WO2005070454A2 - A novel process of hepatitis a vaccine preparation - Google Patents
A novel process of hepatitis a vaccine preparation Download PDFInfo
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- WO2005070454A2 WO2005070454A2 PCT/IN2005/000020 IN2005000020W WO2005070454A2 WO 2005070454 A2 WO2005070454 A2 WO 2005070454A2 IN 2005000020 W IN2005000020 W IN 2005000020W WO 2005070454 A2 WO2005070454 A2 WO 2005070454A2
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
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- a novel process for the Isolation of an Indian strain of Hepatitis A virus, adaptation to tissue culture and further cultivation for vaccine preparation The present invention further relates to a novel process for isolation, cell culture adaptation, characterization and further large scale culture of an Indian isolate of Hepatitis A virus in cell lines.
- Hepatitis A virus HAV particles purified from marmoset liver and inactivated with formaldehyde when injected to the susceptible primates is capable of inducing protection against virulent virus challenge.
- HAV Hepatitis A virus
- the development of Hepatitis A vaccines as live attenuated or inactivated vaccines has originated from the successful in-vitro culture of the virus in cell cultures by Provost and Hilleman (1979) using the HAV serially passaged in the marmosets as inoculum. Isolation of HAV from clinically ill patients' stools samples using cell cultures has been described in the literature (Provost etal., 1981; Frosner et.al., 1979; Flehming 1980).
- cell lines can be used for HAV isolation (Locarini et.al., 1981; Gauss-Muller et.al.,1981).
- the cell lines used include fetal rhesus monkey kidney (Wheeler et.al, 1986), primary African green monkey kidney (AGMK) cells(Daemer et.al., 1981) , transformed rhesus monkey kidney cells( de Chastonay and Siegel,1987) and human diploid fibroblasts (Binn et.al., 1986 and Andre et.al., 1990). Reports also include Vero cells and continuously growing AGMK cells.
- Vero and MRC- 5 cells have been used extensively for the production of viral vaccines like polio, rabies, rubella, Hepatitis A etc.
- the Vero and MRC-5 cells have also been successfully grown on microcarriers and various types of cell bioreactor systems. (van Wazel 1978).
- the invention provides a method of isolation and adaptation to cell culture of an Indian isolate of Hepatitis A virus , its characterization and further adaptation to grow in Nero and MRC-5 cells for production of an inactivated vaccine.
- the later process of harvesting the virus from the culture poses a greater problem since the majority of the virus is cell bound.
- the cells are required to be detached and lysed using suitable down stream process of extraction. Since the process of lysis adds to the contamination of cellular nucleic acids, the purification strategy shall also include selective separation of the D ⁇ A contaminants besides cell debris.
- the present invention outlines the methodology for the commercial scale production of Hepatitis A vaccine using the culture of Nero and MRC5 cells with an Indian isolate of Nero cell adapted Hepatitis A virus ( ⁇ INI ⁇ 97 strain) and a subsequent harvest, purification and inactivation of the virus for production of Hepatitis A vaccine.
- the invention method is also applicable and not limited to the production of other Hepatitis A virus isolates of similar genotypes or serotypes as well as other viruses like Rabies, Rotavirus, Polio etc. using the Vero or MRC5 cells as a substrate. It is also applicable and not limited to other cell substrates like WI-38, and FRhK cells.
- the method describes a commercially viable scale-up option for the production of viral vaccines using cell culture technology.
- Fig. 8 depicts Titration of MVTN97 strain of HAN adapted to BGMK cells by ELISA
- Fig. 9 depicts Reactivity of virus tested in anti-HAN IgM capture ELISA using known anti HAN IgM negative (47) and positive (41) sera with ⁇ NI ⁇ 97 and HM-175 of HAN
- Fig. 10 depicts The reactivities of ⁇ IVI ⁇ 97 strain of HAV derived from BGMK cell line and Vero cell line compared in anti-HAV IgM capture ELISA test Fig.
- This invention is a process for the isolation ,characterization and cell culture adaptation of an Indian isolate of Hepatitis A virus as a candidate strain for vaccine production using Vero and MRC-5 cells.
- the Hepatitis A virus is one of the difficult viruses to adapt for culture in cells. Traditionally, the virus is isolated from the stool of the infected persons by passage in primates like marmoset and then the virus from the animal tissue is sub-cultured using primary cell cultures. From the primary cultures, the virus is further adapted to grow in other cell cultures.
- the present process describes the adaptation of the Indian isolate of Hepatitis A in a culture of Buffalo Green Monkey Kidney (BGMK) cells to establish a persistent infection and its characterization. Subsequent process of adaptation to Vero and MRC-5 cells for large scale culture, inactivation, purification etc. for the production of inactivated Hepatitis A vaccine. The following step-wise description of the process will help in better understanding of the invention. Stage I
- a 10 Years old Male patient suffering from nausea, vomiting, fever and icterous was diagnosed by Sero-immunological tests to be positive for Hepatitis A and negative for Hepatitis E.
- the feacal sample from the patient was also found to be positive for Hepatitis A indicating shedding of virus.
- a 10% suspension of the fecal sample was used as an inoculum to infect BGMK cells and the infected cells were serially sub-cultured at 7-10 days interval to establish a persistant type of infection.
- the cell bound virus at various passage levels was released by freeze thawing a portion of the cells and used for various tests.
- RT-PCR Reverse Transcriptase Polymerase Chain Reaction
- RT-PCR was also carried out on lysates of BGMK cells (passage # 6) inoculated with fecal sample using primers from VP1/2A (hypervariable - 2799- 3296) and P2 (3204-4997) regions of HAV genome. PCR products of 157 bp; 497 bp; and 1793 bp in size were detected on agarose gel respectively.
