Table 3.17. Results of sensory, density and sterility evaluation of 10 JECEVAX vaccine batches
Vaccine batch
Sense | Density (volume) | Aseptic | ||||
N | % obtain | N | g/ml | N | % obtain | |
J-VC0110 | 3 | 100 | 3 | 1.12 | 20 | 100 |
J-VC0210 | 3 | 100 | 3 | 1.13 | 20 | 100 |
J-VC0310 | 3 | 100 | 3 | 1.13 | 20 | 100 |
J-VC010313 | 3 | 100 | 3 | 1.14 | 20 | 100 |
J-VC020713 | 3 | 100 | 3 | 1.13 | 20 | 100 |
J-VC030713 | 3 | 100 | 3 | 1.13 | 20 | 100 |
J-VC040713 | 3 | 100 | 3 | 1.12 | 20 | 100 |
J-VC031114 | 3 | 100 | 3 | 1.14 | 20 | 100 |
J-VC010416 | 3 | 100 | 3 | 1.13 | 20 | 100 |
J-VC010818 | 3 | 100 | 3 | 1.12 | 20 | 100 |
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Table 3.18. Results of chemical index testing of 10 batches of JECEVAX vaccine
Vaccine batch
Thimerosal (µg/ml) | Formaldehyde (µg/ml) | Protein TS(µg/ml) | Residual DNA (ng/ml) | pH | |
Standard (WHO TRS 963) | ≤ 120 µg/ml | ≤ 10 µg/ml | ≤ 80 µg/ml | ≤ 10 ng/ml | 6.8-7.4 |
J-VC0110 | 53.3 | 0.34 | 9.89 | < 5 | 7,061 |
J-VC0210 | 49.3 | 0.71 | 6.3 | < 5 | 7,079 |
J-VC0310 | 48.7 | 0.22 | 5.35 | < 5 | 6,967 |
J-VC010313 | 47.0 | 0.086 | 10.43 | < 5 | 7,078 |
J-VC020713 | 49.0 | 0.11 | 3.16 | < 5 | 7,109 |
J-VC030713 | 49.0 | 0.14 | 5.63 | < 5 | 7.11 |
J-VC040713 | 48.0 | 0.1 | 9.26 | < 5 | 7,113 |
J-VC031114 | 52.0 | 0.27 | 8.46 | < 5 | 7,045 |
J-VC010416 | 45.7 | 0.27 | 4.43 | < 5 | 7,016 |
J-VC010818 | 50.1 | 0.57 | 8.18 | < 5 | 7,023 |
The physical-chemical, sensory and sterility indicators of 10 batches of JECEVAX vaccine all met the requirements according to WHO standards (WHO TRS 963).
3.1.2.3. Immunogenicity results of Japanese Encephalitis vaccine JECEVAX on experimental animals
Table 3.19. Results of immunogenicity assessment (efficacy/neutralizing antibody titer (NTI)) of JECEVAX vaccine batches in experimental animals
Target
HGKTTH of the test vaccine (log10 n ) | HGKTTH of standard vaccine BR209 (log10 n ) | Correlated efficacy (test sample/standard sample BR209) | HG VR (log FPU/ml) | Year of manufacture | |
Method | DDVN IV, 2009, WHO TRS 963 | ||||
J-VC0110 | 2,359 | 2,304 | 1,024 | 7.59 | 2010 |
J-VC0210 | 1,907 | 1,772 | 1,076 | ||
J-VC0310 | 2,260 | 2,196 | 1,029 | ||
J-VC010313 | 2,053 | 1,910 | 1,075 | 7.57 | 2013 |
J-VC020713 | 1,729 | 1,678 | 1,030 | ||
J-VC030713 | 1,700 | 1,678 | 1,013 | ||
J-VC040713 | 1,713 | 1,678 | 1,021 | ||
J-VC031114 | 1,772 | 1,689 | 1,049 | 7.57 | 2014 |
J-VC010416 | 1,867 | 1,629 | 1,146 | 7.58 | 2016 |
J-VC010818 | 2,069 | 1,875 | 1,103 | 7.54 | 2018 |
Medium | 1,943 | 1,841 | 1,057 | 7.57 | / |
Vaccine efficacy was calculated by the neutralization index log10 n , n is the result of quantifying neutralizing antibodies using the 50% necrosis reduction neutralization technique (PRNT 50 ). The neutralizing antibody titers of the 10 JECEVAX vaccine lots were all higher than the neutralizing titers of the standard vaccine (the minimum allowable limit of the reference HGKTTH test according to the JE-CEVAX vaccine (Biken) is ≥ 1.3 log) (Table 3.19).
