Study on the production of measles antibody used for vaccine quality control

Background: With the help of Japan, the Center for Research and Production of vaccines and biologicals, Hanoi has received a WHO standard measles vaccine production technology, including techniques in the examination of vaccine quality. Therefore, it is needed to be initiative on production of measles antibody. Objective: Study on production of measles antibody in rabbits and selecting the appropriate antibody for production of high titre antibody, which meets the standard of vaccine quality control in Vietnam. Subject and methods: Using the measles antigen from Edmonston and AIK-C strains, which were provided by the Kitasato Institute, to produce measles antibody. Making immunoreaction in rabbits and determination of neutralization antibody titre. Results and Conclusion: Measles antigen of Edmonston Vero 7/P2 strain used in the production of measles antibody in rabbit created the highest antibody titre in comparison with AIK-C strain from vero cell and FL cell supplied by the Kitasato Institute of Japan. Antibody titre of Edmonston Vero 7/P2 strain reached up to 1/8192 and met the sera standard required for measles vaccine quality control, it is similar to the measles sera to be produced from the Kitasato Institute.

Study on the stable of gene sequence of seed lot system G1P8 KH0118 during production rotavirus vaccine

Background: Rotavirus strain (KH0118) is used as the primary material to produce original rotavirus vaccine strains with the symbol of G1P8 MS. According to the World Health Organization\u2019s standard, the strain is needed to evaluate the stability of gene throughout analysis of gene and amino acid sequence during vaccine production. Objective: To determine the sequence of genes 4 (VP4), 6 (VP6), 9 (VP7) and 10 (NSP4) with base pair correlative 855:866:1345:745 of seed lot system and vaccine of G1P8 strain and to evaluate the stability of seed lot system during vaccine production. Subject and methods: ARN was divided from the original strain of rotavirus vaccine G1P8 MS, rotavirus vaccine productive strain (G1P8 WS) and rotavirus vaccine (G1P8 VX). Then using primer pairs to determine gene sequence VP4, VP6, VP7, NSP4 and comparing gene and amino acid sequence of the seed lot system. Results and Conclusion: The study demonstrated that, there was no difference for the nucleotide and amino acid sequence from the original strain during production of rotavirus vaccine G1P8 KH0118.

The difference for quantity of nucleotide and amino acid between human rotavirus strain \u2013 Vietnam candidate for rotavirus vaccine production and international standard wild strains

Background: Rotavirus induced diseases is most commonly seen in children between 6 and 36 months old. In developing countries, rotavirus is also a common cause of gastrointestinal inflammation in below 2 years old children. Study on production of vaccine strains is a target that the World Health Organization is providing. Objective: To determine the quantity of different nucleotide and amino acids of genes 4, 6, 9, 10 of the Vietnam seed lot system and vaccine G1P8, G1P4 and G4P6. Subject and methods: By sequence method determine the quantity of different nucleotide and amino acids of gene 4 (VP4), gene 6 (VP6), gene 9 (VP7) and gene 10 (NSP4) of the Vietnam seed lot system and vaccine G1P8 (KH0118), G1P4 (2001019210) and G4P6 (2001019203) in comparison with international standard wild strains such as Ku, DS1, ST3, Hochi, TB-chen. Results and Conclusion: Each strain had a different nucleotide and amino acid sequence and it was characterized by each country. However, these strains had the same general chemical components including nucleic acid and protein. Nucleic acid was a double fiber ARN with 11 genes, and 18 thousand base pairs. 3 proteins with specific antigens were VP4, VP6 and VP7. 57 nucleotide of gene 4 (VP4) of the seed lot system G1P8 were different with Ku (AB22772) strain but there were only 17 different amino acids. For gene 7 (VP7), 70 nucleotide were different between G1P8 strain and Ku (AB222784) strain but there were 15 different amino acids. For gene 6 (VP6), although 141 nucleotide were different between G1P8 strain and Ku (AB222784) strain but there were only 9 different amino acids. For gene 10 (NSP4), 62 nucleotide were different between G1P8 strain and Ku strain (AB222772), making 12 different amino acids.