- Figures 2 A, 2B and 2C herein below are the photographs of the said detection. Detection of HAV-RNA by RT-PCR Fig 2A
- Lane 1,2,3 RNA from Hepatitis A patient's fecal sample and from BGMK cells inoculated with fecal sample at passages 6 and passage#24 respectively (RNA Pol region)
- Lane 4 Molecular weight marker: ⁇ X 174/Hae III
- Lane 1 Molecular weight marker: ⁇ X 174/Hae III
- Lane 2C RNA from BGMK cells inoculated with fecal sample at passage # 6 and passage # 24 respectively (VP1/2A region) Fig. 2C
- Lane 1 Molecular weight marker Lambda/Hind III
- Lane 2 RNA from BGMK cells inoculated with fecal sample at passage#6 (P2 region)
- RNA Polymerase region The dendogram shown in Fig 3 A depicts phylogenetic status of 16 representative strains including currently available hepatitis A vaccine strains in 159bp segment of RNAP region.
- the vaccine strains included HM 175 (Accession No. M14707 HAVRIX, SKB, USA) CR326 (Accession No. M10033, VAQTA, Merck, USA) GBM (Accession No. X75215, AVAXIM, Pasteur Merieux, France).
- the second change T6527C was non synonymous and resulted in the replacement of amino acid, phenylalanine by serine.
- the culture adapted NIVIN97/P6 strain showed A at position 6522 similar to attenuated HM175 strain (Accession No. 16632) and no change in amino acid sequence.
- Fig. 3B
- Figure 4A Phylogram illustrates the genetic relationship between culture adapted NIN (Indian) isolate of HAN ( ⁇ INT ⁇ 97/P6) in relation to other strains characterized previously over a 168 bp fragment of the NP1/2A junction region.
- nucleotide changes There were 12 nonsynonymous nucleotide changes at positions 3453 (A-»G), 3720 (G A), 3889 (C-»T), 3924 (G A), 4086 (A C) 4185 (G A), 4222 (T-»C) 4236 ( A-»G) 4237 (A T) , 4360 (A-»G), 4419 (T C) and 4896 (A ⁇ T) resulting in amino acid substitutions as isoleucine to valine , alanine to threonine , alanine to valine , valine to isolecuine, lysine to threonine, glutamic acid to lysine, phenylalanine to serine, lysine to glutamic acid, lysine to valine, asparagine to serine, tyrosine to histidine and methionine to leucine respectively.
- nucleotide changes there were 5 synonymous nucleotide changes at positions
- Lysates obtained by freezing and thawing of monolayered cells showed presence of antibody coated virus like particles, 27 nm in diameter in Vero cells at passage # 2, 4 and 10 as shown in Figure 6A, 6B and 6C hereunder.
- HAV antigen titre was noted to be between 1:32-1:64.
- Supematants of infected cultures were tested negative by ELISA indicating either absence or presence of antigen below the detection limits of ELISA test.
- RT-PCR was carried out on lysates of infected Vero cell cultures (Virus passage # 10) using HAV specific primers from (i) RNA polymerase region (6436-6593) and (ii) VP1/2A region (2799-3296). PCR products 157 bp and 497 bp in size were detected on agarose gel respectively. Detection of PCR products 157 bp and
- Lane 1 RNA from Vero cells inoculated with BGMK cell culture adapted NIVIN97 strain. Virus Passage. #10 (RNAPol region)
- Lane 2 Molecular weight marker: ⁇ X 174/Hae III
- Lane 3 RNA from Vero cells inoculated with BGMK cell culture adapted NININ97 strain. Virus Passage. #10 (VP1/2A region)
- Infectivity assay Virus recovered from cell lysates was found to be infectious when inoculated into normal Vero cells as evidenced by antigen capture assay.
- NIVIN97 strain of HAV adapted to Vero cell line The reactivities of NIVIN97 strain of HAN derived from BGMK cell line (virus passage 21 and 26) and Nero cell line (virus passage#10) were compared in (i) anti-HAN IgM capture ELISA test using known anti-HAV negative (21) and positive (15) sera and (ii) blocking ELISA test using known anti-HAV negative (20) and positive (20) sera.
- the study results of comparision of the reactivities of ⁇ IVL ⁇ 97 strain of HAN derived from BGMK cell line and Nero cell line in anti-HAN IgM capture ELISA test using anti-HAN negative and positive are shown in figure 10 in Sheet o.
- a proprietory lysis buffer of hypertonic buffer salts and a non-ionic detergent is filled and agitated for 30 mts. This results in complete lysis of all the cells the liquid contains the released virus particles along with the cell lysates.
- the harvest as above is collected into sterile centrifugation bottles. Clear viral harvests are obtained from the supernatant by low speed centrifugation of the harvest at 900 g for 30 mts.
- Stage IV Inactivation of Virus: The clear viral harvest as above is inactivated by the conventional process of formalin addition. Formalin solution (37% w/v) is added to the viral harvest at the rate of 1 : 1500 and stirred for about 12 to 18 hours at 37° C.
- Stage V Purification of inactivated viral antigen:
- the inactivated viral harvest as above contains impurities like cellular DNA and traces of media component proteins of bovine origin. These are separated from the viral antigen using a proprietary process of selective precipitation and re-suspension. According to this method, one part of a proprietary hydrophobic interaction matrix is added to 50 parts of the viral harvest and mixed for 5 minutes. The precipitate is then separated by centrifugation at 900 g for 15 minutes. The precipitate is then resuspended in a chelating buffered solution to selectively dissolve viral proteins.
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Abstract
An Indian isolate of Hepatitis A virus- NIVIN97 has been isolated, adapted to tissue culture, characterized and further propagated using Vero and MRC-5 cell lines for vaccine preparation. The method involves the cell culture adaptation of the virus isolate from clinical sample (faecal) in BGMK cell line initially , characterization of the virus and further adaptation to Vero and MRC-5 cells , scale-up ,inactivation and down stream processing method of the inactivated viral antigens for the preparation of an inactivated vaccine.
Description
A novel process for the Isolation of an Indian strain of Hepatitis A virus, adaptation to tissue culture and further cultivation for vaccine preparation The present invention further relates to a novel process for isolation, cell culture adaptation, characterization and further large scale culture of an Indian isolate of Hepatitis A virus in cell lines.