10/10 vaccine series had neutralizing antibody titers in the test sample/standard sample BR209 correlation >1. Meeting the requirements of WHO (vaccine sample ≥ standard sample).
Vaccine neutralizing antibody titer results
JECEVAX and VX standard model BR209
2,500
2,000
1,500
1,000
500
0
CEJEVAX
BR209
JECEVAX vaccine batch
Antibody titer
neutral (log PRNT 50 )
Figure 3.8. Neutralizing antibody titers of JECEVAX vaccine and BR209 standard vaccine
Relative efficacy results of JECEVAX vaccine compared
1,200
1,150
1,100
1,050
1,000
950
900
with standard (>=1.0)
effective
JECEVAX vaccine correlation
Effective standards
correlate
JECEVAX vaccine batch
Correlated effects
The results of the immunogenicity study on experimental animals (efficacy) of 10 batches of VNNB JECEVAX vaccine showed that they were all higher than the standard sample vaccine BR209, the immunogenicity of JECEVAX achieved from 1.7 - 2.36 log compared to the basic standard> 1.3 log (Figure 3.8 and Table 3.19).
Figure 3.9. Correlative efficacy of JECEVAX vaccine and standard vaccine BR209
The research results in Figure 3.9 show that the relative efficacy of 10 JECEVAX vaccine lots compared to the standard vaccine BR209 are all >1.0 and range from 1.013 - 1.146.
Laboratory-scale research results show that the quality of the BV-WSV-0310 strain and the vaccine produced from this Beijing-1 strain both meet the requirements.
of WHO (WHO TRS 963). That is the scientific basis to convince the state to fund human trials at a 3-phase clinical scale according to WHO guidelines.
3.1.3. Developing quality standards for strains used to produce inactivated VNNB vaccine on Vero cells in Vietnam
Table 3.20. Results of research on quality standards of strains used to produce inactivated VNNB vaccine on Vero cells in Vietnam
STT
Test name | Method | WHO Standards | Building TCCS | Apply | |
1 | Sterility testing | Direct culture on Thioglycolate and soybean casein medium | Aseptic | No bacterial and fungal growth on both media after 14 days of culture | Applicable in strain registration and routine testing of strains before and during vaccine production |
2 | Mycoplasma testing | Direct culture on PPLO agar medium | Not detected Mycoplasma | No growth of Mycoplasma after 21 days of follow-up | Applicable in strain registration and routine testing of strains before and during vaccine production (if necessary) |
3 | Testing for foreign viruses in mice | Intracerebral and intraperitoneal injection, follow-up for 21 days | At least 80% of mice lived healthy lives, without any signs of disease. | At least 80% of mice lived healthy lives, without any signs of disease. | Applicable in strain registration and routine testing of strains before and during vaccine production (if necessary) |
4 | Testing for foreign viruses in rat nests | Intracerebral and intraperitoneal injection, follow-up for 14 days | At least 80% of mice lived healthy lives, without any signs of disease. | At least 80% of mice lived healthy lives, without any signs of disease. | Applicable in strain registration and routine testing of strains before and during vaccine production (if necessary) |
5 | Testing for foreign viruses in guinea pigs | Intraperitoneal injection, follow-up for 42 days | At least 80% of mice lived healthy, internal organs were normal, no signs of disease | At least 80% of mice lived healthy, internal organs were normal, no signs of disease | Applicable in strain registration and routine testing of strains before and during vaccine production (if necessary) |
6
Testing of foreign viruses of the strain on cells | Infection of neutralised virus suspension with anti-VNNB Beijing-1 serum on Vero, MRC5, BHK21 cells After 14 days, remove the supernatant and check for red blood cell adsorption virus. | No cytotoxicity (CPE) and no pink-adsorbed virus in the sample-contaminating cell bottles, no hemagglutinating virus in the sample | No cytotoxicity (CPE) and no pink-adsorbed virus in the sample-contaminating cell bottles, no hemagglutinating virus in the sample | Applicable in strain registration and routine testing of strains before and during vaccine production (if necessary) | |
7 | Identification | ELISA | Not given | Sample containing VNNB virus antigen (OD is yellow) | Applicable in strain registration and routine testing of strains before and during vaccine production |
