (sample 13) have been examined for prolonged period of time ranging for a period up to 5 years, is provided below. The various formulation details is provided below along with their stabilities at 37° C. for up to 4 weeks, 25° C. for at least 6 months, and for 5+3° C. for up to 2 years to 5 years. The studies establish that, the rotavirus vaccine formulations without buffer at dose volumes of 0.5 ml are also stable in nature for prolonged periods of time at refrigerated temperatures for at least 2 years. Rotavirus vaccine formulations are also found to be stable for a period of 5 years at refrigerated temperatures as well.

[0071] Sample 1 is formulated containing rotavirus 116E, Sucrose 50%, Lactose 0.5%, HSA 0.5% and Lactalbumin hydrolysate 0.05%.

Stability of Sample 1 (Table 11):

[0072]

TABLE-US-00015 Temp 0 day 1M 2M 3M 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° C. 6.02 5.32 2.63

[0073] Sample 2 is formulated containing rotavirus 116E. Sucrose 50%, Trehalose 0.5%. HSA 0.5% and Lactalbumin hydrolysate 0.5%.

Stability of Sample 2 (Table 12):

[0074]

TABLE-US-00016 Temp 0 day 1M 2M 3M 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

[0075] Sample 3 is formulated containing rotavirus 116E. Sucrose 50%, Trehalose 1.0%. HSA 0.5% and Lactalbumin hydrolysate 0.5%.

Stability of Sample 3 (Table 13):

[0076]

TABLE-US-00017 Temp 0 day 1M 2M 3M 6M 9M 12M 24M 36M 60M 5 ± 3° C. 6.22 6.24 6.26 6.14 6.20 6.13 6.04 25° C. 6.22 6.18 6.15 5.80 5.34 2.03 37° C. 6.22 5.35 3.22

[0077] Sample 4 is formulated containing rotavirus 116E. Sucrose 40%, Lactose 0.5%, and Lactalbumin hydrolysate 1.0%.

Stability of Sample 4 (Table 14):

[0078]

TABLE-US-00018 Temp 0 day 1M 2M 3M 6M 9M 12M 15M 24M 60M 5 ± 3° C. 6.19 6.18 6.08 6.21 6.24 6.12 25° C. 6.19 5.66 5.10 4.8 3.01 37° C. 6.19 4.23 2.01

[0079] Sample 5 is formulated containing rotavirus 116E. Sucrose 40%, Maltose 5.0% and Lactalbumin hydrolysate 1.0%.

Stability of Sample 5 (Table 15):

[0080]

TABLE-US-00019 Temp 0 day 1M 2M 3M 6M 9M 12M 15M 24M 60M 5 ± 3° C. 6.35 6.37 6.18 6.01 6.17 6.08 25° C. 6.35 5.89 5.11 4.77 2.43 37° C. 6.35 5.31 3.58

[0081] Sample 6 is formulated containing rotavirus 116E, sucrose 40%, Trehalose 0.5% and lactalbumin hydrolysate 1%.

Stability of Sample 6 (Table 16):

[0082]

TABLE-US-00020 Temp 0 day 1M 2M 3M 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

[0083] Sample 7 is formulated containing rotavirus 116E. Sucrose 40%, Trehalose 1.0% and Lactalbumin hydrolysate 1.0%.

Stability of Sample 7 (Table 17):

[0084]

TABLE-US-00021 Temp 0 day 1M 2M 3M 6M 9M 12M 15M 24M 60M 5 ± 3° C. 6.34 5.79 6.00 5.55 6.07 6.09 25° C. 6.34 6.07 5.81 5.9 5.51 4.65 37° C. 6.34 5.32 2.63

[0085] Sample 8 is formulated containing rotavirus 116E, sucrose 50%, Trehalose 0.5%, and lactalbumin hydrolysate 0.5%.

Stability of Sample 8 (Table 18):

[0086]

TABLE-US-00022 Temp 0 day 1 M 2 M 3 M 6 M 15 M 18 M 24 M 36 M 60 M 53° C. 6.34 6.34 6.33 6.31 6.24 6.14 6.08 25° C. 6.34 6.32 6.14 6.01 5.25 37° C. 6.34 5.11 4

[0087] Sample 9 is formulated containing rotaviris 116E, Sucrose 70%. Trehalose 0.5%.

