FORM - 2
THE PATENTS ACT, 1970
(39 OF 1970}
AND
THE PATENTS RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10; rule 13)
"Novel Viral Vaccine Compositions and Methods for Preparing such Vaccines"
Serum Institute of India Ltd., an Institute organized and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028 Maharashtra India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:

STATE OF ART
Group A rotaviruses are firmly established as the most important etiologic agents of dehydrating gastroenteritis in infants and young children worldwide.Severe rotavirus disease is preventable by vaccination.For Asia, Africa and Latin America, it has been estimated that there are between 3-4 billion cases of diarrhoea each year and of those cases about 5-10 million result in death (Walsh, J. A. et al.: N. Engl. J. Med., 301:967-974 (1979)).
Rotaviruses are nonenveloped viruses whose genome comprises 11 segments of double-stranded RNA (dsRNA), contained in the core of the mature, triple-layered particle . Rotavirus serotypes are determined by neutralizing antibody responses to each of the two outer caps id proteins, VP7 (termed G serotype) and VP4 (termed P serotype) . Ten G serotypes and 7 P serotypes have been identified in humans. Since serotypes Gl, G2, G3, and G4 together account for >80% of global human rotavirus strains, some vaccines aim to provide serotype-specific protection against these four serotypes.
However, in certain geographic settings, other G types (e.g., G12, G8, and G9) may be epidemiologically important. Since their first report in 1987 in the United States, rotaviruses of serotype G9 had been rarely detected in the

human population • . Since 1995, however, serotype G9 has been documented in India, Brazil, Italy, the United States, Bangladesh, Malawi, the United Kingdom, France, and Australia . Recent reports from Ireland , the Netherlands , Japan , and Thailand further emphasize the wide geographic distribution of this serotype.
The development of novel multivalent rotavirus formulations containing the emerging serotypes(G9 & others) must comply with a number of requirements, including worldwide distribution potential and stability under a broad range of environmental and storage conditions.
RotaShield (Wyeth-Lederle) was an oral, lyophilized, tetravalent (Gl-4) RRV rhesus/human reassortant vaccine supplied with a liquid diluent comprising sodium bicarbonate and citric acid having antacid property. The said vaccine was withdrawn later.
Rotarix (GSK) is an oral, lyophilized, G1P(8) vaccine supplied with a liquid diluent comprising calcium carbonate having antacid property.
Rotateq(Merck) is an oral, lyophilized ,G1-4&P1 vaccine formulations.

It is a known fact that the lyophilization process has a limiting capacity, and is associated with a high production cost. Furthermore, lyophilized vaccines have a more sophisticated handling for administration as they may require more complex, hence relatively expensive devices such as multichamber/vial vaccines, with the active ingredient in one chamber and the reconstitution liquid in another chamber. Lyophilized vaccines are also associated with higher shipment and storage cost. These options may be inadequate for some countries in the developing world where the administration device has to be financially affordable and where the availability of production and storage infrastructure may be inadequate.
Accordingly liquid vaccine formulations of Rotateq & Rotarix were developed.Rotateq(Merck) also has an oral, liquid ,G1-4&P1 vaccine formulations containing buffers (succinate/phosphate or citrate/phosphate) to protect the viruses from gastric acid .Rotarix (GSK) also has an oral, liquid, G1P(8) vaccine containing Di-sodium adipate having antacid property wherein the formulation has minimum phosphate & found stable at 37°C for 7 days, 25°C for lyear ,4°C for 2years. Refer US 20080166372.
As Rotavirus are conventionally administered orally to

human infants, this route brings several challenges to immunizing rotavirus compositions.Rotavirus is rapidly inactivated in an acidic environment, upon exposure to acid buffer or acidic gastric juice for example (C. Weiss and H.F. Clark, 1985, J. Gen. Virol.,66, 2725-2730; T. Vesikari et al., 1984, The Lancet, page 700 ; R.H.Foster and A.J.Wagstaff, 1998, BioDrugs Feb: 9(2) 155-178). Therefore it is desirable that rotavirus compositions are formulated in a way that they are stable during storage and after administration into the host recipient.
Rotavirus vaccines are primarily intended to be administered to babies, as early as at the age of 4 weeks. A small vaccine dose volume, such as lower than 2ml or even than 1.5 ml dose volume, will be advantageous for that population. Therefore, it is desirable that rotavirus compositions are formulated in a small volume dose.
Previously disclosed methods for concentrating viruses for obtaining higher titre have been outlined below.
J.Emerson et al discusses concentrating polio virus by aluminium hydroxide mediated sedimentation during water purification procedures,wherein the virus is not inactivated.Refer J.Emerson et al "Effect of Aluminum Hydroxide Sedimentation, Sand Filtration and Chlorination

