Methods for enterovirus inactivation, adjuvant adsorption and dose reduced vaccine compositions obtained thereof

Abstract

The present invention is directed to improved methods of Enterovirus inactivation by formaldehyde in presence of tromethamine buffer resulting in maximum recovery of D-antigen. Subsequent adsorption of said sIPV on aluminium hydroxide provides significantly dose reduced sIPV compositions.

Claims

1. A method for producing a composition comprising Enteroviral poliovirus particles, wherein the method comprises the steps of: a) purifying Enteroviral particles in a medium comprising a phosphate buffer; b) exchanging the phosphate buffer of the purified Enteroviral particles of a) for a TRIS buffer; c) stabilizing the purified and buffer-exchanged Enteroviral particles of b); d) inactivating the Enteroviral particles of c) by: (i) adding formalin to the purified Enteroviral particles and stirring for 7 days; (ii) filtering the product of step (i); (iii) adding formalin to the product of step (ii) and stirring for 6 days; and (iv) filtering the product of step (iii); wherein: the TRIS buffer is present at a concentration of 30-70 mM; aggregation of the Enteroviral particles is prevented or reduced; and D-antigen losses are reduced post inactivation by 8 to 10 fold as compared to inactivation in phosphate buffer; and e) adsorbing the inactivated Enteroviral particles on an aluminum salt adjuvant, whereby the percentage adsorption on the aluminum salt adjuvant is at least 95%.

2. The method according to claim 1, wherein the TRIS buffer has a pH of about 6.8 to 7.2 and at a concentration in the range of 30 mM to 70 mM.

3. The method according to claim 1, wherein the aluminum salt adjuvant of step (e) is aluminum hydroxide, aluminum phosphate, or a mixture of both.

4. The method according to claim 3, wherein the aluminum salt adjuvant is aluminum hydroxide having concentration between 1.5 mg/0.5 ml dose and 2.5 mg/0.5 ml dose at a pH of about 6.5.

5. The method according to claim 4, wherein the total aluminum content in the composition is less than 1.2 mg Al.sup.3+ per 0.5 mL dose, wherein at least 0.4 mg is Al.sup.3+ for Type 1, at least 0.2 mg is Al.sup.3+ for Type 2, at least 0.2 mg is Al.sup.3+ for Type 3.

6. The method according to claim 1, wherein the composition is a vaccine.

7. The method according to claim 6, wherein the Enteroviral particles comprise polioviruses of the Sabin serotypes 1, 2 and 3.

8. The method according to claim 6, wherein the Enteroviral particles comprises a poliovirus of a Salk serotype IPV type 1 (Mahoney strain), IPV type 2 (MEF-1 strain), and/or IPV type 3 (Saukett strain).

9. The method according to claim 6, wherein the vaccine is a dose reduced Inactivated Polio Vaccine (IPV).

10. The method according to claim 9, wherein the dose reduced Inactivated Polio vaccine comprises: i) inactivated poliovirus type 1 at a dose less than 15 D-antigen units instead of standard dose of 40 DU; and/or ii) inactivated poliovirus type 2 at a dose less than 18 D-antigen units; and/or iii) inactivated poliovirus type 3 at a dose less than 17 D-antigen units instead of standard dose of 32 DU.

11. The method according to claim 10, wherein the dose reduced inactivated Polio vaccine containing Salk or Sabin polioviruses is prepared by a) purifying poliovirus from a medium; b) stabilizing the purified poliovirus by adding a M-199 medium containing glycine; c) inactivating the polio virus using formaldehyde 0.025% at 37 C. for 5 to 13 days in presence of TRIS buffer at a concentration between 30 mM to 60 mM to prevent or reduce aggregation of the poliovirus particles thereby reducing the D-antigen losses post inactivation by 8 to 10 fold as compared to phosphate buffer; and d) adsorbing the inactivated poliovirus on Alum hydroxide adjuvant having a concentration between 2 to 2.5 mg/dose, whereby percentage adsorption on Alum hydroxide is greater than 95% for Type 1, Type 2 and Type 3.

12. The method according to claim 10, wherein the dose reduced inactivated Polio vaccine containing Salk or Sabin poliovirus is prepared by: a) purifying the poliovirus from a medium containing a poliovirus; b) stabilizing the purified poliovirus by adding a M-199 medium containing glycine; c) inactivating the polio virus using formaldehyde 0.025% at 37 C. for 5 to 13 days in presence of TRIS buffer at a concentration between 30 mM to 60 mM to prevent or reduce aggregation of the poliovirus particles thereby reducing the D-antigen losses post inactivation by 8 to 10 fold as compared to phosphate buffer; and d) absorbing the inactivated poliovirus on Alum hydroxide adjuvant having concentration between 2 to 2.5 mg/dose, wherein the percentage adsorption on Alum hydroxide is greater than 95% for Type 1 and Type 3.

