OA18258A - Improved methods for enterovirus inactivation, adjuvant adsorption and dose reduced vaccine compositions obtained thereof. - Google Patents

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.

Description

IMPROVED METHODS FOR ENTEROVIRUS INACTIVATION, ADJUVANT ADSORPTION AND DOSE REDUCED VACCINE COMPOSITIONS OBTAINED THEREOF

Background of the invention ·

The prevalence of polio virus has largely been decreased by the use of.Oral Polio Vaccine (OPV), based on live-attenuated Sabin polio strains. However, OPV has limitations for the post-eradication era. Therefore, development of Sabin-IPV plays an important rôle in the WHO polio éradication strategy. The use of attenuated Sabin instead of wild-type Salk polio strains will provide additional safety during vaccine production. Moreover, to prevent the emergence of circulating vaccinederived polioviruses (cVDPVs), the use of OPV should be discontinued following polio éradication, and replaced by IPV. These cVDPVs are transmissible and can become neurovirulent (similar to wild polioviruses) resulting in vaccine associated paralytic poliomyelitis.Such strains can potentiallÿ re-seed the world with polioviruses and negate the éradication accomplishments.

IPV is delivered by intramuscular (IM) or deep subcutaneous (SC) injection. IPV is currently available either as a non-adjuvanted stand-alone formulation, or in various combinations, including DT-IPV (with diphtheria and tetanus toxoids) and hexavalentDTPHepB- Hib-IPV vaccines(additionally with pertussis, hepatitis B, and Haemophilus influenzae b.The currently acceptable standard dose of polio vaccines contains D antigehs as 40 Units of inactivated poliovirus type 1 (Mahoney), 8 units of inactivated poliovirus type 2 (MEF;1) and 32 units of inactivated poliovirus type 3 (Saukett) (e.g. Infanrix-IPV™).Existing préparations of stand-alone IPV do not contain adjuvant.

*

Most experts agréé that worldwide use of IPV is préférable because of its proven protective trackrecord and safety. However, when compared to OPV, the cost-prize for IPV is significantly higher . This is mainly due to requirements for: (i) more virus per dose; (ii) additional downstream processing (i.e. concentration, purification and inactivation), and the related QC-testing (iii) loss of antigen or poor recovery in downstream and iv) containment. Until now, the financial challenge has been a major drawback for IPV innovation and implémentation in low and middle-income countries.The production costs of sIPV are currently estimated équivalent to that for IPV, which is about 20-fold more expensive than OPV . The future global demand for IPV following éradication of

polioviruses could increase from the current level. of 80 million doses to 450 million doses per .yëar. Consequently, approaches to stretch supplies of IPV are likely to be required.

Reduced-dose effîcacious vaccine formulations which provide protection against infection using a lower dose of IPV antigen are désirable in situations where the supply of conventional vaccine is insufficient to meet global needs or where the cost of manufacture o.f the conventional vaccine prevents the vaccine being sold at a price which is affordable for developing countries. Also the exposure to lower dose of IPV; compared to the existing marketed formulations could be more safer.Thus,various strategies to make IPV available at more affordable prices need tô be evaluated.

In case of pandémie influenza vaccines the use of adjuvants has permitted dose réduction, increased the availability and reduced cost ofthe vaccine.Therefore, it has been speculated that an adjuvanted vaccine formulation of sIPV would reduce cost and also increase the number of available sIPV doses worldwide.

Globally different research groups hâve been evaluating dose sparing for vaccines (Influenza vaccines in particularjby employing several adjuvants namely Alum, Emulsion, TLR-agonists (MPL, CpG, poly-IC, imiquimod) ,dmLT,l,25- dihydroxyvitamin D3,CAF01, poly [di (carboxylatophenoxy)phosphazene] (PCPP) and Venezuelan equine encephalitis (VEE) replicon particles. Most of the 20 adjuvant types being studied hâve encountered following hurdles i) Unknown safety or classified as toxic by regulatory agencies ii) having limitations regards to route of administration iii) lacking manufacturing reproducibility iv) stability of adjuvant.

Emulsion adjuvants (MF-59,AS03,AF3) hâve been previously reported to provide a strong dose25 réduction effect (> 3Ofold) for Influenza and Hepatitis B vaccines. These adjuvants work by forming a depot at the site of injection, enabling the meted release of antigenic material and the stimulation of antibody producing plasma cells.However, these adjuvants hâve been deemed too toxic for widespread human prophylactic vaccine use and are usually reserved for those sevére and/or terminal conditions such as cancer where there is a higher tolérance of side-effects.