on the Virus of Poliomyelitis"Dec 1942,Universa ty of Michigan.However this method was utilized for poliovirus removal during water purification.
Samuel et al had reported removal of human rotavirus(SA-11) from stool suspensions or tap water by aluminum hydroxide floes,wherein SA-11 was reduced by 1 log or less.Further Samuel et also discussed comparison of rotavirus and poliovirus adsorption to aluminium hydroxide.Refer Samuel et al "Comparison Between Adsorption of Poliovirus and Rotavirus by Aluminum Hydroxide and Activated Sludge Flocs"University of Texas Applied And Environmental Microbiology, Feb. 197 8,Vol. 35, No. 2 p. 3 60-363.However this method was utilized for rotavirus removal during water purification.
US7579008 discloses use of aluminium hydroxide as both,an adjuvant and an antacid in Gl rotavirus composition.This method however does not discuss concentrating rotavirus titre.
There is a need therefore to develop alternative rotavirus formulations, in particular alternative liquid stable formulations that a) are applicable to both inactivated as well as live rotavirus b)have minimum dose volume c)can withstand gastric acidity.

Andreas et al US 2008/02 93123 discloses a novel method of concentrating viruses by utilizing a mixture of glycerol /magnesium salt,sodium salt in combination with tangential flow filtration. It also discusses that high virus concentration contribute significantly to virus instability and that there is a critical need to develop formulations that stabilize relatively high concentrations of virus. However said virus concentrating method does not involve alum adsorption and is only applicable to live recombinant adenovirus containing wild-type p53 gene.
The inventors have surprisingly found a novel method of concentrating live rotaviruses based on aluminium salt gel adsorption that could provide alternative liquid formulations possessing above mentioned advantageous features.
SUMMARY OF THE INVENTION
The present invention provides a stable gel adsorbed liquid rotavirus vaccine ,wherein the composition has following characteristics:
a) gel adsorbed stable rotavirus vaccine for live and inactivated virus vaccines;
b) minimum vaccine dose volume achieved due to virus concentration and stabilization by aluminium salt;
c) Aluminium salt based adsorption and washing with virus

medium results in partial removal of nucleic acid
contaminants; and d) optimal virus release from gel during potency testing & post -vaccine administration.
Also provided is an improved method for concentrating rotavirus by atleast 5 times,wherein an aluminium salt is employed, said method comprising:
a)Growing rotavirus on a suitable cell substrate;
b)Addition of aluminium salt solution so that the concentration of aluminium salt in the preparation reaches a final concentration of about 0.06 % or more;
c)Subjecting the above mixture to vortexing for a duration between 10 to 20 hrs at 4°C;
d)Allowing settling of the mixture obtained in step (c) for a duration between 1 to 2 hrs;
e)Separating the supernatant and sedimented gel after step (d);
f)Washing of the sedimented gel with virus medium atleast once in a ratio between 1:1 to about 1:10 followed by

vortexing for a duration between 1 and 5 hrs at 4°C;
g) Mixing gel obtained in step (f) with an antacid combination in a ratio from about 60:40 to about 80:20.
An important aspect of the instant invention is that the process of concentrating rotavirus by aluminium salt having a concentration between 0.2 and 0.8 mg/ml,preferably between 0.5 and 0.8 mg/ml results in a final virus concentrate having atleast 5 times higher virus titre,more specifically 10 times with simultaneous removal of nucleic acid and protein contaminants.
Yet another aspect of the instant invention is that said aluminium salt is aluminium hydroxide,aluminium phosphate or a mixture of both.
A second aspect of" the instant invention is that washing of sedimented gel is performed by utilizing a basal media selected from the group consisting of BME, MEM, DMEM, DMEM-F-12, IMDM, McCoy's 5 A, Media 199, Ham's F-10, Ham's F-12, MS- 162, MS- 174, and RPMI,preferably MEM.
The immunogenic composition of the present invention contains a mixture of atleast one aluminium hydroxide gel adsorbed rotavirus antigens,basal medium , antacid,

stabilizers , buffers and has a pH of about 7.2 to 7.8, in a 1 ml dose volume.
Another aspect of the instant invention is that said liquid immunogenic composition can be stable for at least 1 month at 36° C , atleast 3 months at 25° C and atleast 12 months at 2-8° C.
Further the vaccine formulations of the present invention preferably include one or more additional components, alone or in a biologically effective combination, which provides enhanced stability characteristics; including but not limited to amino acids,particularly glycine at a concentration between 2 and 5 % w/v.
DETAILED DESCRIPTION OF THE DRAWINGS
Figure 1: Biological titer of virus preparation pre & post-aluminium hydroxide adsorption.
Figure 2: Effect of aluminium hydroxide gel adsorption & virus medium washing steps on concentration of nucleic acid & protein contaminants.
Figure 3: Thermal stability of Gel adsorbed live
rotaviruses.