13. The method according to claim 10, wherein the dose reduced Inactivated Polio vaccine is: i) Sabin single dose composition having Sabin Type 1, Type 2, Type 3 combination that is 5-16-10; ii) Sabin two dose composition having Sabin Type 1, Type 2, Type 3 combination that is 5-16-10; iii) Sabin single dose composition having Sabin Type 1, Type 2, Type 3 combination that is 2.5-8-5; iv) Sabin two dose composition having Sabin Type 1, Type 2, Type 3 combination that is 2.5-8-5; v) Sabin single dose composition having Sabin Type 1, Type 2, Type 3 combination that is 5-8-10; vi) Sabin two dose composition having Sabin Type 1, Type 2, Type 3 combination that is 5-8-10; vii) Salk single dose composition having Sabin Type 1, Type 2, Type 3 combination that is 6.5-16-10; viii) Salk two dose composition having Sabin Type 1, Type 2, Type 3 combination that is 6.5-16-10; ix) Salk single dose composition having Salk Type 1, Type 2, Type 3 combination that is 8-2-5; x) Salk two dose composition having Salk Type 1, Type 2, Type 3 combination that is 8-2-5; xi) Salk single dose composition having Salk Type 1, Type 2, Type 3 combination that is 10-2-5; xii) Salk two dose composition having Salk Type 1, Type 2, Type 3 combination that is 10-2-5; xiii) Salk single dose composition having Salk Type 1, Type 2, Type 3 combination that is 10-2-12; xiv) Salk two dose composition having Salk Type 1, Type 2, Type 3 combination that is 10-2-12; xv) Salk single dose composition having Salk Type 1, Type 2, Type 3 combination that is 5-2-5; xvi) Salk two dose composition having Salk Type 1, Type 2, Type 3 combination that is 5-2-5; xvii) Salk single dose composition having Salk Type 1, Type 2, Type 3 combination that is 10-2-10; xviii) Salk two dose composition having Salk type 1, Type 2, Type 3 combination that is 10-2-10; xix) Salk single dose composition having Salk Type 1, Type 2, Type 3 combination that is 10-2-16; or xx) Salk two dose composition having Salk type 1, Type 2, Type 3 combination that is 10-2-16.

14. The method according to claim 13, wherein the dose reduced Salk or Sabin Inactivated Polio Vaccine does not comprise Type 2.

15. The method according to claim 13, wherein the vaccine comprises of one or more antigens from a pathogen that is Haemophilus influenzae b, Neisseria meningitidis type A, Neisseria meningitidis type C, Neisseria meningitidis type W, Neisseria meningitidis type Y, Neisseria meningitidis type X, Neisseria meningitidis type B, Streptococcus pneumoniae, Salmonella typhi, Hepatitis A, Hepatitis B, diphtheria toxoid, tetanus toxoid, whole cell pertussis, or acellular pertussis.

16. A method for preparing a dose reduced inactivated Polio vaccine containing Salk or Sabin poliovirus, comprising: a) purifying the poliovirus from a medium comprising a poliovirus and a phosphate buffer; b) exchanging the phosphate buffer of the purified Enteroviral particles of a) for a TRIS buffer; c) stabilizing the purified and buffer-exchanged poliovirus of b) by adding a M-199 medium containing glycine; d) inactivating the poliovirus of c) using formaldehyde 0.025% at 37 C. for 5 to 13 days in presence of TRIS buffer at a concentration between 30 mM to 60 mM to prevent or reduce aggregation of the poliovirus particles thereby reducing the D-antigen losses post inactivation by 8 to 10 fold as compared to phosphate buffer; and e) adsorbing the inactivated poliovirus on an Alum hydroxide adjuvant having concentration between 2 to 2.5 mg/dose, whereby percentage adsorption on Alum hydroxide is greater than 95% for Type 1, Type 2 and Type 3.

17. The method according to claim 3, wherein the TRIS has a 40 mM concentration.

18. The method according to claim 4, wherein the aluminum salt adjuvant has a concentration between 2.100 mg/0.5 ml dose and 2.4 mg/0.5 ml dose at a pH of about 6.5.

19. The method according to claim 5, wherein the total aluminum content in the composition is 0.8 to 1.2 mg Al.sup.3+ per 0.5 mL dose.