Further, Aluminum salts hâve been considered safe, are already being used in combination vaccines containing sIPV, hâve the lowest development hurdles and are ' inexpensive to

manufacture.However aluminium adjuvants are not known for permitting signifïcant dosereduction.

One of the most critical steps in the production of vaccines against pathogens, in particular viral vaccines, is viral inactivation. In the case of virus inactivation, formalin is the most frequently used inactivating agent in the manufacture of vaccines.Formaldehyde inactivâtes a virus by irreversibly cross-linking primary amine groups in surface proteins with other nearby nitrogen atoms in protein or DNA through a —CH2-Iinkage.A potential problem with using formalin for viral inactivation is that this involves a sériés of chemical reactions that produce reactive products that 10 can induce cross-linking of viral proteins and aggregation of virus particles. This could hamper the inactivating efficiency of the formalin and could also resuit in the partial destruction of the immunogenicity ofthe antigen in vaccine-Accordingly, ithas been reported previously that formalin inactivation of polioviruses could affect the viràl immunogenicity as well as antigenicity.Refer Morag Ferguson et al Journal of General Virology (1993), 74, 685-690. Most importantly, previously ' 15 disclosed formaldéhyde inactivation methods were particularly carried out in presence of phosphate buffer wherein signifïcant D-antigen losses were observed alongwith epitope modification for SabinType I/II/III (D-antigen recovery post inactivation:22% forsabin type 1,15% for sabin type II, 25% for sabin type 111), thereby failing to preserve the epitopic conformation.lt is therefore possible that antibodies produced by récipients of formalin-inactivated polioviruses (in presence of phosphate buffer) may not contribute to the protective immune response.

By combining formalin and UV-inactivation, scientists tried to overcome the limitations of isolated UV-inactivation or formalin-inactivation, respectively, when inactivating the particularly résilient .poliovirus. See, e.g., McLean, et al., Expériences in the Production of Poliovirus Vaccines, Prog.

Med. Virol., vol 1, pp. 122-164 (1958.) Taylor et al. (J. Immunol. (1957) 79:265-75) describe the inactivation of poliomyelitis virus with a formalin and ultraviolet combination. Molner et al. (Am. J. Pub. Health (1958) 48:590-8) describe the formation of a measurable level of circulating antibodies in the blood of subjects vaccinated with ultraviolet-formalin inactivated poliomyelitis vaccine. Truffelli et al. (Appl. Microbiol. (1967) 15:516-27) report on the inactivation of Adenovirus and

Simian Virus 40 Tumorigenicty in hamsters by a three stage inactivation process consisting of formalin, UV light and p-propiolactone(BPL). Miyamae (Microbiol. Immunol. (1986) 30:213-23) describes the préparation of immunogens of Sendai virus by a treatment with UV rays and formalin.However previously discussed promising alternatives for formaldéhyde like β18258

propiolactone(BPL) hâve been reported to produce an immune complex-reaction when combined with other components of the rabies vaccine. Additionally, it has been shown to produce squamous cell carcinomas, lymphomas and hepatomas in mice.

It is therefore particularly désirable to employ favorable formaldéhyde inactivation conditions that maintain the structural integrity of antigenic structures of Sabin strains as well as utilize safe and cost-effective adjuvants that can resuit in significantly dose reduced (i.e. 8 to 10 fold] slPVfSabin IPV) vaccine compositions· thereby reducing cost of manufacture, increasing vaccine supplies and making vaccines affordable for developing countries.

'

The présent inventors hâve surprisingly found that D-antigen losses post-formaldehyde inactivation could be due to presence of phosphate buffer that unexpectedly causes undesirable aggregation of polio viruses.The instant invention provides an improved process of formaldéhyde inactivation in presence of TRIS buffer thereby ensuring minimal epitopic modifications and 15 subsequently minimizing D-antigen losses. Subsequently significantly dose reduced Sabin IPV vaccine compositions with atleast 8 fold dose réduction for Sabin Type 1 and 3 fold dose réduction for Sabin Type III can be obtained.

Description of Figures:

Fig l:Alum phosphate gel prepared in 0.9% NaCl(pH Vs Zêta potential at different concentrations of Alum phosphate gel) . .