Epidemiology of rotavirus diarrhea in the National Pediatric Hospital

Background: Rotavirus type A is the most common cause of acute gastrointestinal inflammatory in children under 5 years old, especially in age groups 6 and 36 months. Some rotavirus strains are common; seen recently in Vietnam are G1, G2, G3, G4 and G9, P4, P6 and P8. Objective: Surveillance of epidemiological characteristics of rotavirus induced diarrhea in the National Pediatric Hospital from September, 2007 to March, 2008. Subject and methods: Collection of 322 stool specimens of pediatric patients with acute diarrhea (including 213 specimens from male, 109 specimens from female), who were treated in the National Pediatric Hospital. All of these specimens were determined for causes of rotavirus with the enzyme immunoassay (EIA). Results and Conclusion: Among these 322 stool specimens, there were 195 rotavirus positive specimens, accounted for 60.56%. The rate of monthly distribution of rotavirus diarrhea from September, 2007 to March, 2008 were 76%, 56%, 62%, 61%, 64%, 56% and 44%, respectively. Number of rotavirus positive cases in male and female was 56 (26.29%) and 79 (72.48%), respectively. The rate of rotavirus positive children compared to total number of specimens with the age 0-3 months, 3-6 months, 6-12 months, 12-24 months, 24-36 months and over 36 months was 7.69%, 15.9%, 41.54%, 32.82%, 1.54% and 0.51%, respectively. The results of type identification indicated that phenotypes of 37 among 40 specimens were identified (92.5%) in which there were 5 specimens of G1P8 (12.5%), 20 specimens of G3P8 (50%), 1 specimen of G9P8 (2.5%), 2 specimens of G1Pmixed (5%), 9 specimens of G3Pmixed (22.5%), 1 specimen of G unidentified-type P8 (2.5%) and 2 specimens of G3 P unidentified-type (5%).

Stable study of gene sequence of seed lot system G1P4 (2001019210) during production of rotavirus vaccine

Background: Presently, toxicity decreased oral live rotavirus is a candidate for vaccine for the prevention of rotavirus induced diarrhea. According to the World Health Organization, the seed lot system is robustly checked, in which determining the stable of gene sequence. Objective: To determine the sequence of genes 4: 6: 9: 10 with base pair correlative 855: 824: 1314: 734 of seed lot system G1P4 (2001019210) during production of rotavirus vaccine. Subject and methods: Gene 4 (VP4), gene 6 (VP6), gene 9 (VP7) and gene 10 (NSP4) of seed lot system G1P4 were determined for gene sequencing and then comparing the nucleotide sequence as well as deduced amino acids from original strain with the produced strain and vaccine virus. Results and Conclusion: There was no different for nucleotide and deduced amino acid sequence from the original strain during the production of rotavirus vaccine of G1P4 MS (2001019210) to producing strains of G1P4 WS and vaccine strains of G1P4 VX.

Epidemiology of ROTA virus diarrhea in Ho Chi Minh city from 12/2006-11/2007

Background: Acute gastroenterophathy usually caused by the Rota virus for children under 5 years old. Objectives: To present various types of data on epidemiology of ROTA virus diarrhea in Ho Chi Minh city from 12/2006-11/2007. Material and method: The data were collected from 500 stool specimens of diarrhea diagnosed chilren hosptalised at Thuy Dien Pediatric hospital 1, Ho Chi Minh city from December/2006 to November /2007. Results:There were 322 rotavirus-positive specimens, representing 64.4%. The proportions of monthly distribution of cases with diarrhea due to rotavirus were 90.1%, 54.39%, 85.37%, 74.51%, 72.92%, 41.67%, 26.67%, 58.33%, 79.31%, 52.63%, 69.05% and 57.78%, respectively. The numbers of rotavirus-positive cases in male and female were 216 (65.26%) and 106 (62.72%), respectively. The proportions of Rota virus positive children compared to total number of diarrheal cases with age 0-3, 3-6, 6-12, 12-24, 24-36 and over 36 months were 2.80%, 7.76%, 40.06%, 40.68%, 5.28% and 3.42%, respectively.\r\n', u'The results of typing identification indicated that the phenotypes of 98 among 100 specimens were identified (98%) in which there were sixty-one specimens of G1P8 (61%), one specimen of G2P8 (1%), fourteen specimens of G3P8 (14%), four of specimens of G4P8 (4%), eighteen specimens of GmixedP8 (18%). There were only two specimens of GnontypeableP8 (2%). Conclusion: Further studies should be carried out to clear this issue.\r\n', u'

Study on the stability of gene sequences of seed lot system G4P6 (2001019203) during production of rotavirus vaccine