Stability of Sample 9 (Table 19):

[0088]

TABLE-US-00023 Temp 0 day 1 M 2 M 3 M 6 M 15 M 18 M 24 M 36 M 60 M 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° C. 6.38 5.07 4.01

[0089] Sample 10 is formulated containing rotavirus 116E. Sucrose 50%, lactose 0.5%. Maltose 0.5%, HSA 0.5% and Lactalbumin hydrolysate 0.05%.

Stability of Sample 10 (Table 20):

[0090]

TABLE-US-00024 Temp 0 day 1 M 2 M 3 M 6 M 15 M 18 M 24 M 36 M 60 M 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

[0091] Sample 11 is formulated containing rotavirus 116E. Sucrose 50%. Trehalose 0.5%, maltose 0.5%. HSA 0.5%.

Stability of Sample 11 (Table 21):

[0092]

TABLE-US-00025 Temp 0 day 1 M 2 M 3 M 6 M 9 M 12 M 24 M 36 M 60 M 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

[0093] Sample 12 is formulated containing rotavirus 116E. Sucrose 50%. Trehalose 0.5%, HSA 0.5% and Lactalbumin hydrolysate 0.05%.

Stability of Sample 12 (Table 22):

[0094]

TABLE-US-00026 Temp 0 day 1 M 2 M 3 M 6 M 9 M 12 M 24 M 36 M 60 M 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

[0095] Sample 13 is formulated containing rotavirus 116E, sucrose (40%), trehalose (0.5%), lactose (5%), rHSA (0.5%), LAH (1%) and mixed buffer (ammonium acetate+ammonium bicarbonate+diammonium orthophosphate).

Stability of Sample 13 (Tables 23 and 24):

[0096]

TABLE-US-00027 Temp 0 day 1 M 2 M 3 M 6 M 9 M 12 M 24 M 36 M 60 M 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

[0097] Stability of Sample 13 at −20° C.:

TABLE-US-00028 Temp 0 day 1 M 2 M 3 M 6 M 9 M 12 M 24 M 36 M 60 M −20° C. 6.61 6.59 6.59 6.56 6.54 6.52 6.21 6.11 6.13 6.09

[0098] Sample 14, 15 and 16 are formulated with rotavirus 116E, 10% SPG dissolved in DMEM. The samples 14, 15, and 16 are kept at −20° C., and stability checked for 60 months. The vaccine formulations 13, 14, and 15 are found to be very stable at −20° C. for a period of at least 5 years.

TABLE-US-00029 TABLE 25 Vaccine sample 0 Day 3 M 6 M 9 M 12 M 24 M 36 M 45 M 60 M Sample 4.25 4.01 4.08 4.15 4.25 4.02 4.18 4.12 4.08 14 (10{circumflex over ( )}4.0 FFU/0.5 mL) Sample 5.55 5.42 5.68 5.37 5.77 5.89 5.32 5.40 5.32 15 (10{circumflex over ( )}5.0 FFU/0.5 mL) Sample 6.12 6.12 6.06 6.33 6.36 6.27 6.15 6.11 6.05 16 (10{circumflex over ( )}6.0 FFU/0.5 mL)

[0099] Thus, it is established that various rotavirus vaccine compositions of 0.5 ml of dose volume are stable at −20° C. for 5 years, 2-8° C. for 2 years, 25° C. for 6 months, and 37° C. for 1 week. Irrespective of the vaccine formulation, the rotavirus vaccine at 0.5 ml to 1 ml dose volume is also found to be acid stable, as exemplified in embodiment 3 as well at a pH range of 2-4.