Fig 2: Alum phosphate gel prepared in 14Τ7(ρΗ Vs Zêta potential at different concentrations of Alum phosphate gel)

Fig 3: Alum Hydroxide gel prepared in 0.9% AtaC/(pH Vs Zêta potential at different concentrations of Alum hydroxide gel)·

Fig 4: Alum Hydroxide gel prepared in WF1 (pH Vs Zêta potential at different concentrations of Alum hydroxide gel)

Detailed Description:

An important aspect of the instant invention is that said improved process of formàlin inactivation and adsorption on alum sait comprises of following steps:.

a) Adding Sabin IPV purified bulk to TRIS buffer (30 to 50mM) having pH between 6.8 to 7.2,

b) Adding M-199 medium containing glycine (Sgm/l) to mixture of (a),

c) Adding 0.025% formaldéhyde while mixing,

d) Incubating mixture obtained in Step (c) at 37°C from 5 to 13 days on magnetic stirrer,

e) Subjécting post-incubation mixture to intermediate 0.22μ filtration on day 7 and final filtration 5 on day 13,

f) Storing bulk obtained after step (e) at 2-8°C,

g) Performing D-Ag ELISA for D-Antigen unit détermination,

h) Taking the desired volume of autoclaved A1(OH)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 (ΙΟχ M-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 bulkto magnetic stirring overnight at 2-8°C and wherein formalin inactivation of step (a) does not occur in presence of phosphate buffer

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

A preferred aspect of first embodiment is that said formaldéhyde inactivation can occur in presence of TRIS Buffer or TBS(TR1S Buffered saline) having concentration selected from 30mM,40mM and 20 50mM, preferably 40mM 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.

I

A second embodiment of the instant invention is that adsorption of formalin inactivated sIPV can be done on aluminium hydroxide having concentration selected. from 1.5mg/dose, 1.8mg/dose,2.2 mg/dose, preferably between 2mg/dose to 2.4 mg/dose and at a pH selected from 6.2,6.3,6.4 and 25 6.5, preferably 6.5.

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

One aspect of third embodiment is that présent invention provides an improved process of formalin 30 inactivation and aluminium hydroxide adsorption resulting in dose réduction of atleast 8 fold for

Sabin Type 1, 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 formaldéhyde inactivation and aluminium hydroxide. adsorption methods. that resuit 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 lOD-antigen units.

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

One aspect of fourth embodiment is that total aluminium content in the trivalent vaccinefType 1,2 and 3)can be between 800-1000pg, preferably 800pg Al³⁺ per 0.5mL dose .characterized in that atleast 400 pg Al³⁺ for Type 1,atleast 200 pg AI³⁺ for Type 2,atleast 200 pg Al³⁺ for Type 3.

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.

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|7 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 slPV”wherein said non-IPV antigens of combination vaccines can be selected^ from but not limited to diphtheria toxoid, tetanus toxo’id, 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),Neisseriail * .! f meningitidis B bleb or purified antigenes), Hepatitis A antigen(s), Salmonella ty.phi antigen(s),'. · Streptococcus pneumoniae antigeri(s). <

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

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 formaldéhyde.

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.

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 5 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 of0.5mL.

Examples:

¹⁰

Example 1

Purification of Sabin IPV (sIPV)

1) Tangential flow filtration (TFF):

Clarified harvest pool was concentrated to 10X using tangential flow filtration system with lOOKda cassettes(0.5m²) and then diafiltered 3 times of harvest volume with phosphate buffer (40 mM, pH : 7.0) • ‘t

2) Column Chromatography:

The purification was done by Ion Exchange Chromatography (IECJ.10X TFF concentrate was passed through DEAE Sepharose fast flow (Weak- Anion exchanger) packed in column xk25 26 using Akta explorer (GE Healthcarej.Negatively charged impurities was found.to bind to the column whereas polio virus was collected in flow through with phosphate buffer 40 mM.

3) TRIS Buffer exchange:

To minimize the loss of antigen in a quite cumbersome inactivation procedure(13days), purified virus pool was buffer exchanged from phosphate buffer to TRIS buffer (40mM ,pH:

7) with TFF System (100 KDa ,0.1 m2). The purified virus pool was exchanged with three volumes of tris buffer.

Example 2

A) Inactivation of sIPV

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

B) Inactivation of sIPV in TRIS buffer and Phosphate buffer

0.025% formaldéhyde was used for inactivation for 13 days at 37°C.