BACKGROUND OF THE INVENTION
Provost and Hilleman (1978) have demonstrated that Hepatitis A virus (HAV) particles purified from marmoset liver and inactivated with formaldehyde when injected to the susceptible primates is capable of inducing protection against virulent virus challenge. The development of Hepatitis A vaccines as live attenuated or inactivated vaccines has originated from the successful in-vitro culture of the virus in cell cultures by Provost and Hilleman (1979) using the HAV serially passaged in the marmosets as inoculum. Isolation of HAV from clinically ill patients' stools samples using cell cultures has been described in the literature (Provost etal., 1981; Frosner et.al., 1979; Flehming 1980). Several cell lines can be used for HAV isolation (Locarini et.al., 1981; Gauss-Muller et.al.,1981). The cell lines used include fetal rhesus monkey kidney (Wheeler et.al, 1986), primary African green monkey kidney (AGMK) cells(Daemer et.al., 1981) , transformed rhesus monkey kidney cells( de Chastonay and Siegel,1987) and human diploid fibroblasts (Binn et.al., 1986 and Andre et.al., 1990). Reports also include Vero cells and continuously growing AGMK cells. As the virus typically gets established as persistent infection and remains cell bound, the preparation of vaccine in a large scale poses several problems for
optimum yield and requirement of efficient downstream process. Vero and MRC- 5 cells have been used extensively for the production of viral vaccines like polio, rabies, rubella, Hepatitis A etc. The Vero and MRC-5 cells have also been successfully grown on microcarriers and various types of cell bioreactor systems. (van Wazel 1978).
OBJECT OF THE INVENTION:
To identify an Indian strain of Hepatitis A virus suitable for preparation of an inactivated vaccine and its characterization and adaptation to various cell lines to achieve the above objective. SUMMARY OF THE INVENTION:
The invention provides a method of isolation and adaptation to cell culture of an Indian isolate of Hepatitis A virus , its characterization and further adaptation to grow in Nero and MRC-5 cells for production of an inactivated vaccine. The later process of harvesting the virus from the culture poses a greater problem since the majority of the virus is cell bound. In order to obtain the virus the cells are required to be detached and lysed using suitable down stream process of extraction. Since the process of lysis adds to the contamination of cellular nucleic acids, the purification strategy shall also include selective separation of the DΝA contaminants besides cell debris. The present invention outlines the methodology for the commercial scale production of Hepatitis A vaccine using the culture of Nero and MRC5 cells with an Indian isolate of Nero cell adapted Hepatitis A virus (ΝINIΝ97 strain) and a subsequent harvest, purification and inactivation of the virus for production of Hepatitis A vaccine.
The invention method is also applicable and not limited to the production of other Hepatitis A virus isolates of similar genotypes or serotypes as well as other viruses like Rabies, Rotavirus, Polio etc. using the Vero or MRC5 cells as a substrate. It is also applicable and not limited to other cell substrates like WI-38, and FRhK cells. The method describes a commercially viable scale-up option for the production of viral vaccines using cell culture technology. The method also outlines the down stream processing details for the harvesting of the desired viral antigens. BRIEF DESRIPTION OF THE ACCOMPANYING DRAWINGS: Fig. 8 depicts Titration of MVTN97 strain of HAN adapted to BGMK cells by ELISA
Fig. 9 depicts Reactivity of virus tested in anti-HAN IgM capture ELISA using known anti HAN IgM negative (47) and positive (41) sera with ΝιNIΝ97 and HM-175 of HAN Fig. 10 depicts The reactivities of ΝIVIΝ97 strain of HAV derived from BGMK cell line and Vero cell line compared in anti-HAV IgM capture ELISA test Fig. 11 depicts The reactivities of NIVIN97 strain of HAV derived from BGMK cell line and Vero cell line compared in blocking ELISA test DETAILED DESCRIPTION OF THE INVENTION: This invention is a process for the isolation ,characterization and cell culture adaptation of an Indian isolate of Hepatitis A virus as a candidate strain for vaccine production using Vero and MRC-5 cells. The Hepatitis A virus is one of the difficult viruses to adapt for culture in cells. Traditionally, the virus is isolated from the stool of the infected persons by passage in primates like
marmoset and then the virus from the animal tissue is sub-cultured using primary cell cultures. From the primary cultures, the virus is further adapted to grow in other cell cultures. The present process describes the adaptation of the Indian isolate of Hepatitis A in a culture of Buffalo Green Monkey Kidney (BGMK) cells to establish a persistent infection and its characterization. Subsequent process of adaptation to Vero and MRC-5 cells for large scale culture, inactivation, purification etc. for the production of inactivated Hepatitis A vaccine. The following step-wise description of the process will help in better understanding of the invention. Stage I
A 10 Years old Male patient suffering from nausea, vomiting, fever and icterous was diagnosed by Sero-immunological tests to be positive for Hepatitis A and negative for Hepatitis E. The feacal sample from the patient was also found to be positive for Hepatitis A indicating shedding of virus. A 10% suspension of the fecal sample was used as an inoculum to infect BGMK cells and the infected cells were serially sub-cultured at 7-10 days interval to establish a persistant type of infection. The cell bound virus at various passage levels was released by freeze thawing a portion of the cells and used for various tests. 1.0 Antigen Capture ELISA 1.1 Cell ly sates from infected cultures were subjected to ELISA for detection and titration of HAV antigen according to the method described earlier (Chitambar et al (1994) Indian J Med Res. 99, 115-120). The cell lysates from normal cultures were included as negative controls in the tests. The ratio of test
sample Optical Density (OD) and known negative sample OD (S/N) equal to or greater than 2.1 was considered positive. The S/N ratio was directly proportional to the concentration of virus in the cell lysates and indicated differential amount of virus produced at different passage levels. The HAN antigen litres in the cell lysates at passage levels 4,6 and 7 were > 1 : 256. The Fig. 8 in the accompanying drawing represents the said study. 1.2. Serial passages of the virus resulted in persistent infection of BGMK cell line and indicated continuous production of virus. Table 1 herein below represents the said study. Table 1
Production of ΝINιΝ97stι-ain of HAV isolated in BGMK cell line
Passage Number S/N Ratio in antigen Titre capture ELISA
P-4 (1 : 2 dim) 38.7 1 : 256
P-6 (l : 2 diln) 64.9 > 1 : 256
P-7 (l : 2 diln) 101.: > 1 : 256
P-10 23.9 1 : 128
P-l l 19.2 1 : 128
P-12 7.4 1 : 32
P-13 9.5 1 : 64
P-14 19.4 1 : 128
P-15 18.4 1 : 64
P-16 10.6 1 : 64
1-17 23.9 1 : 256
P-19 12.5 1 : 32
P-21 9.8 1 : 64
P-23 28.5 1 : 256
P-24 8.0 1 : 64
P-25 4.9 1 : 16 I P-26 10.3 > 1 : 256
2. Anti-HAV IgM capture ELISA:
Cell lysates from infected cultures (passage # 4) showed no reaction with known anti-HAV IgM negative serum and strong positive reaction with known anti- HAV IgM positive serum (S/CO > 2).
