PDF(Pubmed)
Abstract：
BACKGROUND: Rabies is a fatal zoonotic disease whose pathogenesis has not been fully elucidated, and vaccination is the only effective method for protecting against rabies virus infection. Most inactivated vaccines are produced using Vero cells, which are African green monkey kidney cells, to achieve large-scale production. However, there is a potential carcinogenic risk due to nonhuman DNA contamination. Thus, replacing Vero cells with human diploid cells may be a safer strategy. In this study, we developed a novel 2BS cell-adapted rabies virus strain and analysed its sequence, virulence and immunogenicity to determine its application potential as a human diploid cell inactivated vaccine.
RESULTS: The 2BS cell-adapted rabies virus strain 2aG4-B40 was established by passage for 40 generations and selection of plaques in 2BS cells. RNA sequence analysis revealed that mutations in 2BS cell-adapted strains were not located at key sites that regulate the production of neutralizing antibodies or virulence in the aG strain (GQ412744.1). The gradual increase in virulence (remaining above 7.0 logLD50/ml from the 40th to 55th generation) and antigen further indicated that these mutations may increase the affinity of the adapted strains for human diploid cells. Identification tests revealed that the 2BS cell-adapted virus strain was neutralized by anti-rabies serum, with a neutralization index of 19,952. PrEP and PEP vaccination and the NIH test further indicated that the vaccine prepared with the 2aG4-B40 strain had high neutralizing antibody levels (2.24 to 46.67 IU/ml), immunogenicity (protection index 270) and potency (average 11.6 IU/ml).
CONCLUSIONS: In this study, a 2BS cell-adapted strain of the 2aG4 rabies virus was obtained by passage for 40 generations. The results of sequencing analysis and titre determination of the adapted strain showed that the mutations in the adaptive process are not located at key sequence regions of the virus, and these mutations may enhance the affinity of the adapted strain for human diploid cells. Moreover, vaccines made from the adapted strain 2aG4-B40 had high potency and immunogenicity and could be an ideal candidate rabies virus strain for inactivated vaccine preparation.
RESULTS: The 2BS cell-adapted rabies virus strain 2aG4-B40 was established by passage for 40 generations and selection of plaques in 2BS cells. RNA sequence analysis revealed that mutations in 2BS cell-adapted strains were not located at key sites that regulate the production of neutralizing antibodies or virulence in the aG strain (GQ412744.1). The gradual increase in virulence (remaining above 7.0 logLD50/ml from the 40th to 55th generation) and antigen further indicated that these mutations may increase the affinity of the adapted strains for human diploid cells. Identification tests revealed that the 2BS cell-adapted virus strain was neutralized by anti-rabies serum, with a neutralization index of 19,952. PrEP and PEP vaccination and the NIH test further indicated that the vaccine prepared with the 2aG4-B40 strain had high neutralizing antibody levels (2.24 to 46.67 IU/ml), immunogenicity (protection index 270) and potency (average 11.6 IU/ml).
CONCLUSIONS: In this study, a 2BS cell-adapted strain of the 2aG4 rabies virus was obtained by passage for 40 generations. The results of sequencing analysis and titre determination of the adapted strain showed that the mutations in the adaptive process are not located at key sequence regions of the virus, and these mutations may enhance the affinity of the adapted strain for human diploid cells. Moreover, vaccines made from the adapted strain 2aG4-B40 had high potency and immunogenicity and could be an ideal candidate rabies virus strain for inactivated vaccine preparation.
摘要：
背景：狂犬病是一种致命的人畜共患疾病，其发病机制尚未完全阐明，接种疫苗是预防狂犬病病毒感染的唯一有效方法。大多数灭活疫苗是使用Vero细胞生产的，非洲绿猴肾细胞，实现规模化生产。然而,由于非人类DNA污染，存在潜在的致癌风险。因此，用人二倍体细胞替代Vero细胞可能是更安全的策略。在这项研究中,我们开发了一种新的2BS细胞适应狂犬病病毒株，并分析了其序列，毒力和免疫原性，以确定其作为人二倍体细胞灭活疫苗的应用潜力。
结果：通过传代40代并在2BS细胞中选择噬斑，建立了适应2BS细胞的狂犬病病毒株2aG4-B40。RNA序列分析揭示2BS细胞适应菌株中的突变不位于调节aG菌株中的中和抗体产生或毒力的关键位点(GQ412744.1)。毒力(从第40代到第55代保持在7.0logLD50/ml以上)和抗原的逐渐增加进一步表明,这些突变可以增加适应菌株对人二倍体细胞的亲和力。鉴定试验显示,抗狂犬病血清中和了适应2BS细胞的病毒株,中和指数为19,952。PrEP和PEP疫苗接种和NIH试验进一步表明，用2aG4-B40菌株制备的疫苗具有较高的中和抗体水平（2.24至46.67IU/ml），免疫原性(保护指数270)和效力(平均11.6IU/ml)。
结论：在这项研究中，通过传代40代获得2aG4狂犬病病毒的2BS细胞适应株。适应株的测序分析和滴度测定结果表明，适应过程中的突变不位于病毒的关键序列区域，这些突变可以增强适应菌株对人二倍体细胞的亲和力。此外，由适应株2aG4-B40制成的疫苗具有较高的效力和免疫原性，可以作为灭活疫苗制备的理想候选狂犬病病毒株。
结果：通过传代40代并在2BS细胞中选择噬斑，建立了适应2BS细胞的狂犬病病毒株2aG4-B40。RNA序列分析揭示2BS细胞适应菌株中的突变不位于调节aG菌株中的中和抗体产生或毒力的关键位点(GQ412744.1)。毒力(从第40代到第55代保持在7.0logLD50/ml以上)和抗原的逐渐增加进一步表明,这些突变可以增加适应菌株对人二倍体细胞的亲和力。鉴定试验显示,抗狂犬病血清中和了适应2BS细胞的病毒株,中和指数为19,952。PrEP和PEP疫苗接种和NIH试验进一步表明，用2aG4-B40菌株制备的疫苗具有较高的中和抗体水平（2.24至46.67IU/ml），免疫原性(保护指数270)和效力(平均11.6IU/ml)。
结论：在这项研究中，通过传代40代获得2aG4狂犬病病毒的2BS细胞适应株。适应株的测序分析和滴度测定结果表明，适应过程中的突变不位于病毒的关键序列区域，这些突变可以增强适应菌株对人二倍体细胞的亲和力。此外，由适应株2aG4-B40制成的疫苗具有较高的效力和免疫原性，可以作为灭活疫苗制备的理想候选狂犬病病毒株。