Background: Currently, the World Health Organization is encouraging developing countries to establish a seed lot system of rotavirus vaccine for production of this vaccine. Objectives: To determine gene sequences of rotavirus strain that was used for vaccine production and to evaluate its stability. Materials and method: Master seed (G4P6MS), Working seed (G4P6WS) and vaccine strain (G4P6VX) of Rotavirus were used for analysis at the US Center for Disease Control and Prevention (CDC). Results: 855 base pairs of gene 4 (VP4); 1195 base pairs of gene 6 (VP6); 824 base pair of gene 9 (VP7) and 715 base pairs of gene 10 (NSP4) from seed lot system and vaccines of G4P6 strain were determined. The results demonstrated this seed lot system is completely stable during vaccine production. There is no difference for nucleotide and amino acid sequence in this seed lot system. Conclusion: G4P6 strain (2001019203) is completely stable during vaccine production.

IgG immune response of Macaca mulatta monkeys after immunization with human rotavirus strains that were used for vaccine production

Background: Rotavirus acute diarrhea is a common disease in children aged 6 to 24 months, accounting for 50-70% of hospitalizations in Vietnam. Vaccines recommended by the WHO are quite expensive, so vaccination for this disease isn\u2019t widely used in Vietnam. Objectives: To evaluate the immune responses of 3 human rotavirus strains in Macaca mulatta monkeys. Subjects and method: 32 healthy monkeys aged 6-12 monthswere divided into 4 groups that received orally the G1P8 strains (Master seed- MS and Working seed- WS), G1P4 strains (MS and WS), G4P6 strains (MS and WS),and placebo. All monkeys were evaluated on general status, gastrointestinal symptoms and blood samples taken for immune analysis. Results: By ELISA technique, the Master Seed (MS) and Working Seed (WS) of Rotavirus, including G1P8 (KHI008), G1P4 (2001019210) and G4P6 (2001019203) strains showed high titer of IgG antibody in monkey at least four-fold after 3 doses of immunization. Conclusion: These 3 rotavirus strains produced by the Center for Research and Production of Vaccines and Biologicals could be candidates for vaccine production.

Sequencing VP4, VP7, NSP1, NSP4 genes of human rotavirus strain G1P8

Background: Rotavirus is the main cause of acute viral gastroenteritis in children under 5 years old. The virus leads to over 600000 children deaths a year in the world, 80% of which occur in the developing countries. In Viet Nam, 50%-70% the children\u2019s hospitalizations for acute diarrhea were resulted from rotavirus infection. Objective: To sequence nucleotides and amino acids of VP4, VP7, NSP1, and NSP4 genes of 5 passages of human rotavirus strain G1P8. Materials and method: A study was conducted in rotavirus sample of 5 passages of human rotavirus strain G1P8: B17A3; B17.3; B17.3 pp32vero15; B17.3 pp36TKP2; B17.3 pp43.7vero in Centre for Disease Control and Prevention, Atlanta, United State. Methods: using NucliSen Kit for detection of ARN; RT-PCR; sequencing genes by ABI 3100 machine. Results and Conclusion: Sequencing nucleotides and amino acids of VP4, VP7, NSP1, and NSP4 genes of 5 passages of human rotavirus strain G1P8 showed that: the number of nucleotide mutations ofVP4, VP7, NSP4 genes occurring among the passages were 3 (at nucleotit 175, 419, 790), 1 (at nucleotit 644), 3 (at nucleotit 134, 254, 482), respectively. All these mutations resulted in changes in amino acid composition. No mutation was found in NSP1 gene.

Determine the quality of vero cells using for rota vaccine development

Background: Vero cell (ATCC) is from kidney of Blue Monkey in Africa. Because of its strong points such as non tumor form, non exotic virus infection, this cell strain is commonly used for vaccin development in the world. Objective: To determine the quality of vero cell supplying by Japan Polio-myelitis Research Center and WHO vero cell supplying by the Company for Vaccine and Biological Production No.1 in use for develop rota vaccine. Subjects and method:A study was conducted in 2 kinds of vero cell (one ATCC 134 generation supplying by Japan Polio-myelitis Research Center and another WHO ATTC 137 generation supplying by the Company for Vaccine and Biological Production No.1) using standard methods. Results and Conclusion: Both these ATCCs had no exotic agents in generation from 134 to 137. The vero working cell bank for vaccine development has been established by the POLYVAC by using standard methods, in accordance with the WHO regulations. The vero working cells established by POLYVAC had the same quality as that of Vabiotech cell bank. Rota virus strains multiplied well on WHO ATTC 137 generation and ATCC 134 generation supplying by Japan Polio-myelitis Research Center.