8 | Identification | PCR (GTTG) | Not given | % E gene similarity ≥ 95% compared to standard strain | Applicable in strain registration or routine inspection |
9 | Titration virus on BHK21/Ver o cells | Formation of necrotic clusters (PFU) on BHK21 cells/ Vero | Not given | ≥ 6 log PFU/ml (or ≥ 106 PFU/ml) | Applicable in strain registration and routine testing of strains before and during vaccine production |
Table 3.21. Establishing standards for the number of generations of transfer, transferred cells and storage temperature of original strains and production strains
STT
Standard office | Recommendation | ||
Original strain | Production strain | ||
1 | Number of transplants | - Maximum 6 generations, including transplanted generations in Vietnam - Maximum 47 generations, based on Strain Profile from manufacturer (NIH, Japan) | - Maximum 7 generations, including transplanted generations in Vietnam - Maximum 48 generations, based on Strain Profile from manufacturer (NIH, Japan) |
2 | Type of cell culture transfer | Cell type: Vero | |
Number of cell lifetimes: Maximum 150 | |||
3 | Storage conditions | Prioritize storage conditions in order: - Liquid nitrogen - - 20 o C/3 months - 2-8 o C/3 days | |
Table 3.22. Development of standards for genetic stability and titer of parent and production strains
STT
Basic standards | Recommendation | |
1 | Genetically stable | E antigen nucleotide sequence homology with Reference JEV Beijing-Kanonji standard (≥95%) |
The amino acid sequence of the E antigen biosynthetic gene region is similar to the Reference JEV Beijing-Kanonji standard sample (≥95%) | ||
The nucleotide sequence of the E antigen is similar to Japanese Encephalitis Virus strains isolated in Vietnam (≥85%) | ||
The amino acid sequence of the E antigen biosynthetic gene region is similar to Japanese Encephalitis Virus strains isolated in Vietnam (≥85%) | ||
2 | Stable in price | Viral titer maintained ≥ 6.0 logPFU/ml (or ≥ 10 6 PFU/ml) |
From the results of culturing and producing the original strain BV-MSV-0210 and the production strain BV-WSV-0310 of VNNB virus from the time of establishment to the time of research, the recommended standard for the maximum number of culturing generations of the original strain is 6 generations and the production strain is 7 generations. The cell type used for culturing VNNB virus is Vero cells with the maximum number of subculture generations being 150 generations. The original strain BV-MSV-0210 and the production strain BV-WSV-0310 will gradually decrease in virus titer when stored over time. The preferred storage conditions are liquid nitrogen, - 60 o C/3 months and - 20 o C/3 days, respectively (Table 3.21).
3.2. Discussion
3.2.1. Stability of titer and genetics of Japanese Encephalitis virus strain after production and storage conditions
The development history of Japanese encephalitis vaccine has gone through many stages with different production techniques such as inactivated vaccine cultured from mouse brain, inactivated vaccine cultured on Vero cells, live attenuated vaccine cultured on Vero cells [4, 25, 33]. Regardless of the technology used in production, the quality of the virus strain used in production is a decisive factor in ensuring the quality of the vaccine, especially the ability to generate immunity to protect the body [5]. Based on the epidemiological situation of the disease, the VNNB virus strain is selected, isolated and produced into
Batches of pure virus strains, fully meeting the conditions for vaccine strains, are preserved for a long time to serve vaccine production. Batches of original standard virus strains are permitted by WHO to be distributed to countries and/or manufacturers. Here, the original virus strain (MSV) and the production virus strain (WSV) continue to be cultured through many generations and preserved in suitable conditions such as liquid nitrogen to serve the vaccine production process for a long time. The storage process at different temperature conditions has certain risks affecting the stability of the titer and genetics of the virus strain [5, 51]. Therefore, during the post-production preservation of strains, strains need to be periodically tested for factors related to virus titer, genetics and some properties that affect the quality of the strain used in vaccine production.