20. A method for producing a composition comprising Enteroviral poliovirus particles, wherein the method comprises the steps of: a) purifying Enteroviral particles comprising: (i) concentrating a solution comprising the Enteroviral particles using tangential flow filtration; (ii) diafiltering the product of step a-i with a medium comprising a phosphate buffer; and (iii) concentrating the product of step a-ii using column chromatography and a phosphate buffer; b) exchanging the phosphate buffer of the product of step a-iii for a TRIS buffer having a concentration of 30 to 50 mM of TRIS and a pH of 6.8 to 7.2; c) stabilizing the purified and buffer-exchanged Enteroviral particles by adding a medium comprising glycine to the product of step b; d) inactivating the Enteroviral particles of c) by: (i) adding formalin to the purified Enteroviral particles and stirring for 7 days; (ii) filtering the product of step (i); (iii) adding formalin to the product of step (ii) and stirring for 6 days; and (iv) filtering the product of step (iii), wherein: aggregation of the Enteroviral particles is prevented or reduced; and D-antigen losses are reduced post inactivation by 8 to 10 fold as compared to phosphate buffer; and e) adsorbing Enteroviral particles on an Al(OH)3 adjuvant, whereby the percentage adsorption on the adjuvant is at least 95%.

21. The method of claim 20, wherein step a comprises 40 mM of a phosphate buffer at a pH of 7.0 and step c comprises 0.5% glycine.

Description

DESCRIPTION OF FIGURES

(1) FIG. 1: Alum phosphate gel prepared in 0.9% NaCl (pH Vs Zeta potential at different concentrations of Alum phosphate gel)

(2) FIG. 2: Alum phosphate gel prepared in WFI (pH Vs Zeta potential at different concentrations of Alum phosphate gel)

(3) FIG. 3: Alum Hydroxide gel prepared in 0.9% NaCl (pH Vs Zeta potential at different concentrations of Alum hydroxide gel)

(4) FIG. 4: Alum Hydroxide gel prepared in WFI (pH Vs Zeta potential at different concentrations of Alum hydroxide gel)

DETAILED DESCRIPTION

(5) An important aspect of the instant invention is that said improved process of formalin inactivation and adsorption on alum salt comprises of following steps: a) Adding Sabin IPV purified bulk to TRIS buffer (30 to 50 mM) having pH between 6.8 to 7.2, b) Adding M-199 medium containing glycine (5 gm/l) to mixture of (a), c) Adding 0.025% formaldehyde while mixing, d) Incubating mixture obtained in Step (c) at 37 C. from 5 to 13 days on magnetic stirrer, e) Subjecting post-incubation mixture to intermediate 0.22 filtration on day 7 and final filtration on day 13, f) Storing bulk obtained after step (e) at 2-8 C., g) Performing D-Ag ELISA for D-Antigen unit determination, h) Taking the desired volume of autoclaved Al(OH).sub.3 to get the final concentration of Alum(Al++) between 0.8 to 1.2 mg/dose in a 50 ml Container, i) Adding sIPV bulk with adjusted D-Ag unit and making up the volume with diluent (10M-199+0.5 Glycine %), j) Adjusting the final formulation pH and obtaining final formulation with pH between 6 and 6.5, k) Subjecting the formulation bulk to magnetic stirring overnight at 2-8 C. and wherein formalin inactivation of step (a) does not occur in presence of phosphate buffer

(6) A first embodiment of instant invention is that said buffer to be used during formaldehyde inactivation can be selected from the group consisting of TRIS, TBS, MOPS, HEPES, and bicarbonate buffers.

(7) A preferred aspect of first embodiment is that said formaldehyde inactivation can occur in presence of TRIS Buffer or TBS (TRIS Buffered saline) having concentration selected from 30 mM, 40 mM and 50 mM, preferably 40 mM and at a pH selected from 6.8, 6.9, 7, 7.1 and 7.2, preferably between 6.8 and 7.2 wherein said inactivation does not utilize any phosphate buffer.

(8) A second embodiment of the instant invention is that adsorption of formalin inactivated sIPV can be done on aluminium hydroxide having concentration selected from 1.5 mg/dose, 1.8 mg/dose, 2.2 mg/dose, preferably between 2 mg/dose to 2.4 mg/dose and at a pH selected from 6.2, 6.3, 6.4 and 6.5, preferably 6.5.

(9) A third embodiment of instant invention is that said improved process of formalin inactivation and aluminium hydroxide adsorption can result in D-Antigen recovery post-inactivation between 50% and 80% and percent adsorption of aluminium hydroxide can be between 85 and 99%.

(10) One aspect of third embodiment is that present invention provides an improved process of formalin inactivation and aluminium hydroxide adsorption resulting in dose reduction of atleast 8 fold for Sabin Type I, atleast 3 fold for Sabin Type III as compared to standard dose of 40 DU-8DU-32DU. Second aspect of third embodiment is that instant invention provides improved formaldehyde inactivation and aluminium hydroxide adsorption methods that result in vaccine compositions comprising of i) inactivated poliovirus type 1 at a dose of atleast 5D-antigen units, ii) inactivated poliovirus type 2 at a dose of atleast 8D-antigen units and iii) inactivated poliovirus type 3 at a dose of atleast 10D-antigen units.