Table 1: D-Antigen Content ,Formalin inactivation in presence of. TRIS buffer and Phosphate buffer

-	D-Antigen content (40mM Phosphate buffer during Inactivation)	D-Antigen content (40mM Tris buffer during Inactivation)
Type 1	52.70 DU/ml	408.19 DU/ml
Type 2	22.63	180.20
Type 3	4.21	21.50

When formaldéhyde 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 formaldéhyde inactivation in presence of TRIS buffer resulted in minimum loss of D-antigen.

Table 2: Different concentrations of TRIS Buffer used during inactivation

	30mM	40mM	50mM
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 40mM was found to be most efficient in terms of DAntigen content préservation for sIPV 1,2 and 3.

C) D-antigen content détermination by ELISA.

Day 1 : Plate coating:

1. lOOul of spécifie bovine anti polio was pippeted in PBS per well

2. Microtiter plate was sealed and incubated overnight at room température.

Day 2: Blocking:

1. The plates were washed (Washing/dilution buffer -0.05% tween 20 in lx PBS)3 times.

2. 300ul block buffer (1% BSA in PBS) was pipetted per well.

3. The plate was sealed and incubated for 45minutes at 37±1°C.

Sample addition:

1. The plate was washed 3 times.

2. lOOul of sample diluent was added in ail wells except well of row A.

3. lOOul standard was added to first two wells of column 2 and 3.

4. lOOul sample was added to first two wells of column 4-12.

5. Prediluting sample to a suitable concentration.

6. lOOul sample diluents was added to first two wells of column 1.

•7. Serial two fold dilution were made down the column by transferring lOOul from each well to adjacent well of the same column and discarding lOOul from the last well.

8. Incübatingat37°cfor2hr.

9. Plates were kept overnight at 4°C;

Day 3: Monoclonal antibody addition:

1. The plate was washed 3 times.

2. lOOul dilutedf 1:240) type spécifie monoclonal antibodies were added.

3. The plates were sealed and incubated for 2 hours at 37°C.

Conjugate:

l.The plate were washed 3 times

2.,lOOul diluted conjugate( Typel-1:2400 ,Type2- 1:1500, Type3 -1: 4800)was added.

3.The plate was sealed and incubated for 1 hour at 37°C.

Substrate addition:

1. lOOul TMB substrate was added to ail wells.

2. Mixture incubated at room température for 10 minutes.

3. Réaction was stopped by adding lOOul 2M H2S04.

4. Plate was read at 450/630nm.

5. D antigen concentration was calculated using KC4 software.

Example 3

Adsorption o f sIP V:

1. Autoclavéd 1% stock of AI(0H)3 and A1PO₄ was used for the préparation of formulations.

2. Desired volume of A1(OH)3/A1PO4 was taken to get the required concentration of alum in a 100 ml glass bottle.

3.1nactivated polio virus bulk with known D-Àg 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 ovemight at 2-8°C.

Example 4

Preformulation Studies

Different concentrations of Al(0H)3 & A1PO₄ were prepared in 0.9% saline and in WFI to check size and zêta potential with respect to change in pH.

It was observed that zêta potential of A1PO₄ decreases (negativity) with increase in pH from 5 to 7.5 in presence of WFI as well as in saline (Refer Figure 1 and 2).

Whereas, zêta potential of Al(0H)3 in saline remains constant,independent of pH and A1(OH)3 sait concentration(Refer Figure 3 and 4).

Example 5

Adsorption studies of sIPV on Alum phosphate and Alum hydroxide

Table 3: Sabin Type 1,2&3 (Titer 10⁶⁰/dose) adsorption on alum (Alum phosphate and Alum

Hydroxide)

	Sample	Titer (per does)	Virus Particles (in K)	% free in SUP	% adsorbed on gel
Type l,A10H3	Control	5.45	284	NA
A1+++ 125ug/dose	4.15	14	4.98	95.02

	A1+++ 250ug/dose	3.85	7	2.49	97.51
A1+++ 500ug/dose	3.8	6.3	2.24	97.78
Type 1, Α1Ρ04	Control	5.84	691	NA
A1+++ 125ug/dose	3.49'	3	0.43	' 99.57
A1+++ 250ug/dose	3.09	1.2	0.17	99.83
A1+++ 500ug/dose	2.94	0.87	0.12	99.87
Type 2,A1OH3	Control	5.49	309	NA
A1+++ 125ug/dose	3.59	3.89	1.25	98.75
A1+++ 250ug/dose	3.49	3.09	1	99
A1+++ 500ug/dose	3.49	3.09	i-	99
Type 2, A1PO4	Control	5.49	309	NA
A1+++ 125ug/dose	. 3.15.	1.41	0.45	99.5
A1+++ 250ug/dose	3.09	1.23	0.39	99.6
A1+++ 500ug/dose	3.09	1.23	0.39	99.6'
Type 3, A10H3	Control	5.59	389	NA
AI+++ 125ug/dose	4.14	13.8	3.54	96.47
A1+++ 250ug/dose	3.94	8.7	2.23	97.77