3. Immune Electron Microscopy ( IEM ) :
Patient's fecal sample and cells infected with fecal sample were examined for the presence of virus particles.10% fecal suspension and cell lysates derived from persistently infected BGMK cells with virus passage # 24 were centrifuged at 25,000 m for 2 hrs in ultracentrifuge (Beckman Model L5-50) using SW 28 rotor after removal of debris. The pellets were subjected to IEM by the technique described previously (Kapikian et al (1972) J Virol 10, 1075-1081) using human serum (1 :20) known to contain anti-HAV antibodies. Antibody coated virus like particles, 27 nm in diameter, were visualized in both, fecal specimen as shown in Fig. 1A and cell lysates as shown in Fig. IB hereunder confirming the presence of HAV.
FiglA Fig IB
4. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR): RT-PCR was performed on the patient's fecal sample and lysates of BGMK cells (passages # 6 and 24) inoculated with the same fecal sample to detect HAV RNA according to the method described previously (Chitambar et al. (2001) Hepatology Res. 19, 237-246). For all runs of RT-PCR negative controls and HM-175 strain derived positive controls were subjected to the entire protocol. The primers used were specific to RNA polymerase (conserved - 6436-6593) region. RT-PCR was also carried out on lysates of BGMK cells (passage # 6) inoculated with fecal sample using primers from VP1/2A (hypervariable - 2799- 3296) and P2 (3204-4997) regions of HAV genome. PCR products of 157 bp; 497 bp; and 1793 bp in size were detected on agarose gel respectively. Figures 2 A, 2B and 2C herein below are the photographs of the said detection. Detection of HAV-RNA by RT-PCR
Fig 2A
Lane 1,2,3: RNA from Hepatitis A patient's fecal sample and from BGMK cells inoculated with fecal sample at passages 6 and passage#24 respectively (RNA Pol region)
Lane 4: Molecular weight marker: φX 174/Hae III
Fig 2B
Lane 1 : Molecular weight marker: φX 174/Hae III
Lane 2,3: RNA from BGMK cells inoculated with fecal sample at passage # 6 and passage # 24 respectively (VP1/2A region)
Fig. 2C
Lane 1 : Molecular weight marker Lambda/Hind III
Lane 2: RNA from BGMK cells inoculated with fecal sample at passage#6 (P2 region)
5. Sequencing: PCR amplified DNA fragments were purified by using Wizard (Promega, USA) mini-columns. Both the strands were sequenced using a Taq dye terminator cycle sequencing kit (Perkin Elmer, USA) and an automatic sequencer. 6. Genomic characterization of isolated NIVIN97 strain of HAV : The Phylogenetic status of NIV isolate of HAV [wild type (NIVIN97) and culture adapted NIVIN97 (P6)] was assessed employing the software MEGA version 2.1 For analysis, Jukes-Cantor (JC) distance was utilized employing the neighbor joining (NJ) algorithm. The reliability of different phylogenetic groupings was evaluated by using the bootstrap test (1000 bootstrap replications) available in MEGA. Scales indicated in figures 4,5 and 6 represent genetic distance. The internal node numbers represent percent bootstrap support. 6.1 RNA Polymerase region :
The dendogram shown in Fig 3 A depicts phylogenetic status of 16 representative strains including currently available hepatitis A vaccine strains in 159bp segment of RNAP region. The vaccine strains included HM 175 (Accession No. M14707 HAVRIX, SKB, USA) CR326 (Accession No. M10033, VAQTA, Merck, USA) GBM (Accession No. X75215, AVAXIM, Pasteur Merieux, France). Indian (NIV) isolate of HAV prior to (NTVIN97) and after adaptation in in vitro cell culture (NIVIN97/P6) exhibited genetic relatedness with wild type strains from Australia (HM175), North Africa (MBB) and China (LA-1) and differentiated itself from human strains from Germany (GBM), Italy (F.G.), Japan (AH) and simiar strains from Kenya and USSR. Fig.3A
Comparison of sequences of wild type (NINιN97) and culture adapted (NININ 97/P6) NIV strain and available hepatitis A vaccine strains within 159 nucleotide
segment of RNAP region according to the wild type HM175 HAN sequence (Cohen et al (1987) J Nirol 61, 50-59) is shown in Fig 3B. The percent nucleotide identity shown by wild type and culture adapted ΝIN strain was 98.4% and 99.4% respectively with HM175 strain. Wild type ΝJNIΝ97 strain showed 2 changes at nucleotide level, T6522A and T6527C. First change was synonymous (no change in amino acid). The second change T6527C was non synonymous and resulted in the replacement of amino acid, phenylalanine by serine. The culture adapted NIVIN97/P6 strain showed A at position 6522 similar to attenuated HM175 strain (Accession No. 16632) and no change in amino acid sequence. Fig. 3B
M14707(Australia) CAA CCT GTC CAA AAG ATG AAT TGA GAC CAT TAG AGA AAG TGT TGG AAT CAA AAA CAA GAG
M16632(USA) NIVIN97(India)
NIVIN97/P6(India)
X75215(Germany) ... .T T
M14707(Australia) CTA TTG ATG CTT GTC CTC TGG ATT ACT CAA TTT TGT GCC GAA TGT ATT GGG GTC CAG CTA M16632(USA) A NIVIN97(India) A ... .C NIVIN97 P6(India) A X75215(Germany) C. ..T TA C .A. .T
M14707(Australia) TTA GTT ATT TTC ATT TGA ATC CAG GTT TCC ATA CAG GTG
M16632(USA)
NIVIN97(India) - NIVIN97/P6(India)
Λ. /5 15(vιermany) (J. .1
6.2 VP1/2A region: Figure 4A Phylogram illustrates the genetic relationship between culture adapted NIN (Indian) isolate of HAN (ΝINTΝ97/P6) in relation to other strains characterized previously over a 168 bp fragment of the NP1/2A junction region.