(11) A fourth embodiment of instant invention is that said aluminium salt adjuvant is an aluminium hydroxide having concentration between 1.5 mg/0.5 ml dose and 2.5 mg/0.5 ml dose, preferably between 2.100 mg/0.5 ml dose and 2.4 mg/0.5 ml dose at a pH of about 6.5.

(12) One aspect of fourth embodiment is that total aluminium content in the trivalent vaccine (Type 1, 2 and 3) can be between 800-1000 g, preferably 800 g Al.sup.3++ per 0.5 mL dose, characterized in that atleast 400 g Al.sup.3+ for Type 1, atleast 200 g Al.sup.3+ for Type 2, atleast 200 g Al.sup.3+ for Type 3.

(13) Another aspect of fourth embodiment is that said dose reduced polio virus vaccine composition can consist of Type 1 and Type 3 and is devoid of Type 2 wherein the dose volume can be between 0.1 and 0.4 ml.

(14) The dose reduced vaccine compositions prepared by instant methods can be i) Standalone sIPV wherein the antigens may comprise of sIPV type 1 or sIPV type 2 or sIPV type 3, or sIPV types 1 and 2, or sIPV types 1 and 3, or sIPV types 2 and 3, or sIPV types 1, 2 and 3 or ii) Combination Vaccines containing sIPV wherein said non-IPV antigens of combination vaccines can be selected from but not limited to diphtheria toxoid, tetanus toxoid, whole cell pertussis antigen(s), acellular pertussis antigen(s), Hepatitis B surface antigen, Haemophilus influenzae b antigen(s), Neisseria meningitidis A antigen(s), Neisseria meningitidis C antigen(s), Neisseria meningitidis W-135 antigen(s), Neisseria meningitidis Y antigen(s), Neisseria meningitidis X antigen(s), Neisseria meningitidis B bleb or purified antigen(s), Hepatitis A antigen(s), Salmonella typhi antigen(s), Streptococcus pneumoniae antigen(s).

(15) The non-IPV antigen(s) may be adsorbed onto an aluminium salt such as aluminium hydroxide, an aluminium salt such as aluminium phosphate or onto a mixture of both aluminium hydroxide and aluminium phosphate, or may be unadsorbed.

(16) Poliovirus may be grown in cell culture. The cell culture may be a VERO cell line or PMKC, which is a continuous cell line derived from monkey kidney. VERO cells can conveniently be cultured microcarriers. After growth, virions may be purified using techniques such as ultrafiltration, diafiltration, and chromatography. Prior to administration to patients, the viruses must be inactivated, and this can be achieved by treatment with formaldehyde.

(17) Compositions may be presented in vials, or they may be presented in ready filled syringes. The syringes may be supplied with or without needles. A syringe will include a single dose of the composition, whereas a vial may include a single dose or multiple doses (e.g. 2 doses). In one embodiment the dose is for human. In a further embodiment the dose is for an adult, adolescent, toddler, infant or less than one year old human and may be administered by injection.

(18) Vaccines of the invention may be packaged in unit dose form or in multiple dose form (e.g. 2 doses). The said multidose composition can be selected from a group consisting of 2 dose, 5 dose and 10 dose. For multiple dose forms, vials are preferred to pre-filled syringes. Effective dosage volumes can be routinely established, but a typical human dose of the composition for injection has a volume of 0.5 mL.

EXAMPLES

Example 1

(19) Purification of Sabin IPV (sIPV)

(20) 1) Tangential Flow Filtration (TFF): Clarified harvest pool was concentrated to 10 using tangential flow filtration system with 100 Kda cassettes (0.5 m.sup.2) and then diafiltered 3 times of harvest volume with phosphate buffer (40 mM, pH: 7.0)

(21) 2) Column Chromatography: The purification was done by Ion Exchange Chromatography (IEC). 10TFF concentrate was passed through DEAE Sepharose fast flow (Weak-Anion exchanger) packed in column xk-26 using Akta explorer (GE Healthcare). Negatively charged impurities was found to bind to the column whereas polio virus was collected in flow through with phosphate buffer 40 mM.

(22) 3) TRIS Buffer Exchange: To minimize the loss of antigen in a quite cumbersome inactivation procedure (13 days), purified virus pool was buffer exchanged from phosphate buffer to TRIS buffer (40 mM, pH: 7) with TFF system (100 KDa, 0.1 m2). The purified virus pool was exchanged with three volumes of tris buffer.

Example 2

(23) A) Inactivation of sIPV 10 concentrated M-199 with 0.5% glycine was added so as to achieve final concentration 1. Inactivation agent formalin (0.025%) was added into purified virus bulk while constant mixing. Inactivation was carried out at 37 C. while continuous stirring for 13 days containing 0.22 u filtration on 7th day and 13th day.

(24) B) Inactivation of sIPV in TRIS Buffer and Phosphate Buffer

(25) 0.025% formaldehyde was used for inactivation for 13 days at 37 C.