Titre and safety of rotavirus master seed G1P8 (KH0118)

Background: Rotavirus vaccines used to prevent acute diarrhea among children under 5 years old. In Vietnam, with the assistance of the World Health Organization (WHO), Centers for disease control and prevention (CDC), Atlanta, United States, Ministry of Health and Ministry of science and technology researched to create rotavirus master seed G1P8 which would use to produce Rota virus in Vietnam. Objectives: to evaluate titre and safety of rotavirus master seed G1P8 in vitro and in experimental animals. Subjectives and Method: experimental studies in vitro and experimental animals. Rotavirus master seed G1P8 (KH0118) series 1 (MS-P5) and series 2 (MS-P5) produced in 2005, preserved 80oC, were determined by immunofluorescence method (with unit of FFU/ml). The strains were tested in experimental animal\u2019s safety like the safe requirements of World Health Organization on live-attenuated vaccine (OPV). Results: no detection for exotic virus strains in virus suspension by PCR and titres of rotavirus master seed were similar and were more than 107 FFU / ml. 10 intervened rabbits were healthy life during follow up period, 8/10 these rabbits increased weight after follow up period, only two rabbits reduced weight but increase of mean weight of all 10 rabbits was 106%. 5 intervened guinea-pig increased mean weight (158%) and healthy during follow-up period. 4 experimental white mice increased mean weight (195%), higher than control groups. It was safety in experimenting on baby mice. Conclusions: two lots of rotavirus master seed G1P8 produced in POLYVAC had high titre and safety both in vitro and in experimental animals.

The safety and immune response of rotavirus master seeds in monkeys

Background: Group A Rotavirus is the main cause of diarrhea in human especially in children under 5 years old. Rotavirus master seeds were established from group A Rotavirus (mainly 3 strains: G1P8, G1P4 and G4P6) causing acute diarrhea for children in Vietnam. The master seeds must meet the potency and safety in the laboratory as well as animal experiments under the guidance of WHO. Objectives: To determine the safety and immune response of rotavirus master seeds in monkeys to confirm their safety and effect in preclinical stage. Subjects and method: Baby Macacca mulatta monkeys had average weight of 1.5 kg (provided by monkey ranch in Reu island in Quang Ninh province) were tested and determined neutral antibody by immunofluorescence technique. Results: The rotavirus master seeds: G1P8 (KH0118); G1P4 (2001019210) and G4P6 (2001019203) had good safety and immune response with high neutral antibody after 3 dose vaccination in baby Macacca mulatta monkeys. Conclusion: The rotavirus master seeds would be a base for diarrhea vaccine production in Viet Nam under the guidance of the World Health Organization.

Assessment on Quality of single polio vaccine type 1 produced on primary monkey cells

Background: \r\n', u'October 2000, Vietnam was acknowledged as the country to successfully eradicate the polio by WHO.This success was partly due to the oral polio vaccine (OPV) produced on the primary money cells by the Centre of Research, Production of vaccines and biologicals, Ha Noi. In 2006, the Centre developed the single polio vaccine type 1 from primary monkey cells.\r\n', u'Objectives: \r\n', u'To evaluate the safety and antibody titre .\r\n', u'Subjects and method: \r\n', u'6 lots of single polio vaccine type 1 (ISO- 90, Antibodies of Polio type 1,2,3; the standard sample F113 from Japanese research on Polio Institute\ufffd?\r\n', u'Using the tests (T- maker, D maker, PFU, CCID50) to check the safety of single polio vaccine type 1. \r\n', u'Results:\r\n', u'After 14 days, 6/6 lots of viruses were observed via the microscope that they stayed in well developed, and of no serious adverse affects.There was no appearance of degenerated cells. \r\n', u'Conclusion:\r\n', u'6/6 lots of single polio vaccine type 1 produced on Macca mulltta monkey kidney cells with the first time passage at POLYVAC in 2006 are safe and high antibody titre.\r\n', u'