Based on phylogenetic analysis within 168bp region of NP1/2A (Fig 4A) Indian ΝINIΝ97/P6 strain of HAV was placed in genotype IB. This analysis incoφorated 47 HAV isolates. These included hepatitis A vaccine strains: HM175 [wild type (Genotype IB, Accession No. M14707) and attenuated (Genotype IB, Accession No. Ml 6632)]; CR326 (Genotype IA, Accession No. Ml 0033) and GBM (Genotype IA, Accession No. X75215). Culture adapted NININ97/P6 strain (P6) showed 98.8 % nucleotide identity in comparison to control (HM175, Accession No. M14707) strain and G at positions 3149 and 3152 with no change in amino acid sequence. Nucleotide identity comparison to control (HM175, Accession No. M14707) strain and G at positions 3149 and 3152 is shown in Fig.4B below. Fig. 4B
M14707(Australia) GAA TCA ATG ATG AGC AGA ATT GCA GCT GGA GAC TTG GAG TCA TCA GTG GAT GAT CCT AGA M10033(CostaRica) ..G ..T T
M16632(USA) .T
NIVIN97 P6(India)
X75215(Germany) ..G ..T T
M14707(Australia) TCA GAG GAA GAT AAA AGA TTT GAG AGT CAT ATA GAA TGC AGG AAG CCA TAT AAA GAA CTG M10033(CostaRica) C .G T A T.. M16632(USA) NIVIN97/P6(India)
X75215(Germany) G ..C ,G T A C T..
M14707(AustraIia) AGA TTA GAA GTT GGG AAA CAA AGA CTC AAG TAT GCT CAG GAA GAA TTG M10033(CostaRica) G ..G T ..A G ...
M16632(USA)
NIVIN97/P6(India) G ..G
X75215(Germany) G ..G A G ...
6.3 P2 Region: Comparison of sequences within 1738 nucleotide fragment of P2 region of culture adapted NIN strain (ΝINIΝ97/P6) and other HAN strains has further confirmed the close genetic relationship between Australian (HM 175 Accession No. Ml 4707), North African (MBB, Accession No. M20273) and Indian isolate (NININ97/P6) of HAV. Fig 5Aand 5B represents the said comparitive study. The percent nucleotide identity of culture adapted NIVIN97/P6 strain with control (HM 175, Accession No. Ml 4707) strain was 99% (Fig.5B). There were 12 nonsynonymous nucleotide changes at positions 3453 (A-»G), 3720 (G A), 3889 (C-»T), 3924 (G A), 4086 (A C) 4185 (G A), 4222 (T-»C) 4236 ( A-»G) 4237 (A T) , 4360 (A-»G), 4419 (T C) and 4896 (A^T) resulting in amino acid substitutions as isoleucine to valine , alanine to threonine , alanine to valine , valine to isolecuine, lysine to threonine, glutamic acid to lysine, phenylalanine to serine, lysine to glutamic acid, lysine to valine, asparagine to serine, tyrosine to histidine and methionine to leucine respectively. In addition there were 5 synonymous nucleotide changes at
positions 3629 (A T), 3677 (T C), 3729 (T-»C), 3782 (C*T) and 4583 (T^A) without affecting amino acid sequence. Fig. 5A
Fig 5B.