(26) TABLE-US-00001 TABLE 1 D-Antigen Content, Formalin inactivation in presence of TRIS buffer and Phosphate buffer D-Antigen content (40 mM D-Antigen content (40 mM Phosphate buffer during Tris buffer during Inactivation) Inactivation) Type 1 52.70 DU/ml 408.19 DU/ml Type 2 22.63 180.20 Type 3 4.21 21.50

(27) When formaldehyde inactivation methods were particularly carried out in presence of phosphate buffer, significant D-antigen losses were observed for Sabin Type I. Whereas it was found that formaldehyde inactivation in presence of TRIS buffer resulted in minimum loss of D-antigen.

(28) TABLE-US-00002 TABLE 2 Different concentrations of TRIS Buffer used during inactivation 30 mM 40 mM 50 mM Type 1 500 DU/ml 576.80 DU/ml 585 DU/ml Type 2 140 DU/ml 165.16 DU/ml 155 DU/ml Type 3 16 DU/ml 21.17 DU/ml 19 DU/ml TRIS Buffer at a concentration of 40 mM was found to be most efficient in terms of D-Antigen content preservation for sIPV 1, 2 and 3.

(29) C) D-Antigen Content Determination by ELISA.

(30) Day 1: Plate Coating: 1. 100 ul of specific bovine anti polio was pippeted in PBS per well 2. Microtiter plate was sealed and incubated overnight at room temperature.

(31) Day 2: Blocking: 1. The plates were washed (Washing/dilution buffer 0.05% tween 20 in 1PBS) 3 times. 2. 300 ul block buffer (1% BSA in PBS) was pipetted per well. 3. The plate was sealed and incubated for 45 minutes at 371 C.

(32) Sample Addition: 1. The plate was washed 3 times. 2. 100 ul of sample diluent was added in all wells except well of row A. 3. 100 ul standard was added to first two wells of column 2 and 3. 4. 100 ul sample was added to first two wells of column 4-12. 5. Prediluting sample to a suitable concentration. 6. 100 ul sample diluents was added to first two wells of column 1. 7. Serial two fold dilution were made down the column by transferring 100 ul from each well to adjacent well of the same column and discarding 100 ul from the last well. 8. Incubating at 37 C. for 2 hr. 9. Plates were kept overnight at 4 C.

(33) Day 3: Monoclonal Antibody Addition: 1. The plate was washed 3 times. 2. 100 ul diluted (1:240) type specific monoclonal antibodies were added. 3. The plates were sealed and incubated for 2 hours at 37 C.

(34) Conjugate: 1. The plate were washed 3 times 2. 100 ul diluted conjugate (Type1-1:2400, Type2-1:1500, Type3-1:4800) was added. 3. The plate was sealed and incubated for 1 hour at 37 C.

(35) Substrate Addition: 1. 100 ul TMB substrate was added to all wells. 2. Mixture incubated at room temperature for 10 minutes. 3. Reaction was stopped by adding 100 ul 2M H2SO4. 4. Plate was read at 450/630 nm. 5. D antigen concentration was calculated using KC4 software.

Example 3

(36) Adsorption of sIPV: 1. Autoclaved 1% stock of Al(OH).sub.3 and AlPO.sub.4 was used for the preparation of formulations. 2. Desired volume of Al(OH).sub.3/AlPO.sub.4 was taken to get the required concentration of alum in a 100 ml glass bottle. 3. Inactivated polio virus bulk with known D-Ag Unit was added and volume make up was done with diluent. 4. Final formulation pH was adjusted to 6.5 with 1 N HCl/NaOH. 5. The formulation bulk was kept on magnetic stirrer overnight at 2-8 C.

Example 4

(37) Preformulation Studies

(38) Different concentrations of Al(OH).sub.3 & AlPO.sub.4 were prepared in 0.9% saline and in WFI to check size and zeta potential with respect to change in pH.

(39) It was observed that zeta potential of AlPO.sub.4 decreases (negativity) with increase in pH from 5 to 7.5 in presence of WFI as well as in saline (Refer FIGS. 1 and 2).

(40) Whereas, zeta potential of Al(OH).sub.3 in saline remains constant, independent of pH and Al(OH).sub.3 salt concentration (Refer FIGS. 3 and 4).