DETAILED DESCRIPTION
Prior to setting forth the invention, it may be helpful to an understanding thereof to first set forth definitions of certain terms that will be used hereinafter.
Definitions
The term "live or inactivated rotavirus antigen" for inclusion in the claimed composition refers to a monovalent rotavirus strain, i.e. containing a single rotavirus strain, or be multivalent, i.e. containing at least two or more rotavirus strains.Typically, the vaccine contains atleast one rotavirus serotype selected form a group of Gl G2, G3, G4, G5, G6, G7, G8, G9, G10, G11, G12, G13 and G14.
The term "antacid" refers to MylantaTM, calcium carbonate (CaC03) , magnesium carbonate, aluminium carbonate, aluminium phosphate, mix of aluminium hydroxide and magnesium carbonate, aluminium- magnesium- hydrycarbonate , aluminium hydroxide- magnesium carbonate- sorbitol- manitol , hydroxy- aluminium- sodium- carbonate , dihydroxy-aluminium- potassium- carbonate, magaldrate , hydrotalcite, almagcit, magnesium- aluminium- silicate- hydrate.
Preferably the immunogenic composition of the present

invention contains a) atleast one aluminium hydroxide gel adsorbed rotavirus antigen, at a minimum concentration of 5.2 log10 FFU/ml/Serotype in combination with a citrate bicarbonate buffer in a suitable ratio,b) basal medium,c) phosphate and has a pH of about 7.2 to7.8, in a 1 ml dose volume.
The rotavirus antigen of the claimed composition may be produced according to routine production techniques. Typically rotavirus antigen preparations may be derived from tissue culture methods used to propagate the virus or express recombinant rotavirus antigens. Suitable cell substrates for growing the virus include for example monkey kidney cells such as VERO or cells from a clone of VERO, VERO-like cells, monkey kidney cells such as AGMK cells MDCK, other cells lines of monkey kidney origin such as BSC-1, LLC-MK2 and MA104, suitable pig cell lines, or any other mammalian cell type suitable for the production of rotavirus for vaccine purposes. Suitable cell substrates also include human cells e.g. MRC-5 cells. Suitable cell substrates are not limited to cell lines; for example primary cells are also included.
For the instant invention mixture of sodium bicarbonate and citric acid is particularly suitable as antacid.Thus the concentrated gel containing virus is mixed with citrate

bicarbonate buffer in a ratio selected from 60:40,70:30 and 80:20,preferably 80:20.
Typically the volume of a dose of vaccine according to the invention will normally be 2.5 ml or lower, typically between 0.5 ml and 2.5 ml. In a specific aspect of the invention, a suitable vaccine dose will normally be 1.5 ml or suitably any volume smaller than 2.5 ml such as a volume of 2 ml or less, that is suitable for oral administration to babies or infants. In particular the dose volume will be such that the technical feasibility of the formulation is possible and there is no detrimental effect on the immunogenic potential of the formulation.
The rotavirus vaccine composition of the instant invention can be administered by oral or injectable route.
The immunogenic composition of the invention may also be formulated to contain other antigens, in particular antigens from other suitable live viruses for protection against other diseases, for example poliovirus. Said additional active ingredients suitable for oral administration may be given either in admixture with the rotavirus composition, or alternatively may be coadministered (i.e. in a separate dose but on the same occasion) with the rotavirus composition claimed herein.

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Example 1
Gel adsorption of Live Rotaviruses
The sterilized stock solution (9mg/ml) of aluminium hydroxide was kept at 4°C till the temperature of solution reached at 4°C. 6.66 ml of this solution was added to the 100ml of live rotavirus culture (titer 5.825 log10 CCID50/ml) so that the final concentration of aluminium hydroxide was 0.6mg/ml. This mixture was then incubated at 4°C for 15 to 20 hrs on rocker. The container then was kept in vertical position to settle the gel at bottom for 1 to 2 hrs. The supernatant was then removed and 10 to 15ml of gel was collected. The sample were removed before pre and post gel adsorption from gel and supernatant respectively.
This gel was then washed thrice with 50ml of minimum essential medium (MEM) followed by vortexing and samples after every wash were analyzed.
Table 1: Biological titer of virus preparation pre & post-