M14707(Australia) AAG GGA CTG TTT TCA CAA GCC AAA ATT TCT CTT TTT TAT ACT GAG GAG CAT GAA ATA ATG
M16632(USA) NIVIN97/P6(India)
X75215(Germany) ..A ..G G.T G ..T
M14707(Australia) AAG TTT TCC TGG AGA GGT GTG ACT GCT GAT ACT AGA GCT TTA AGG AGG TTT GGA TTC TCT M16632(USA) NIVIN97 P6(India) X75215(Germany) ..A T A G ..A ..A
M14707(Australia) TTG GCC GCA GGC AGA AGT GTG TGG ACT CTT GAA ATG GAT GCT GGG GTT CTT ACT GGG AGA
M16632(USA)
NIVIN97/P6(India) X75215(Germany) A T ..T G A A ..G
M14707(Australia) CTG ATT AGA TTG AAT GAT GAG AAA TGG ACA GAA ATG AAG GAT GAC AAG ATT GTT TCA TTG
M16632(USA) NIVIN97/P6(India) G
X75215(Germany) T.. G.C A T A
M14707(Australia) ATT GAA AAG TTT ACA AGT AAC AAA TAT TGG TCC AAA GTG AAT TTC CCA CAT GGG ATG TTG M16632(USA)
NIVIN97/P6(India)
M14707(Australia) GAT CTT GAA GAA ATT GCT GCC AAT TCT AAG GAT TTT CCT AAC ATG TCT GAA ACG GAT TTG M16632(USA) NIVIN97 P6(India) X75215(Germany) A C ..A ..T G ..A
M14707(Australia) TGT TTC TTG CTG CAT TGG TTA AAT CCA AAG AAA ATT AAT TTA GCA GAT AGA ATG CTT GGA
M16632(USA)
NIVIN97 P6(India) T X75215(Germany) CA T CG C G
M14707(Australia) TTG TCT GGA GTT CAG GAA ATT AAA GAA CAA GGT GTT GGA TTA ATA GCA GAG TGT AGA ACT
M16632(USA) NIVIN97/P6(India) C
X75215(Germany) G G CG
M14707(Australia) TTC TTA GAT TCT ATT GCT GGA ACT TTA AAA TCT ATG ATG TTT GGA TTT CAT CAT TCT GTG M16632(USA)
NIVIN97/P6(India) A C
X75215(Germany) G G ... CG C
M14707(AustraIia) ACT GTT GAA ATT ATA AAC ACT GTG CTC TGT TTT GTT AAG AGT GGA ATT TTG CTT TAT GTA M16632(USA) NIVIN97/P6(India) T X75215(Germany) T T ..C G ..C T
M14707(Australia) ATA CAA CAA TTG AAT CAG GAT GAA CAT TCT CAC ATA ATT GGT TTG TTG AGA GTC ATG AAT
M16632(USA)
NIVIN97 P6(India) X75215(Germany) C ..A C T
M14707(Australia) TAT GCA GAT ATT GGT TGT TCA GTT ATT TCA TGT GGC AAA GTT TTT TCC AAA ATG CTG GAA
M16632(USA) ... .T C NIVIN97 P6(India) ... .T A
X75215(Germany) ... .T C ..C T A ...
M14707(Australia) ACA GTC TTT AAT TGG CAA ATG GAC TCC AGA ATG ATG GAG TTA AGG ACT CAG AGT TTT TCC M16632(USA)
NIVIN97/P6(India)
X75215(Germany) T CG ..A C
M14707(AustraIia) AAC TGG TTA AGA GAT ATT TGT TCT GGG ATC ACC ATT TTT AAA AAC TTC AAG GAT GCA ATT
M16632(USA) C NIVIN97/P6(India) X75215(Germany) ..T C A ..A ..T ..T ..C GT ..T ..A
M14707(Australia) TAT TGG CTT TAT ACA AAA TTA AAG GAC TTT
TAT GAA GTG AAT TAT GGC AAG AAG AAG GAC
M16632(USA) T
NIVIN97/P6(India) C X75215(Germany) A T A A T
M14707(Australia) ATT TTA AAT ATT CTT AAA GAT AAC CAA CAA
AAA ATA GAG AAA GCC ATT GAG GAA GCC GAT
M16632(USA)
NIVIN97/P6(India) X75215(Germany) G.. C.T C C ..T .GG A A ..G a* ^
M14707(Australia) GAA TTT TGC ATT TTG CAA ATC CAA GAT GTG GAA AAA TTT GAA CAG TAT CAG AAA GGG GTT
M16632(USA) A C NIVIN97/P6(India) A C GT
M14707(Australia) GAC TTG ATA CAA AAA TTG AGA ACT GTT CAT TCA ATG GCT CAG GTT GAT CCA AAT TTA ATG M16632(USA) NIVIN97/P6(India)
GG.
M14707(Australia) GTT CAT TTG TCA CCT TTG AGA GAT TGT ATA GCA AGA GTT CAT CAG AAA CTT AAA AAC CTT
M16632(USA)
NIVIN97/P6(India) G. ...
••!_ **vτ •• X
M14707(Australia) GGA TCT ATA AAT CAG GCA ATG GTA ACG AGA TGT GAG CCA GTT GTT TGT TAT TTA TAT GGC
M16632(USA) , NIVIN97/P6(India) C
X75215(Germany) T G ..A C G AT. ...
M14707(AustraIia) AAA AGA GGG GGA GGA AAG AGC TTA ACA TCA ATT GCA TTG GCA ACC AAA ATT TGT AAA CAT M16632(USA)
NIVIN97/P6(India) N
X75215(Germany) T ..G ..T
M14707(Australia) TAT GGT GTT GAG CCT GAA AAG AAT ATC TAT ACT AAA CCT GTG GCT TCA GAT TAC TGG GAT M16632(USA) NIVIN97 P6(India) X75215(Germany) A G ..A T ..C C T
M14707(Australia) GGA TAT AGT GGA CAA TTA GTT TGC ATC ATT GAT GAT ATT GGC CAA AAC ACA ACA GAT GAG
M16632(USA) A
NIVIN97/P6(India) A .Λ. /3.ώx3l rcf u αXT I *« X ••v-> ••• ••• ••• ••• ••• •• X ••-cV ••• ••• ••• •■ ^ •■■ ••• •• X ••• ••• ••• **r
M14707(Australia) GAT TGG TCA GAT TTT TGT CAG TTA GTG TCA GGA TGT CCA ATG AGA TTA AAC ATG GCC TCT
M16632(USA)
NIVIN97/P6(India) X75215(Germany) C A G ..T T ...
M14707(AustraIia) CTT GAG GAG AAG GGT AGG CAT TTT TCT TCT
CCT TTT ATA ATA GCA ACT TCA AAT TGG TCA
M16632(USA) NIVIN97/P6(India)
X75215(Germany) C ..A C
M14707(Australia) AAT CCA AGT CCA AAA ACA GTT TAT GTT AAG GAA GCA ATT GAC CGC AGA CTC CAT TTC AAG M16632(USA) NIVIN97 P6(India) X75215(Germany) G T ..T ..G ..T T ...