Example 5

(41) Adsorption Studies of sIPV on Alum Phosphate and Alum Hydroxide

(42) TABLE-US-00003 TABLE 3 Sabin Type 1, 2&3 (Titer 10.sup.6.0/dose) adsorption on alum (Alum phosphate and Alum Hydroxide) Virus Titer (per Particles % free in % adsorbed Sample does) (in K) SUP on gel Type 1, Control 5.45 284 NA AlOH.sub.3 Al+++ 4.15 14 4.98 95.02 125 ug/dose Al+++ 3.85 7 2.49 97.51 250 ug/dose Al+++ 3.8 6.3 2.24 97.78 500 ug/dose Type 1, Control 5.84 691 NA AlPO.sub.4 Al+++ 3.49 3 0.43 99.57 125 ug/dose Al+++ 3.09 1.2 0.17 99.83 250 ug/dose Al+++ 2.94 0.87 0.12 99.87 500 ug/dose Type 2, Control 5.49 309 NA AlOH.sub.3 Al+++ 3.59 3.89 1.25 98.75 125 ug/dose Al+++ 3.49 3.09 1 99 250 ug/dose Al+++ 3.49 3.09 1 99 500 ug/dose Type 2, Control 5.49 309 NA AlPO.sub.4 Al+++ 3.15 1.41 0.45 99.5 125 ug/dose Al+++ 3.09 1.23 0.39 99.6 250 ug/dose Al+++ 3.09 1.23 0.39 99.6 500 ug/dose Type 3, Control 5.59 389 NA AlOH.sub.3 Al+++ 4.14 13.8 3.54 96.47 125 ug/dose Al+++ 3.94 8.7 2.23 97.77 250 ug/dose Al+++ 3.54 3.4 0.87 99.13 500 ug/dose Type 3, Control 5.59 389 NA AlPO.sub.4 Al+++ 5.34 218 56.04 43.96 125 ug/dose Al+++ 5.24 173 44.47 55.53 250 ug/dose Al+++ 5.16 144 37.01 62.9 500 ug/dose

(43) It was found that Sabin polio virus type-3 shows only 50-60% adsorption with aluminium phosphate (AlPO.sub.4). Whereas, Sabin polio virus type-3 shows atleast 90% adsorption with Al(OH).sub.3. Thus, Alum hydroxide was found to be more efficient as compared to Alum phosphate with respect to adsorption of Sabin Type 1, 2 and 3.

Example 6

(44) Immunogenicity Studies of Alum Adsorbed sIPV

(45) To check immune response of adjuvanted sIPV in rat (Sera Neutralisation Test) SNT test was carried out. Sera was separated and used to test the presence of neutralizing antibodies for type specific polio virus. Control sera used to validate the test. Virus back-titration was also performed to get the number of challenge virus particles added.

(46) Animal Model: Wistar rat (8 weeks, approx 200 gm) 50% male and 50% female per group.

(47) Route of Inoculation: Intra Muscular.

(48) Volume: 0.5 ml

(49) Blood withdrawal: on day 21.

(50) Site of bleeding: Retro-Orbital plexus.

(51) TABLE-US-00004 TABLE 4 Type 1 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 15 Comm. 5 DU 2.5 DU 1 DU 5 DU 2.5 DU 1 DU ve IPV 1.15 mgOH 1.15 mgOH 1.15 mgOH 1.8 mgPO4 1.8 mgPO4 1.8 mgPO control Rat SNT Sera Sera Sera Sera Sera SNT Sera Sera Sera No +ve Titer SNT Titer SNT Titer SNT Titer SNT Titer +ve Titer SNT Titer SNT Titer 1 1 (1:2) 8 (1:256) 1 (1:2) 4 (1:16) 5 (1:32) 5 (1:32) 2 (1:4) 0 (<1:2) 2 1 (1:2) 5 (1:32) 1 (1:2) 7 .sup.(1:128) 8 (1:256) 4 (1:16) 1 (1:2) 0 (<1:2) 3 0 (<1:2) 7 (1:128) 3 (1:8) 0 (<1:2) 4 (1:16) 6 (1:64) 0 (<1:2) 0 (<1:2) 4 0 (<1:2) 11 (1:2048) 2 (1:4) 2 (1:4) 1 (1:2) 5 (1:32) 0 (<1:2) 0 (<1:2) 5 7 (1:128) 3 (1:8) 7 (1:128) 5 (1:32) 6 (1:64) 4 (1:16) 1 (1:2) 0 (<1:2) 6 4 (1:16) 7 (1:128) 7 .sup.(1:128) 1 (1:2) 5 (1:32) 6 (1:64) 3 (1:8) 0 (<1:2) 7 3 (1:8) 5 (1:32) 4 (1:16) 1 (1:2) 8 (1:256) 7 .sup.(1:128) 0 (<1:2).sup. 0 (<1:2) 8 1 (1:2) 7 (1:128) 3 (1:8) 2 (1:4) 6 (1:64) 0 (<1:2) 0 (<1:2).sup. 0 (<1:2) 9 3 (1:8) 8 (1:256) 2 (1:4) 3 (1:8) 8 (1:256) 4 (1:16) 4 (1:16) 0 (<1:2) 10 3 (1:8) 7 (1:128) 4 (1:16) 5 (1:32) 6 (1:64) 2 (1:4) 2 (1:4) 0 (<1:2)

(52) It was surprisingly found that Alum hydroxide adjuvanted Type 1 Sabin IPV having 5 DU/dose gave better seroconversion as compared to Salk IPV vaccine with 40 DU/dose and Alum phosphate adjuvanted Sabin IPV having 5 DU/dose.