	A1+++ 500ug/dose	3.54	3.4	0.87	99.13
Type 3, A1PO4	Control	5.59	389	NA
A1+++ 125ug/dose	5.34	218	56.04	43.96
A1+++ 250ug/dose	5.24	173	44.47	55.53
A1+++ 500ug/dose	5.16	144	37.01	62.9

It was found that Sabin polio virus type-3 shows only 50-60% adsorption with aluminium phosphate (AlPOri-Whereas, Sabin polio virus type-3 shows atleast 90% adsorption with Al(OH)3.Thus, Alum hydroxide was found to be more efficient as compared to Alum phosphate with 5 respect to adsorption of Sabin.Type 1,2 and 3.

Example 6

Immunogenicity studies of Alum Adsorbed sIPV

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 spécifie polio virus. Control sera used to yalidate the test. Virus back-titration was also performed to getthe number of challenge virus particles added.

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

Route of Inoculation: Intra Muscular.

Volume: 0.5 ml

Blood withdrawal: on day 21.

Site of bleeding: Retro-Orbital plexus.

Table 4:Type 1

Rat No	Group 1	Group 2	• Group 3	Group 4	Group 5	Group 6	Group 7	Group 15
Connu. IPV	5 DU 1.15mgOH	2.5DU 1.15mgOH	1DU 1.15mgOH	5DU 1.8mgPO4	2.5DU 1.8mgPO4	1DU 1.8mgP0	-ve control
SNT +ve	Sera Titer	SN T	Sera Titer	SN T	Sera Titer	SN T	Sera Titer	SNT	Sera Titer	SNT +ve	Sera Titer	SN	Sera Titer	SN	Sera Titer
1	1	(1:2)	8	(1:25 6)	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)	l	(1:2)	7	(1:1? PT	8	(1:25 AT	4	(1:16)	1	(1:2)	0	£<1:2 T
3	0	(<1:2)	7	(1:12 8)	3	(1:8)	0	£<1:2 )	4	(1:16)	6	(1:64)	0	C<1- 2)	0	(<1:2 )
4	0	(<1:2)	11	(1:20 48)	2	(1:4)	2	(1:4)	1	(1:2)	5	(1:32)	0	(<1: 2)	0	(<1:2 )
5	7	(1:12 8)	3	(1:8)	7	(1:12 8)	5	(1:32 )	6	(1:64)	4	(1:16)	1	(1:2)	0	C<1:2 )
6	4	(1:16)	7	(1:12 QT	7	(1:12 QT	1	(1:2)	5	(1:32)	6	(1:64)	3	(1:8)	0	£<1:2 T
7	3	(1:8)	5	(1:32)	4	(1:16 T ’	1	(1:2)	8	(1:25 AT	7	(1:128 T	0	(<1: 23	0	£<1:2 T
8	1	(1:2)	7	(1:12 QT	3	(1:8)	2	(1:4)	6	(1:64)	0	(<1:2)	0	(<1:	0	£<1:2 T '
9	3	(1:8)	8.	(1:25	2	(1:4)	3	(1:8)	8	(1:25 AT	4	(1:16)	4	(1:1 AT	0	(<1:2 T
10	3	(1:8)	Ί	(1:12 8)	4	(1:16 )	5	(1:32 )	6	(1:64)	2	(1:4)	2	(1:4)	ό	£<1:2 )

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

Table 5:Type 2

Rat No	Group 1	Group 2	Group 3
Al (OH) 3 Adjuvànted
4DU( O.ômgÔH)	8DU( 0.6mgOH)	16DU O.ômgOH
SNT +ve	Sera Titer	SNT +ve	Sera Titer	SNT +ve	Sera 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)

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

Table 6:Type 3

RatNô	Group 1	Group 2	Group 3
Al (OH) 3 Adjuvanted
10DU O.ômgOH	5DU O.ômgOH	2.5DU O.ômgOH
SNT +ve	SeraTiter	SNT +ve	SeraTiter	SNT +ve	Sera Titer
. 1	3 ,	(1:θ)	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 a	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	(1:128	1	(1:2)

It was found that Type 3 sIPV having lODU/dose with adjuvant gave équivalent sero conversion as compared to Salk IPV vaccine with 32DU/ dose.