Rotavirus 116E Liquid Formulations with Real Time and Accelerated Stability

[0100] Below mentioned are the compositions using various sugars and proteins which are selected to evaluate the suitability, compatibility and to assure stability to the formulations. Various combinations of buffers are tried out to make the composition stable and also to make the formulation resistant to gastric acidity. In the below mentioned formulations sucrose was used in the range of 30 to 70% with one more secondary sugar i.e. trehalose and one protein Lactalbumin hydrolysate was used. No Human Albumin was used.

Example 4

[0101]

TABLE-US-00030 TABLE 26 B. No Composition T/F-07P Sucrose-about 70% Trehalose-about 0.5% LAH-about 0.5%

TABLE-US-00031 TABLE 27 Stability results Temp. Target Approx. titer 0 Day 1 M 2 M 3 M 6 M 9 M 12 M 24 M 36 M 37° C. 6.0 6.68 6.10 5.58 4.26 25° C. 6.0 6.68 6.50 6.20 6.04 5.32 4.47 2-8° C. 6.0 6.68 6.62 6.95 6.84 6.64 6.78 6.37 6.72 6.05

[0102] The above-mentioned formulation is stable for 36 months at 2-8° C., at 25° C. for 6 months and at 37° C. for 2 months. Due to high concentration of sucrose this formulation is very stable even though there is no human albumin.

Example 5

[0103]

TABLE-US-00032 TABLE 28 Formulations without HAS and low sucrose B. No Composition T/F-6008A Sucrose-about 30% Lactose-about 0.5% LAH-about 1.0% T/F-6009A Sucrose-about 30% Maltose-about 5.0% LAH-about 1.0% T/F-6013A Sucrose-about 30% Trehalose-about 0.5% LAH-about 1.0%

TABLE-US-00033 TABLE 29 Stability at about 2-8° C. B. No Target titer 0 day 1 M 6 M 12 M 15 M 24 M T/F-6008A 6.0 6.19 6.18 6.38 6.21 6.24 6.12 T/F-6009A 6.0 6.35 6.37 6.18 6.01 6.17 6.08 T/F-6013A 6.0 6.19 6.15 6.1 6.03 6.22 6.13

TABLE-US-00034 TABLE 30 Stability at about 25° C. B. No Target titer ODay 1 M 2 M 3 M 4 M 6 M 8 M T/F-6008A 6.0 6.19 5.66 5.1 4.8 4.74 3.01 1.52 T/F-6009A 6.0 6.35 5.89 5.11 4.77 4.49 2.43 2.44 T/F-6013A 6.0 6.19 5.87 5.43 5.12 4.61 3.25 1.66

TABLE-US-00035 TABLE 31 Stability at about 37° C. B. No Target titer 0 Day 1 wk 3 wk 6 wk 8 wk T/F-6008A 6.0 6.19 5.01 4.23 2.8 Nil T/F-6009A 6.0 6.35 5.62 5.31 3.73 Nil T/F-6013A 6.0 6.19 5.23 4.02 2.84 Nil

[0104] The above formulations are having sucrose at low concentration i.e. at 30% in combination with another sugar and one protein. Stable at 2-8° C. for 24 months at 25° C. except for T/F-6013A the other two formulations are stable for 2 months and 6013A is for 3 months. This is due to addition of Trehalose instead of lactose or maltose. Stable at 37° C. for 1 week.

Example 6: Formulations with Three Sugars and Two Proteins

[0105] Below mentioned are the compositions wherein there is a combination of three sugars and two proteins. Combinations are with sucrose, maltose, lactose and trehalose. In both the formulations LAH and human albumin were used (Table 32).

TABLE-US-00036 B. No Composition T/F-07I Sucrose-about 40% Lactose-about 0.5% Maltose-about 0.5% HSA-about 0.35% LAH-about 0.5% T/F-07J Sucrose-about 40% Trehalose-about 0.5% Maltose-about 0.5% HAS-about 0.35% LAH-about 0.5%

TABLE-US-00037 TABLE 33 Stability at about 2-8 C. Target B. No titer 0 day 1 M 6 M 15 M 18 M 24 M 36 M T/F-07I 6.0 6.34 6.44 6.33 6.41 6.24 6.14 6.08 T/F-07J 6.0 6.28 6.24 6.27 6.31 6.15 6.08 6.12