(53) TABLE-US-00005 TABLE 5 Type 2 Group 1 Group 2 Group 3 Al(OH)3 Adjuvanted 4 DU(0.6 mgOH) 8 DU(0.6 mgOH) 16 DU 0.6 mgOH Rat Sera Sera Sera No SNT +ve Titer SNT +ve Titer SNT +ve Titer 1 3 (1:8) 4 (1:16) 7 (1:128) 2 4 (1:16) 6 (1:64) 5 (1:32) 3 0 (<1:2) 3 (1:8) 5 (1:32) 4 3 (1:8) 4 (1:16) 6 (1:64) 5 5 (1:32) 7 (1:128) 6 (1:64) 6 6 (1:64) 4 (1:16) 9 (1:512) 7 4 (1:16) 7 (1:128) 4 (1:16) 8 5 (1:32) 3 (1:8) 8 (1:256) 9 7 (1:128) 8 (1:256) 8 (1:256) 10 5 (1:32) 3 (1:8) 8 (1:256)

(54) Type 2 sIPV having 8 DU/dose with adjuvant gave equivalent sero conversion as compared to Salk IPV vaccine with 8 DU/dose.

(55) TABLE-US-00006 TABLE 6 Type 3 Group 1 Group 2 Group 3 Al(OH)3 Adjuvanted 10 DU 0.6 mgOH 5 DU 0.6 mgOH 2.5 DU 0.6 mgOH Rat Sera Sera Sera No SNT +ve Titer SNT +ve Titer SNT +ve Titer 1 3 (1:8) 2 (1:4) 0 (<1:2) 2 0 (<1:2) 5 (1:32) 1 (1:2) 3 2 (1:4) 3 (1:8) 1 (1:2) 4 4 (1:16) 2 (1:4) 0 (<1:2) 5 4 (1:16) 2 (1:4) 1 (1:2) 6 4 (1:16) 1 (1:2) 1 (1:2) 7 9 (1:512) 0 (<1:2) 2 (1:4) 8 7 (1:128) 2 (1:4) 2 (1:4) 9 1 (1:2) 0 (<1:2) 1 (1:2) 10 5 (1:32) 7 .sup.(1:128 1 (1:2)

(56) It was found that Type 3 sIPV having 10 DU/dose with adjuvant gave equivalent sero conversion as compared to Salk IPV vaccine with 32 DU/dose.

(57) TABLE-US-00007 TABLE 7 Maximum dose reduction observed for individual Sabin Type 1, 2 & 3 after studies. sIPV Standard dose *SIIL Dose Dose reduction Type 1 40 DU 5 DU ~8 Folds Type 2 8 DU 8 DU Equivalent Type 3 32 DU 10 DU ~3 Folds

(58) SIIL: Serum Institute of India In House dose reduced IPV preparation.

(59) 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.

(60) I. Immune Response of the Adjuvanted SABIN Polio Viruses.

(61) We observed that if the viruses are adjuvanted with Al (OH).sub.3 shows excellent dose sparing.

(62) If we consider single dose regimen for immunization then 5-16-10 D-Ag are best combination for Sabin's polio type 1, 2 and 3 respectively.

(63) If we consider two doses for immunization then 2.5-8-5 gives excellent immunity.

(64) TABLE-US-00008 Type 1 Type 2 Type 3 D-Antigen 5 16 10 units of 2.5 8 5 Sabin's 7.5 16 10 strain

(65) TABLE-US-00009 5-16-10 With Alum 2.5-8-5 With Alum 7.5-16-10 With Alum Single Dose Double Dose Single Dose Double Dose Single Dose Double Dose Rat Type No 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 8 4 10 12 7 12 5 5 3 7 7 12 8 4 10 9 8 10 2 6 7 12 11 7 12 5 5 8 5 9 8 9 2 9 12 6 12 3 6 2 9 6 8 12 5 4 3 8 7 12 11 0 10 8 7 12 4 6 4 9 7 7 12 6 3 11 9 7 12 7 7 7 7 6 12 5 8 4 11 9 10 12 10 0 7 10 7 12 11 6 10 9 8 12 6 6 4 11 10 8 12 6 3 7 9 8 12 6 6 11 9 6 12 7 6 6 8 11 9 12 5 4 8 8 6 12 6 5 10 11 6 12 8 6 6 9 9 7 12 7 3 7 10 9 11 6 4 9 9 5 10 9 7 8 5 10 6 12 5 5 11 10 9 12 7 4 12 10 5 11 10 5 3 5 11 9 12 7 5 8 10 10 12 10 3 9 9 7 12