adsorption

Process Parameters Expt-1
CCID50/ml Expt-2
CCID50/ml Expt-3
CCID50/ml
Pre-gel adsorption 5.825 5.4 6
Post-gel adsorption 6.625 6.4 6.825
Virus concentration was estimated by CCID50 assay.It was observed that post-gel adsorption , viral titers increased to about 10 fold (i.e. 6.625 log10 CCID50/ml) from the pre-gel adsorption viral titer (i.e. 5.825 log10 CCID50/ml.Also analysis of supernatant indicated that there was no detectable level of virus concentration in supernatant. Example 2
Effect of aluminium hydroxide gel adsorption & virus medium washing steps on concentration of nucleic acid & protein contaminants Table 2:

Process Parameters Protein DNA
Pre-adsorption) (gel 0.989 μg/ml 5 μg/ml
Post - (gel adsorption + virus medium wash 3 times) 0.8 61 μg/ml 3 μg/ml
Residual DNA was estimated using Qubit and QUANT iT dsDNA BR assay. The adsorption alongwith subsequent washing with virus medium removed substantial amount of residual

proteins and DNA.
Example 3
Thermal stability of Gel adsorbed live rotaviruses
Stability of Gel adsorbed live rotaviruses were studied at 2 to 8°C, 25°C and 36°C from 1 month to about 1 year.
Following were the composition details: aluminium hydroxide gel adsorbed rotavirus antigen, at a minimum concentration of 5.2 log10 FFU/ml/Serotype in combination with a citrate bicarbonate buffer(1.92 mg/dose citric acid,5mg/dose sodium bicarbonate) in 80:20 ratio,b) 8.48 gm/dose MEM,c)0.7 mM phosphate,d)pH of about 7.2 to 7.8, in a 1 ml dose volume.
Table 3:

Duration Temperature

2-8°C 25°C 36°C
Initial 6 6 6
1 month 5.7 5.4 5.1
3 months 5.7 5 4
6 months 5.75 4.8 0
12 months 5.5 4.7 0
The liquid gel-adsorbed vaccine was found to be stable for
at least 1 month at 36° C , atleast 3 months at 25° C and
atleast 12 months at 2-8° C.
Example 4
Gel adsorption of Inactivated Rotaviruses:

Similar study was carried out using inactivated rotaviruses. Results indicated that the viruses can be concentrated 10 fold by adsorbing on the aluminium hydroxide gel. Quantification of DNA indicated a marginal decrease from 0.989 to 0.861ug/ml after three washes of gel adsorbed vaccine with virus medium.
Example 5
Thermal stability of Gel adsorbed inactivated rotaviruses
Stability of the inactivated gel adsorbed vaccine was studied for 1 month. The inactivated rotaviruses were detected by ELISA and the total protein concentration was estimated using Bradford assay.
The vaccine was found to be stable at 2 to 8oC for at least 1 month , at 25°C for at least 1 month and at 36°C for atleast 1 week.
Example 6
Virus release from the gel
In the gel adsorbed vaccine, it is well known fact that aluminium gel act as an adsorbent/ as well as adjuvant. Thus this preparation can be used for final vaccine preparation. When such live rotavirus vaccines will be administered by oral route, the vaccine gel will pass through the stomach and exposed to acidic environment, thus

resulting in dissolving of gel and subsequent release of virus.
In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
We claim,
1. A novel and cost-e'f f icient method of concentrating a virus preparation comprising:
a)Growing virus on a suitable cell substrate; b)Adding aluminium salt to the virus preparation of step (a)so that the a final concentration of aluminium salt in the preparation reaches a level of about 0.06% or more; c)Subjecting the above mixture to vortexing for a duration between 10 to 20 hrs at 4°C;
d)Allowing settling of the mixture obtained in step (c) for a duration between 1 to 2 hrs;
e)Separating the supernatant and sedimented gel after step(d); f)Washing of the sedimented gel with virus medium atleast