Table 7: Maximum dose réduction observed for individual Sabin Type 1, 2 & 3 after studies.

sIPV	Standard dose	*SIIL Dose	Dose réduction
Type 1	40DU	5DU	~8 Folds
Type 2	8DU	. 8DU	Equivalent
Type 3	32DU	10DU	—3 Folds

SIIL:Serum Institute of India In House dose reduced IPV préparation.

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 ofthe invention and should not be taken as limiting the scope ofthe invention. Rather, the scope. of the invention is defined by the following claims. We therefore claim as our 5 invention ail that cornes within the scope and spirit of these claims.

I. Immune response of the adjuvanted SABIN polio viruses.

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

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

Ifwe consider two doses for immunization then 2.5-8-5 gives excellent immunity.

	Type 1	Type 2	Type 3
D-Antigen units of Sabin's strain	5	16	10
2.5	8	5
7.5	16	10

Supporting Experimental Data for Sabin

Rat No	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
Type 1	Type 2	Type 3	Type 1 1	Type 2	Type 3	Type 1	Type 2	Type 3	Type 1	Type 2	Type 3	Type 1: /	Type 2'	Type 3 ..	Type 1 .'	Type 2	Type SS/-/
1	8	4	10	12	7	12	5	5	3	7	7	12	8	4 /;	10 :	9	8
2	6	7	12	11	7	12	5	5	8	5	9	8	9/	/2-.·/	/)-9.- :·/	. -125/	6
3	6	2	9	6	8	12	5	4	3	8	7 ·	12	./11/	,/.0:/	10	8.	7)7/¾	S12/./
4	6	4	9	7	7	12	6	3	11	9	7	12	/--7-/.	--7,-,/	///75-	/ 7')//	/)6//	/1S/5-
5	8	4	11	9	10	12	10	0	7	10	/-7//	<..12/ÿ	//it/	6	10
6	6	4	11	10	8	12	6	3	7	9	//8¾)	-/12;/	/gg/g	6 .	gïl·®	/ÉOi		^%2/H
7	6	6	8	11	9	12	5	4	8	8	//6-//	/,12./	/•.ί'/'Α··''??/·	7/5///	-//10//.	11		w
8	6	6	9	9	7	12	7	3	7	10	9	1®		il®	i®e	//S9W|	WH
9	7	8	5	10	6	12	5	5	11	10	/,9/..	. 12 >	/:7//	,Μ/	12	10	ÙXSM* :^.;·<φ'5ζΛ^λ··	lâWœ
10	5	3	5	11	9	12	7	5	8	10	10	512-/	/10/		9	-.:-95.¾	W/I

*y> À,—

Rat No	Positive Control - Tri. SalkIPV	Négative Control
Single Dose	Double Dose	Single Dose	Double Dose
Type 1	Type 2	Type 3	Type 1	Type 2	Type 3	Type 1	Type 2	Type 3	Type 1	Type 2	Type 3
1	1	4	2	4	7	9	0	0	0	θ	0	0 ’ -
2	0	3	0	3	4 Λ·	8	0	0	0	0	0	0
3	0	5	4	2	4	9	0	0	0	;/10777	0	0
4	0	5	3	6	i 7	7	0	0	0		0 7	0. ·.
5	NA	NA	NA	NA	NA		0	0	0	- 0	0	:7A97W
6	2	6	5	1	7	/6//-	0	0	0	0	0	· · 077
7	0	6	2	7	10 '	'•Tel·?:!·	0	0	0	7/0..7-	‘ 0	//:0/7
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 : 7

'.........' .......20 .. . „ . . .

Μ ....... .......... ...........

II. Immune response of the adjuvanted SALK polio viruses.

We observed that if the viruses are adjuvanted with Al (0H)₃ shows excellent dose sparing.