M14707(Australia) GTT GAA GTT AAA CCT GCT TCA TTT TTC AAA AAT CCT CAC AAT GAT ATG TTG AAT GTT AAT
M16632(USA)
NIVIN97 P6(India) X75215(Germany) C
M14707(Australia) TTA GCT AAA ACA AAT GAT GCA ATC AAA GAT ATG TCT TGT GTT GAT TTG ATA ATG GAT GGA
M16632(USA) NIVIN97/P6(India) T
X75215(Germany) ..G T ..G ..C CA
M14707(Australia) CAT AAT GTT TCA TTG ATG GAT TTG CTC AGT TCT TTA GTC ATG ACA GTT GAA ATT AGA A M16632(USA)
NIVIN97 P6(India)
X75215(Germany) A A ..T C G G .
7. Validation of the reactivity of isolated, culture adapted NTvTN97 strain of HAV BGMK cell line adapted NIVIN97 strain of HAN was grown at a large scale. Pool of virus at passages 13, 14 and 15 was exposed to formalin at 1: 4000 dilution for 72 hrs. The reactivity of virus was tested in anti-HAV IgM capture ELISA using known anti-HAV IgM negative (47) and positive (41) sera. There
were no false negative or positive reactions with N1NLN97 when compared with that of Australian strain (HM-175) of HAV. Fig. 9 in sheet one of the accompanying drawing represents the said study. Stage II The lysate of the 24th passage from BGMK infected culture was used as an inoculum to infect Vero cells and the infected cells were serially sub-cultured through 10 passages at 7-10 days interval to establish a persistant type of infection. The cell bound virus at various passage levels was released by freeze thawing a portion of the cells and used for various tests. 1. Immune Electron Microscopy (IEM)
Lysates obtained by freezing and thawing of monolayered cells showed presence of antibody coated virus like particles, 27 nm in diameter in Vero cells at passage # 2, 4 and 10 as shown in Figure 6A, 6B and 6C hereunder.
Fig 6A Fig 6B
X87,000 X87,000
X105,000 2. Antigen Capture ELISA
Presence of HAV antigen was monitored in Vero cells at each passage level by antigen capture ELISA. Sample OD / Negative control OD (S/N) ratio values were in the range of 8.65 - 31.35 as shown in Table II hereunder. HAV antigen titre was noted to be between 1:32-1:64. Supematants of infected cultures were tested negative by ELISA indicating either absence or presence of antigen below the detection limits of ELISA test. Table II
Production of NIVIN97 strain of HAV in Vero cell line
Serial Passage Level S/N Ratio* in Antigen & Period of cultivation Capture ELISA
P-1/3WK 10.36-13.48
P-2/2WK 8.65
P-3/3-4WK 12.96-31.35
P-4/2WK 18.89-19.25 P-5/3WK 28.63
P-6/3WK 29.72
P-7/2WK 20.88
P-9/2WK 16.79
P-10/-2WK 15.62
* S/ N ratio : Test sample OD / Neg. Control OD
3. RT-PCR:
RT-PCR was carried out on lysates of infected Vero cell cultures (Virus passage # 10) using HAV specific primers from (i) RNA polymerase region (6436-6593) and (ii) VP1/2A region (2799-3296). PCR products 157 bp and 497 bp in size were detected on agarose gel respectively. Detection of PCR products 157 bp and
497bp in size on agarose gel carried out on lysates of infected Vero cell cultures is shown in figure 7 hereunder. Fig 7
Detection of
Lane 1: RNA from Vero cells inoculated with BGMK cell culture adapted NIVIN97 strain. Virus Passage. #10 (RNAPol region)
Lane 2: Molecular weight marker: φX 174/Hae III
Lane 3: RNA from Vero cells inoculated with BGMK cell culture adapted NININ97 strain. Virus Passage. #10 (VP1/2A region)
4. Infectivity assay : Virus recovered from cell lysates was found to be infectious when inoculated into normal Vero cells as evidenced by antigen capture assay.
5. Validation of the reactivity of NIVIN97 strain of HAV adapted to Vero cell line: The reactivities of NIVIN97 strain of HAN derived from BGMK cell line (virus passage 21 and 26) and Nero cell line (virus passage#10) were compared in (i) anti-HAN IgM capture ELISA test using known anti-HAV negative (21) and positive (15) sera and (ii) blocking ELISA test using known anti-HAV negative (20) and positive (20) sera. The study results of comparision of the reactivities of ΝIVLΝ97 strain of HAN derived from BGMK cell line and Nero cell line in anti-HAN IgM capture ELISA test using anti-HAN negative and positive are shown in figure 10 in Sheet o. 2 of the accompanying drawings. Similarly the test results of blocking ELISA test using known anti-HAN negative and positive sera are shown in figure 11 in Sheet Νo.2 of the accompanying drawings. No false negative or positive reactions were noted in both tests and for both the preparations of HAN. Nero cells persistently infected with Ν1NIΝ97 strain of HAN were grown in Roux bottles and 225 cm2 plastic culture flasks and roller bottles under the conditions described in Stage II. Similar adaptation was also done using the MRC-5 cells Stage III : Virus harvest
HAV is known to be predominantly cell bound. Hence the virus is harvested by lysis of the cells. The cells are washed once with Phosphate buffered saline by gentle perfusion for an hour. Then a proprietory lysis buffer of hypertonic buffer salts and a non-ionic detergent is filled and agitated for 30 mts. This results in complete lysis of all the cells the liquid contains the released virus particles along with the cell lysates. The harvest as above is collected into sterile centrifugation bottles. Clear viral harvests are obtained from the supernatant by low speed centrifugation of the harvest at 900 g for 30 mts. Stage IV : Inactivation of Virus: The clear viral harvest as above is inactivated by the conventional process of formalin addition. Formalin solution (37% w/v) is added to the viral harvest at the rate of 1 : 1500 and stirred for about 12 to 18 hours at 37° C. Again a second dose of formalin at the same rate is then added and the stirring at 37° C continued for additional 36 hours for complete inactivation. Stage V : Purification of inactivated viral antigen: The inactivated viral harvest as above contains impurities like cellular DNA and traces of media component proteins of bovine origin. These are separated from the viral antigen using a proprietary process of selective precipitation and re-suspension. According to this method, one part of a proprietary hydrophobic interaction matrix is added to 50 parts of the viral harvest and mixed for 5 minutes. The precipitate is then separated by centrifugation at 900 g for 15 minutes. The precipitate is then resuspended in a chelating buffered solution to selectively dissolve viral proteins. Another centrifugation at 900 g for 15 minutes separates the insoluble cellular and other proteins from the viral proteins which
are obtained in the supernatant. The supernatant so obtained is buffer exchanged and further concentrated using a 100 K tangential flow membrane filtration devise.