(66) TABLE-US-00010 Positive Control-Tri. Salk IPV Negative Control Single Dose Double Dose Single Dose Double Dose Rat Type Type Type Type Type Type Type Type Type Type Type Type No 1 2 3 1 2 3 1 2 3 1 2 3 1 1 4 2 4 7 9 0 0 0 0 0 0 2 0 3 0 3 4 8 0 0 0 0 0 0 3 0 5 4 2 4 9 0 0 0 0 0 0 4 0 5 3 6 7 7 0 0 0 0 0 0 5 NA NA NA NA NA NA 0 0 0 0 0 0 6 2 6 5 1 7 6 0 0 0 0 0 0 7 0 6 2 7 10 9 0 0 0 0 0 0 8 0 5 1 5 8 8 0 0 0 0 0 0 9 0 4 2 5 7 9 0 0 0 0 0 0 10 4 5 4 0 0 0 0 0 0 0 0 0

(67) II. Immune Response of the Adjuvanted SALK Polio Viruses.

(68) We observed that if the viruses are adjuvanted with Al (OH).sub.3 shows excellent dose sparing.

(69) If we consider single dose regimen for immunization then 8-2-5 D-Ag are best combination for Salk's polio type 1, 2 and 3 respectively.

(70) If we consider two doses for immunization then 5-2-5 gives excellent immunity.

(71) TABLE-US-00011 Type 1 Type 2 Type 3 D-antigen 8 2 5 units for 5 2 5 Salk Strain

(72) TABLE-US-00012 Salk 8-2-5 With Alum Salk 5-2-5 With Alum Single Dose Double Dose Single Dose Double Dose Type Type Type Type Type Type Type Type Type Type Type Type 1 2 3 1 2 3 1 2 3 1 2 3 3 8 1 9 10 6 2 8 1 10 11 12 5 6 2 9 12 8 2 6 2 10 10 10 4 7 5 12 11 12 4 5 2 9 10 11 7 5 6 11 11 10 6 7 1 12 12 9 8 6 3 10 12 11 5 5 5 8 9 6 5 8 4 10 10 9 2 8 4 11 10 8 2 7 2 8 9 8 3 6 6 8 12 8 4 5 1 9 12 7 6 9 2 9 9 9 5 6 2 8 9 6 2 8 1 10 8 10 3 9 1 12 10 10 1 7 3 12 12 11 Positive Control-Tri. Salk IPV Negative Control Single Dose Double Dose Single Dose Double Dose Type Type Type Type Type Type Type Type Type Type Type Type 1 2 3 1 2 3 1 2 3 1 2 3 2 8 2 2 6 1 0 0 0 0 0 0 3 10 0 4 5 5 0 0 0 0 0 0 4 7 3 5 7 8 0 0 0 0 0 0 0 4 7 5 7 5 0 0 0 0 0 0 4 5 4 3 9 11 0 0 0 0 0 0 0 6 3 8 9 7 0 0 0 0 0 0 2 10 2 7 8 8 0 0 0 0 0 0 NS NS NS 9 10 9 0 0 0 0 0 0 3 8 4 3 7 6 0 0 0 0 0 0 1 9 6 3 8 8 0 0 0 0 0 0
1. Supporting Experimental Data for Salk (10-2-5) Single Dose

(73) TABLE-US-00013 Polio Salk vaccine 10-2-5 With Adjuvant Type 1 Type 2 Type 3 1 5 7 3 5 6 4 9 3 4 8 2 3 9 5 1 7 7 3 9 7 3 11 6 2 9 5 1 10 6
2. Supporting Experimental Data for Salk (10-2-12) Single Dose

(74) TABLE-US-00014 Polio Salk vaccine 10-2-12 With Adjuvant Type 1 Type 2 Type 3 3 6 9 2 10 9 4 9 8 1 9 10 2 9 11 2 7 9 4 9 9 3 8 11 1 10 10 2 6 12
3. Supporting Experimental Data for Salk (5-8-10) Single Dose

(75) TABLE-US-00015 Polio Salk vaccine 5-8-10 With Adjuvant Type 1 Type 2 Type 3 1 8 9 2 10 7 1 9 8 0 11 10 2 9 11 1 10 8 0 9 9 3 11 12 0 9 10 1 8 7
4. Supporting Experimental Data for Salk (7.5-16-10) Single Dose

(76) TABLE-US-00016 Polio Salk vaccine 7.5-16-10 With Adjuvant Type 1 Type 2 Type 3 1 11 8 2 12 9 1 10 7 4 11 10 3 10 8 0 9 11 3 9 10 2 11 12 0 12 9 1 10 7
5.

(77) TABLE-US-00017 Positive Reference control (IPV08-143) Type 1 Type 2 Type 3 1 8 4 2 9 5 0 2 2 0 3 0 1 4 6 2 4 3 2 5 2 3 9 5 1 6 3 3 6 5