Study on characteristic of human rotavirus strains G9P8 on cell culture

Background: In recent years, the rotavirus diarrhea increased significantly in Vietnam, an estimated 50%-70% of children\u2019s hospitalization for diarrhea in 2005. The virus subtype causing disease mainly in Vietnam today was not only G1, G2, G4, like other countries in the world but also the rare strain was G9. Objectives: to isolate G9 rotavirus strains on MA104 cell culture; to selecting G9 rotavirus which developing well on MA104 cells. Subjectives and Method: an experimental research in the laboratory. 20 stool samples derived from 20 children with acute diarrhea caused by rotavirus G9P8 (using RT-PCR method). The samples were processed according to standards of the Centers for disease control and prevention (CDC), Atlanta, USA. Results: eight out of 20 human rotavirus strains G9P8 were positive on Ma104 cell suspension after 3 consecutive cultures (OD indexes of these sample were over 0.100,sample No 2 had the highest OD (1.347)). Sample No 2 was chosen for the first time cloning (25 clones, 17/25 with OD>0.100). And clone No 16 was selected for the second purifying (25 clones, 24/25 with OD>0.100). Ten out of 25 clones in the second time were adapted on monolayer Ma104 cell culture and only clone No 14 and No 18 with highest OD (2.648 and 2.644, respectively) will be used for next studies. Conclusions: cloning method was a basic method for adapting rotavirus clone on cell cultured. Rate of G9P8 rotavirus strain which isolated on Ma104 cells\ufffd?suspension was relatively high. Among the second time clones, 2/10 samples adapted on Ma104 cells with high OD.

Safety and potency of rotavirus master seed G4P6 (2001019203)

Background: Rota vaccine is used to prevent diarrhea in children under 5 years old. Two vaccines are being used in developed countries: Rotarix (GSK) and RotaTeq (Merk). Rotarix vaccine was produced from master seed G1P8 and RotaTeq vaccine was from the coordination of human rotavirus strains G1, G2, G3, G4 and cow rotavirus strain. Thanks to the help of WHO, Ministry of Health and Ministry of Science and Technology, Center for research and production of vaccines and biologicals \ufffd?Ha Noi made study of creating rotavirus master seed G4P6 for Rota vaccine production in Vietnam. Objectives: To evaluate the safety and potency of rotavirus master seed G4P6 in the laboratory and experimental animals. Subjects and method: Rotavirus master seed G4P6 (2001019203) lot 1 (MS-PL5) and lot 2 (MS-PL5) produced in 2005, preserved at -800C were determined potency by Immunofluorescence (IF) method and tested for safety on rabits and rats. Results:2 lots of Rotavirus master seed G4P6 that had been produced in Center for research and production of vaccines and biologicals \ufffd?Ha Noi had high titre and safety in the laboratory and experimental animals. Conclusion: The results were the basis of Rota vaccine production in Vietnam.

Study on the multiplicity of infection (MOI) of human rotaviruses strains in vero cells

Background: \r\n', u'Rotavirus is the major important cause of infectious infantile gastroenteritis and diarrhea, especially the children aged 6 months to 24 months. Some efforts made to provide safe water and cultivate the hygiene have not reduced the diarrhea caused by rotaviruses. Develope a vaccine agaisnt this virus is of high importance. A question about the production of this kind of vaccine targeted athow to make human rotaviruses strains adapt cultured cells.\r\n', u'Objectives\r\n', u'To determine the multiplicity of infection (MOI) of human rotaviruses strains in vero cell.\r\n', u'Subjects and method: \r\n', u'The multiplicity of infection of Human rotaviruses strains (G1 P8, G1P4, and G4P6) were selected and the CDC- Atlanta US supplied the vero cell.Use vero cells cultivate, temper the vero cell and, applying direct immunofluorescence to determine the multiplicity of G1P8, G1P4, C4P6.\r\n', u'Results:\r\n', u'The MOI of G1P8, G1P4 and C4P6 are 0.02 ffu/cell, 0.15ffu/cell, and 0.35 ffu/cell, respectively.\r\n', u'Conclusion:\r\n', u'Using the vero cell to multiple the rotaviruses is the effective part during the production of vaccines against rotaviruses. The MOI of human rotaviruses strains G1P8, G1P4 and G4P6 are in order:0,02 ffu/cell, 0,15ffu/cell, and 0,35% ffu/cell. \r\n', u'