once in a ratio between 1:1 and 1:10 ,followed by vortexing
for a duration between 1 and 5 hrs at 4°C;
g) Mixing gel obtained in step (f) with an antacid in a
suitable ratio;
wherein the viral titer post-adsorption is greater than the
initial viral titer at pre-adsorption.
2. A method according to claim 1,wherein the said aluminium salt is selected from the group consisting of:aluminium hydroxide,aluminium phosphate and a combination thereof.
3. A method according to claim 1,wherein the said aluminium salt gel adsorption and washing with virus medium results in partial removal of nucleic acid contaminants.
4. A method according to claim 1 , wherein the said virus is selected from an inactivated virus, an attenuated virus, and a live virus.
5. A method according to claim 4 ,wherein said virus is
selected from the group consisting of adenovirus, herpes
simplex, varicella zoster, cytomegalovirus, Epstein Barr
virus, hepatitis B virus, influenza virus, human papilloma
viruses, parainfluenza virus, measles virus, respiratory
syncytial virus, poliovirus, Coxsackie virus, rhinovirus,
hepatitis A virus, vaccinia, variola major, variola minor,

rotavirus, human T lymphotropic virus-1 , human immunodeficiency virus (HIV), rabies virus, rubella virus, Yellow fever virus, arbovirus and combinations thereof.
6. A method according to claim 1,wherein aluminium salt is
added to the virus preparation at a final concentration of
about 0.2 mg/ml to 0.8mg/ml.
7. A method according to claim 1 , wherein said virus medium is selected from the group consisting of BME, MEM, DMEM, DMEM-F-12, IMDM, McCoy's 5 A, Media 199, Ham's F-10, Ham's F-12, MS- 162, MS- 174, and RPMI.
8. A method according to claim 1 , wherein said gel adsorbed and concentrated virus preparation is mixed with an antacid in a ratio selected from 60:40,70:30 and 80:20.
9. A method according to claim 1 , wherein said antacid is
selected from the group consisting of citrate bicarbonate
buffer,Mylanta™,calcium carbonate (CaC03) , magnesium
carbonate, aluminium carbonate, aluminium phosphate, mix of
aluminium hydroxide and magnesium carbonate, aluminium-
magnesium- hydrycarbonate , aluminium hydroxide- magnesium
carbonate- sorbitol- manitol , hydroxy- aluminium- sodium-
carbonate ,dihydroxy- aluminium- potassium- carbonate,
magaldrate , hydrotalcite, almagcit, magnesium- aluminium-
silicate- hydrate.

10. A novel and cost-efficient method of concentrating a
rotavirus preparation comprising:
a)Growing rotavirus on a Vero cell substrate; b)Adding aluminium hydroxide to the virus preparation of step (a)so that the a final concentration of aluminium hydroxide in the preparation reaches a level of about 0.0 6
%;

c)Subjecting the above mixture to vortexing for a duration
between 15 to 20 hrs at 4°C;
d)Allowing settling of the mixture obtained in step (c)
for a duration between 1 to 2 hrs;
e)Separating the supernatant and sedimented gel after
step(d) ;
f) Washing of the sedimented gel with MEM for 3 times in a
ratio of 1:5 ,followed by vortexing for a duration between
2 and 5 hrs at 4°C;
g) Mixing gel obtained in step (f) with citrate bicarbonate
buffer in a ratio of 80:20;
wherein the viral titer post-adsorption is atleast 10 fold greater than the initial viral titer at pre-adsorption.
11. A method according to claim 10,wherein the said
aluminium hydroxide gel adsorption and washing with virus
medium results in partial removal of nucleic acid
contaminants.

12. A method according to claim 1 or 10, wherein the final composition containing gel adsorbed and concentrated virus is stable for atleast 12 months at 2-8°C ,atleast 3 months at 25°C and atleast 1 month at 36°C.
13. A method according to claim 1 or 10 , wherein said final rotavirus vaccine composition obtained is provided in a dose volume of between 0.2 ml and 2.0 ml.
14. A method according to claim 10 , wherein the said virus is selected from an inactivated rotavirus, an attenuated rotavirus, and a live rotavirus.
15. A method according to claim 14, wherein the rotavirus is a human or animal rotavirus.
16.A method according to claim 15, wherein the rotavirus is selected from a group consisting of group A rotavirus, group B rotavirus, group C rotavirus, group D rotavirus, group E rotavirus, group F rotavirus and group G rotavirus.
17. A stable liquid rotavirus vaccine composition of 1 ml
dose volume containing a) atleast one aluminium hydroxide
gel adsorbed and concentrated rotavirus antigen, at a
concentration of 5.2 log10 FFU/ml/Serotype in combination
with citrate bicarbonate buffer in a 80:20 ratio;b) 8.48
gm/dose Minimum Essential Medium ;c)0.7 mM phosphate and
d)pH from about 7.2 to 7.8.
18. The vaccine composition of claim 17, wherein the
vaccine composition is suitable for administration by

routes selected from a group consisting of oral,buccal, sublingual, nasal,mucosal and vaginal.