This is the first study to comprehensively and systematically evaluate the quality of the parent strain and production strain used in the production of inactivated VNNB vaccine on Vero cells in Vietnam. In this study, the parent strain (BV-MSV-0210) and production strain (BV-WSV-0310) of VNNB virus were subcultured from the Beijing-1 Kanonji standard strain. After production, the parent strain (BV-MSV-0210) and production strain (BV-WSV-0310) were stored in liquid nitrogen to produce VNNB vaccine JECEVAX. Identification verification is one of the indispensable tests when evaluating the quality of strains after production and during storage. Immediately after production, this test was also carried out in parallel with the original strain batch BV-MSV-0210 and the production strain batch BV-WSV-0310 and compared with the original standard strain (P0) (Reference JEV Beijing-1) provided by Kanonji, Japan, which was used to produce the two strain batches used in the study. Total RNA was extracted and purified from each sample, using the QIAamp Viral RNA Mini kit (Qiagen). The quality of the double-stranded DNA library was checked with a Bioanalyzer using a High Sensitivity Chip (Agilent Technologies). The results of all tests showed that the nucleotide sequences and amino acid sequences of the E antigen biosynthesis gene region of these two strains were 100% similar and compared to the reference strain Beijing-1 Kanonji, they were also 100% similar. With this result, it can be initially confirmed that the original batches of strains and the VNNB vaccine production strains of Vabiotech after 7 generations of subcultivation remained stable and unchanged. This result also confirmed that the production and preservation processes in this study ensured the quality of the original batches of strains and
Production strain. Confirm that the conditions of cell lines, cell culture generations, equipment, and environment in Vietnam meet the requirements for producing parent strains and production strains for inactivated VNNB vaccines produced on Vero cells.
Stability of viral titer
The inoculum tubes of the original strain batch BV-MSV-0210 and the production strain batch BV-WSV-0310 were also tested for virus titer immediately after production and periodically during the 10 years of strain storage after production. There are many methods for testing virus titer, our study applied two popular methods applied in many studies on VNNB vaccine production, which are the LD 50 titer titration method on mice and the method of creating necrotic clusters (PFU) on cells [9, 10, 25]. These are two methods that have been developed and perfected in Vietnam during the process of perfecting vaccine production and testing technology. For the LD 50 titration method on mice, the strain diluted from 10-5 to 10-8 will be infected on the brain of 11-13 g white mice, 0.03 ml/brain x 10 mice/diluted dose. Raise & monitor for 14 days, read and record the number of dead mice from the 3rd day after infection. Calculate the LD 50 result according to the Reed-Muench formula. Observe the typical neurological signs of mice after infection with the VNNB virus strain such as ruffled fur, some mice also showed signs of hip paralysis, slightly dragging their legs from the 3rd to the 4th day. On the 5th day, there were additional symptoms of blindness and paralysis of the two extended hind legs that became increasingly severe at the diluting doses of 10-5 , mice at higher dilutions had the above symptoms slower. And from the 6th day onwards, mice began to die in large numbers. From the 11th to the 14th day, almost no mice died, but compared to the unvaccinated control mice, they were thinner and lost more weight. The phenomena observed and described in this study are also consistent with these testing methods in Vietnam and around the world [61]. This method is called the direct challenge method on experimental mice, first described by T. Muira in the results of the testing method published in 1970 [61, 62]. Thus, it can be affirmed that the titration test using the LD 50 method on Swiss mice in Vietnam is currently suitable for application in routine testing of this vaccine.
Another method also applied in testing VNNB virus titer is the method of creating necrotic patches on Vero and/or BHK-21 cells (PFU).