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

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

	Type 1	Type 2	Type 3
D-antigen units for Salk Strain	8	2	5
5	2	5

Supporting experimental data for Salk

Salk 8-2-5 With Alum	Salk 5-2-5 With Alum
Single Dose	Double Dose	Single Dose	Double Dose
Type 1	Type 2	Type 3	Type 1	Type 2	Type 3	Type 1	Type 2	Type 3	Typel '	Type 2	Type 3 :
3	8	1	9	10	6	2	8	1	'· 10 y	: 11
5	6	2	9	12	8	2	6	2.	10	10	10
4	7	5	12	11	12. ?.?	4	5	2	9 ;	10	.îix:
7	5	6	11	.11	10 Λ	6	7	1	12	12	97.
8	6	3	10	12	· 11 ·	5	5	5	8	9	6 :
5	8	4	10	10 .	. Λ.9.Χ j	2	8	4	Il <	10	8 T;
2	7	2	8	9	s	3	6	6	8	12	θ
4	5	1	9	12	7 ··.·.	6	9	2	9	9	9
5	6	2	8	9	6	2	8	1	10	• 8	107 .
3	9	1	12	10	10	1	7	3	12	12	11

r

Positive Control - Tri. Salk IPV	Négative Control
Single Dose	Double Dose	Single Dose	Double Dose
Type 1	Type 2	Type 3	Type 1	Type 2	Type 3	Type 1	Type 2	Type 3	Type 1	Type 2	Type 3'
2	8	2	2	6		0	0	0	.0	0	0
3	10	0	4	S	5: \	0	0	0	0	0 .	0 .
4	7	3	5	7	8	0	0	0	0	0	0
0	4	7	S	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

Claims (17)

1. Claims

   1. A method for producing a composition comprising Enteroviral particles, wherein the 5 method comprises the steps of:

   a) producing a medium containing the Enteroviral particles;

   b) purification of the Enteroviral particles from the medium;

   c) stabilization of purified Enteroviral particles;

   d) formalin inactivation of the Enteroviral particles whereby during at least a part of the 15 inactivation a buffer other than a phosphate buffer is présent at a concentration sufficient to prevent or reduce aggregation of the Enteroviral particles thereby reducing D-antigen losses post inactivation by 8 to 10 fold as compared to a phosphate buffer; and

   e) adsorption of Enteroviral particles on an aluminium sait adjuvant whereby a percentage adsorption on the aluminium sait adjuvant is at least 95%.
2. 2. A method according to claim 1, wherein the buffer of step (d) is selected from the group consisting of TRIS, TBS, MOPS, HEPES, and bicarbonate buffers.
3. 3. A method according to claim 2, wherein the buffer is a TRIS buffer having a pH of about 6.8 to 7.2 and at a concentration in the range of 30mM - 70mM, preferably 40mM.
4. 4. A method according to claim 1, wherein the aluminium sait adjuvant of step (e) is selected from the group consisting of aluminium hydroxide, aluminium phosphate, and a mixture of both.
5. 5. A method according to claim 4, wherein said aluminium sait adjuvant is an aluminium hydroxide having a concentration between 1.5mg/0.5 ml dose and 2.5 mg/0.5 ml dose, preferably between 2.1 mg/ 0.5ml dose and 2.4mg/0.5 ml dose at a pH of about 6.5.
6. 6. A method according to claim 5, wherein total aluminium content in the composition comprising Enteroviral particles is 0.8 - 1.2mg, preferably 0.8mg Al³⁺ per 0.5mL dose, or wherein, when the composition includes Sabin or Salk Type 1, Type 2, or Type 3 poliovirus as the Enteroviral particles, total aluminium content in the composition comprising Enteroviral particles is at least 0.4 mg Al³⁺ for Type 1, at least 0.2mg Al³⁺ for Type 2, and at least 0.2mg Al³⁺ for Type 3.
7. 7. A method according to any one of claims 1 to 6, wherein the composition comprising Enteroviral particles is a vaccine.
8. 8. A method according to any one of claims 1 to 7, wherein the Enteroviral particles are of an Enterovirus of polioviruses.
9. 9. A method according to claim 8, wherein the Enteroviral particles comprise polioviruses of the Sabin serotypes 1,2 and 3.
10. 10. A method according to claim 8, wherein the Enteroviral particles comprise polioviruses of Salk serotypes IPV type 1 (Mahoney strain), IPV type 2 (MEF-1 strain), and/or IPV type 3 (Saukett strain).
11. 11. A method according to any one of claims 1 to 10, wherein the composition comprising Enteroviral particles is a dose reduced Inactivated Polio Vaccine (IPV).
12. 12. A method according to claim 11, 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 42DU; and/ or ⁵ 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 15 D-antigen units instead of standard dose of 32DU.
13. 13. A method according to claim 11 or 12, wherein said dose reduced Inactivated Polio Vaccine is selected from the group consisting of

    i) Sabin single dose composition having Sabin Type 1, Type 2, Type 3 combination 15 selected from 5-16-10;