Claims
1. A process for the preparation of Hepatitis A vaccine from a Hepatitis A virus collected from a patient as sample, characterized as genotypically different but serologically similar to the existing vaccine virus strain, grow the virus as a persistent infection in cell culture followed by plurality of subcultures and subsequent use of the lysates of the persistently infected cells for infecting large quantity of vaccine approved cell substrates, breaking of the cells using a lysis buffer herein desribed, inactivation of the virus and purification of the inactivated viral harvests using HiMAX technology followed by Ion exchange chromatography to prepare the final vaccine.
2. The process as claimed in claim 1 wherein the Hepatitis A strain isolated has genetically exhibited relatedness( 85 to 98% in the important neucleotide sequences) with wild type strains from Australia , North Africa and China and differentiated itself from human strains from Germany , Italy , Japan and simian strains from Kenya and USSR but exhibited close serological relatedness with the available vaccine strain HM-175.
3. The process as claimed in claim 1 wherein the Hepatitis A strain is essentially an Indian origin strain( NIVIN97).
4. The process of claimed in claim 1 wherein the number of subcultures during the virus adaptation to a cell line ranges from 2 to 30.
5. The process of claimed in claim 1 and 4 wherein the Hepatitis A virus in any cell culture is allowed to grow from 7 to 26 days
6. The process of claimed in claim 1 and 5 wherein the Hepatitis A virus in any cell culture is allowed to grow at least 10 days.
7. The process of claimed in claim 1 and 3 wherein the cell line for adaptation and subsequent large scale culture is selected from BGMK, AGMK, VERO, MRC5, WI-38 or FRhK cells
8. The process of claimed in claim 1 and 4 wherein the cell lysis for virus release is carried out using lysis buffer.
9. The process of claimed in claim 8 wherein the lysis buffer is a phosphate buffer containing sodium chloride at 0.5 to 1 M and Tween 20 at 0.02 to 0.1 % concentrations.
10. The process of claimed in claim 1 and 4 wherein the inactivated Hepatitis A virus is purified using the HIMAX technology.
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| EP05709163A EP1709162A2 (en) | 2004-01-27 | 2005-01-19 | A novel process of hepatitis a vaccine preparation |
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| IN60/CHE/2004 | 2004-01-27 | ||
| IN60CH2004 | 2004-01-27 |
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| WO (1) | WO2005070454A2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816786A (en) * | 2010-04-30 | 2010-09-01 | 长春生物制品研究所 | Inactivated hepatitis A vaccine and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0583142A2 (en) * | 1992-08-07 | 1994-02-16 | Merck & Co. Inc. | Hepatitis A virus vaccine |
| US5549896A (en) * | 1991-05-08 | 1996-08-27 | Schweiz. Serum- & Impfinstitut Bern | Hepatitis a virus strain, method for the isolation of new hepatitis a virus strains and hepatitis a vaccines |
| WO2000023574A2 (en) * | 1998-10-16 | 2000-04-27 | Smithkline Beecham Biologicals S.A. | Hepatitis a vaccines |
| WO2003049766A2 (en) * | 2001-12-10 | 2003-06-19 | Baxter Healthcare S.A. | Method of purifying hepatitis a virus particles and vaccine preparation |
-
2005
- 2005-01-19 EP EP05709163A patent/EP1709162A2/en not_active Withdrawn
- 2005-01-19 WO PCT/IN2005/000020 patent/WO2005070454A2/en not_active Application Discontinuation
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5549896A (en) * | 1991-05-08 | 1996-08-27 | Schweiz. Serum- & Impfinstitut Bern | Hepatitis a virus strain, method for the isolation of new hepatitis a virus strains and hepatitis a vaccines |
| EP0583142A2 (en) * | 1992-08-07 | 1994-02-16 | Merck & Co. Inc. | Hepatitis A virus vaccine |
| WO2000023574A2 (en) * | 1998-10-16 | 2000-04-27 | Smithkline Beecham Biologicals S.A. | Hepatitis a vaccines |
| WO2003049766A2 (en) * | 2001-12-10 | 2003-06-19 | Baxter Healthcare S.A. | Method of purifying hepatitis a virus particles and vaccine preparation |
Non-Patent Citations (3)
| Title |
|---|
| ANDRE F E ET AL: "INACTIVATED CANDIDATE VACCINES FOR HEPATITIS A" PROGRESS IN MEDICAL VIROLOGY, KARGER, BASEL, CH, vol. 37, no. 1, 1990, pages 72-95, XP000904877 ISSN: 0079-645X * |
| KOFF R S: "Hepatitis A" LANCET THE, LANCET LIMITED. LONDON, GB, vol. 351, no. 9116, 30 May 1998 (1998-05-30), pages 1643-1649, XP004833427 ISSN: 0140-6736 * |
| VAIDYA SUNIL R ET AL: "Polymerase chain reaction-based prevalence of hepatitis A, hepatitis E and TT viruses in sewage from an endemic area." JOURNAL OF HEPATOLOGY, vol. 37, no. 1, July 2002 (2002-07), pages 131-136, XP002340517 ISSN: 0168-8278 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816786A (en) * | 2010-04-30 | 2010-09-01 | 长春生物制品研究所 | Inactivated hepatitis A vaccine and preparation method thereof |
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| WO2005070454A3 (en) | 2005-11-03 |
| EP1709162A2 (en) | 2006-10-11 |
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