TABLE-US-00038 TABLE 34 Stability at about 25° C. Target B. No titer 0 Day 1 M 2 M 3 M 6 M 8 M 11 M T/F-07I 6.0 6.34 6.42 6.14 6.01 5.25 4.57 2.82 T/F-07J 6.0 6.28 6.34 6.21 6.21 5.44 4.71 2.87

TABLE-US-00039 TABLE 35 Stability at about 37° C. B. No Target titer 0 Day 1 wk 3 wk 6 wk 8 wk T/F-07I 6.0 6.34 6.11 5.11 4.57 4.00 T/F-07J 6.0 6.28 6.21 4.95 4.02 3.54

[0106] The above-mentioned formulations are stable at 2-8° C. for 36 months and at 25° C. stable up to 6 months and at 37° C. stable for 3 weeks.

Stability is good at 25° C. because of the presence of three sugars and two proteins.

Antacid Capacity:

[0107] Rotavirus passes through highly acidic conditions and formulations are to be designed in such a way that virus is protected against the gastric acidity. To evaluate the acid neutralizing capacity of the formulations 0.1 N HCl was used to simulate the stomach acidity. ANC of these formulations is approx. 1 or 2 minutes almost similar to ROTAVAC® But with respect to stability these formulations are highly stable at 2-8° C. compared to ROATAVAC®.

[0108] The vaccine formulation was diluted with water injection up to 10 ml, then added 4 ml of 0.1 N HCL, then 0.5 ml of 0.1N HCL added per minute until pH reaches 4.0 ANC is defined as the time in min taken to maintain the pH above 4.0.

TABLE-US-00040 TABLE 36 Experiment with CB Buffer and ROTAVAC ® Time in minutes pH Initial pH of CB Buffer 8.20 pH of 2.5 mL CB Buffer + 0.5 mL Vaccine (0 min) 8.25 pH After addition of 7.0 mL WFI 8.55 pH after addition of 4.0 mL of 0.1N HCl(0 Min) 7.61 1 7.68 2 7.60 3 7.50 4 7.20 5 6.87 6 6.81 7 6.68 8 6.31 9 5.97 10 5.52 11 4.76 12 3.87 ANC (Acid neutralization capacity) of ROTAVAC ® in combination with Citrate Bicarbonate Buffer is 12 minutes as shown in the table above. In case of ROTAVAC ® 5D also the time taken to reach pH 5.0 is just 1 minute and to reach pH 4.0 is for 4 minutes.

[0109] Comparison between ROTAVAC® and ROTAVAC® 5D with respect to ANC based on the information shown in the table below it is evident that in presence of Citrate Bicarbonate Buffer also the titers are low in case of ROTAVAC® when compared with ROTAVAC® 5D at pH 4.0 and 5.0 (Table 37).

TABLE-US-00041 Time in Time in S. No PH 4.0 minutes PH 5.0 minutes ROTAVAC ® 5D 10{circumflex over ( )}5.02 4 10{circumflex over ( )}5.08 1 ROTAVAC ® 10{circumflex over ( )}3.26 13 10{circumflex over ( )}3.99 11

[0110] Therefore, applicants wish to mention that irrespective of the vaccine composition details, any rotavirus vaccine comprising the live attenuated rotavirus 116E is capable to withstand the strong acidic environment in the human infant stomach and produce the required immune response against the rotavirus infections. The vaccine formulations presented in this patent application are only meant for the purposes of explaining the breadth and depth of the invention. It is impractical to provide all possible vaccine formulation details by the applicant, although the applicant has given numerous formulation strategies in the Embodiment and the Examples. Therefore, all possible obvious variations by a person skilled in the art for rotavirus vaccine formulation strategy comprising the rotavirus 116E strain are also covered under this invention. It is to be understood and accordingly construed that, the vaccine compositions which are not specifically included in this patent application comprising the live attenuated rotavirus 116E is also covered under the scope of this invention.

[0111] Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims. Furthermore, the term “comprising of” includes the terms “consisting of” and “consisting essentially of.”