    ii) Sabin two dose composition having Sabin Type 1, Type 2, Type 3 combination selected from 5-16-10;

    iii) Sabin single dose composition having Sabin Type 1, Type 2, Type 3 combination 20 selected from 2.5-8-5;

    iv) Sabin two dose composition having Sabin Type 1, Type 2, Type 3 combination selected from 2.5-8-5;

    v) Sabin single dose composition having Sabin Type 1, Type 2, Type 3 combination 25 selected from 5-8-10;

    vi) Sabin two dose composition having Sabin Type 1, Type 2, Type 3 combination selected from 5-8-10;

    vii) Salk single dose composition having Salk Type 1, Type 2, Type 3 combination 30 selected from 7.5-16-10;

    viii) Salk two dose composition having Salk Type 1, Type 2, Type 3 combination selected from 7.5-16-10;

    ix) Salk single dose composition having Salk Type 1, Type 2, Type 3 combination ₃₅ selected from 8-2-5;

    x) Salk two dose composition having Salk Type 1, Type 2, Type 3 combination selected from 8-2-5;

    xi) Salk single dose composition having Salk Type 1, Type 2, Type 3 combination , _ selected from 10-2-5;

    xii) Salk two dose composition having Salk Type 1, Type 2, Type 3 combination selected from 10-2-5;

    xiii) Salk single dose composition having Salk Type 1, Type 2, Type 3 combination selected from 10-2-12;

    xiv) Salk two dose composition having Salk Type 1, Type 2, Type 3 combination selected from 10-2-12;

    xv) Salk single dose composition having Salk Type 1, Type 2, Type 3 combination selected from 5-2-5; and xvi) Salk two dose composition having Salk Type 1, Type 2, Type 3 combination selected from 5-2-5.
14. 14. A method for preparing a dose reduced inactivated Polio vaccine containing Salk or Sabin polioviruses, comprising the steps of:

    a) producing a medium containing the polioviruses;

    b) purification of the polio viruses from the medium;

    c) stabilization of purified polioviruses by addition of M-199 medium containing glycine;

    d) inactivation of the polio viruses by using formaldéhyde 0.025% at 37°C for 5 to 13 days in the presence of a TRIS buffer at a concentration between 30mM and 60mM to prevent or reduce aggregation of the poliovirus particles thereby reducing D-antigen losses post inactivation by 8 to 10 fold as compared to a phosphate buffer ; and

    e) adsorption of inactivated polioviruses on an aluminium hydroxide adjuvant having a concentration between 2 and 2.5 mg/dose, whereby a percentage adsorption on the aluminium hydroxide is greater than 95% for Type 1, Type2 and Type 3.
15. 15. A method for preparing a dose reduced inactivated Polio vaccine containing Salk or Sabin polioviruses, comprising the steps of:

    a) producing a medium containing the polioviruses;

    b) purification of the polio viruses from the medium;

    c) stabilization of purified polioviruses by addition of M-199 medium containmg glycine;

    d) inactivation of the polio viruses by using formaldéhyde 0.025% at 37°C for 5 to 13 days in the presence of a TRIS buffer at a concentration between 30mM and 60mM to prevent or reduce aggregation of the poliovirus particles thereby reducing D-antigen losses post inactivation by 8 to 10 fold as compared to a phosphate buffer ; and

    e) adsorption of inactivated polioviruses on an aluminium hydroxide adjuvant having a concentration between 2 and 2.5 mg/dose, whereby a percentage adsorption on the aluminium hydroxide is greater than 95% for Type 1 and Type 3.
16. 16. A method according to claim 14 or claim 15_z wherein said dose reduced Salk or Sabin Inactivated Polio Vaccine does not comprise Type 2.
17. 17. The method of any one of claims 7,14,15 or 16,,wherein the vaccine is a multivalent vaccine consisting of dose reduced IPV, the vaccine comprising one or more antigens from a pathogen selected from the groupt consisting of: Haemophilus inftuenzae 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, Streptococcus agalactiae, Salmonella typhi, Hepatitis A, Hepatitis B, RSV, Hepatitis C, diphtheria toxoid, tetanus toxoid, whole cell pertussis, acellular pertussis, Staphylococcus aureus, anthrax, Vibrio choiera, Zika, Ebola, Chikungunya, dengue, malaria, measles, mumps, rubella, BCG, Japanese encephalitis, Rotavirus, smallpox, Shigella, yellow fever, typhoid, CMV, Shingles, Varicella virus, HPV, HSV, and HIV.