Background: The Vero cell rabies vaccine is currently the most widely used human rabies vaccine. However, owing to the presence of residual host cell DNA (HCD) in the final product and the potential tumorigenicity of the DNA of high-passage Vero cells, the WHO not only sets a limit on the number of times cells used in production can be passaged, but also imposes strict requirements on the amount of residual HCD in the final vaccine product. Objectives: To systematically reduce the HCD level in the final vaccine product, multiple purification steps are included in the vaccine production process. This study investigated the effectiveness of key production steps in antigen recovery and DNA removal. Methods: The residual HCD fragment content and size distribution were detected using fluorescence quantitative PCR (qPCR) and capillary gel electrophoresis (CGE), and the rabies virus glycoprotein antigen content was detected using enzyme-linked immunosorbent assay (ELISA). The antigen recovery rate and HCD removal rate in each key process were calculated to evaluate the scientific basis and effectiveness of each production step. Additionally, the stability of the process was studied using multiple commercial batches of the product. Results: The results revealed that the total antigen recovery rate in the production process described in this report was no less than 8.5%, and the effective removal rate of residual HCD was not lower than 99.99%. Moreover, the amount of residual HCD in the final product was far below the quality standard of 2 ng/dose, and most of the residual HCD fragments were smaller than 200 bp. The results of the process stability studies on multiple commercial batches showed that the bulk human rabies vaccine produced by this process had excellent safety and efficacy and that the production process was stable and thus suitable for large-scale batch production. Conclusions: The production process described in this study achieved effective recovery of viral antigens and efficient removal of residual HCD, and the process was stable and controllable, enabling the continuous and stable production of vaccine products that meet WHO recommendations and the relevant requirements of the current edition of the Chinese Pharmacopeia. In addition, this study provides theoretical guidance for optimizing the vaccine production process.

Objectives: This study evaluated the 12-month persistence of rabies virus-neutralizing antibody (RVNA) following different immunization regimens of freeze-dried human rabies vaccine (Vero cells) in individuals aged 10–60 years within the Chinese population. Methods: Number of 600 participants from phase III clinical trials who completed the fullimmunization were randomly assigned into one of three groups, four-dose experimental group, five-dose experimental group, and five-dose control group. The experimental group received the vaccine from Shandong Yeedu Biotechnology Co., while the control group used the vaccine from Liaoning Chengda Biotechnology Co., Ltd. The antibody-positive rate and geometric mean concentration (GMC) were calculated, and analysis of variance was used to compare the results among the three groups. Results: The study included 200 participants in the four-dose experimental group, 186 in the five-dose experimental group, and 214 in the five-dose control group. Twelve months post-immunization, the overall RVNA-positive rates were 97.00%, 93.55%, and 94.86% in the four-dose experimental, five-dose control, and five-dose experimental groups, respectively. The GMCs of RVNA were 2.50, 2.05, and 2.04 IU/mL for the respective groups. The age-stratified analysis exhibited high positivity rates across age groups (≤21, 21–50, and ≥50), with rates ranging from 88.4% to 100%, and GMCs were between 1.75 and 2.61 IU/mL. Conclusions: Twelve months after full immunization, the overall RVNA-positive rate remained high across all dosing regimens, demonstrating satisfactory immunogenicity persistence. This indicates that both the four-dose and five-dose regimens are effective in maintaining long-term immunity against rabies.

The large-scale production of a human diploid cell (HDC) vaccine (HDCV) for rabies is limited by several technical challenges. Kanghua Biological Products Co., Ltd., has successfully used microcarrier technology for the large-scale culture of HDCs in bioreactors to develop a lyophilized and purified HDCV. In this blinded, randomized, parallel-group study conducted between July and October 2014 in Mianzhu, Sichuan Province, China, we monitored the safety and immunogenicity of this vaccine in a healthy population vaccinated according to the Essen post-exposure immunization schedule. A hamster kidney cell vaccine was used as the control. Adverse reactions were monitored 0.5, 6, 24, 48, and 72 h post vaccination to assess safety. Neutralizing antibodies in venous blood were measured on day 7, 14, and 72 to evaluate the immunogenicity of the vaccine while follow-up monitoring continued for 1 month. No serious adverse reactions were observed in any volunteer. The incidence rates of systemic and local adverse reactions were, respectively, 10.6% and 2.9% in the test group and 20.0% and 13.6% in the control group. After the third injection, the positive conversion rates of antibodies in the test and control groups were 100% and 98.82%, respectively. In addition, the average antibody titers on day 7, 14, and 42, were respectively, 1.71, 2.72, and 1.29 times higher than those in the control group. These results indicate that HDCV had a better safety profile and higher immunogenicity than the hamster kidney cell rabies vaccine. Trial registration number: 20130602.

We have obtained an attenuated rabies virus CTN181-3. In this paper, we make a comprehensive studies on CTN181-3. CTN181-3 showed no pathogenicity by i. c. or o. i. inoculation in 3-week-old mice, lower pathogenic in 2-week-old mice, and no virulence by o. i. inoculation in 8-week-old golden hamsters. CTN181-3 showed high immunogenicity, which produced high level neutralizing antibodies, 100% sero-conversation and >5.0 IU/ml GMT by one dose i. m. or o. i. vaccination in mice and golden hamsters. Cellular immune response by one dose i. m. or o. i. inoculation was detected. Especially in PEP, reduced dose of vaccination resulted in 50% (one dose) and 100% (2 doses) protections in golden hamsters. Molecular basis of the attenuation indicated that eight substitutions compared to its parental virus strain CTN-1, among them the two substitutions at the G276 (Leu-*Val) and L1496 (Met-*Trp) were the critical attenuated site. The phenotypic and genotypic characteristics of CTN181-3 were highly stable, no reversion was occurred when the virus was multiple passaged in suckling mice brains, guinea pig submandibular glands or BSR/Vero cell cultures. The gene homology compared to the Chinese rabies isolates showed much higher than rabies vaccine strains used in China, suggesting CTN181-3 is a promising and suitable oral rabies vaccine candidate strain.

Recently optimized freeze-dried rabies vaccines (Vero cell) are promising for rabies prevention; however, comparisons of Essen 5-dose and Zagreb 4-dose regimens are needed for clinical applications. This phase III clinical trial aimed to assess the safety and immunogenicity of candidate freeze-dried rabies vaccines developed for human use by Shandong Yeedo Biotechnology (Registration No.: CTR20182016, http://www.chinadrugtrials.org.cn/index.html). In total, 40 participants in stage I and 1797 subjects in stage II with an age range of 10-60 years were recruited. Both 4-dose and 5-dose experimental vaccines were well-tolerated in stage 1, with similar overall adverse event (AE) rates. In stage 2, systemic fever (35.39%) and local pain (48.91%) were common, and the rate of Grade 3 AEs was approximately 2.5%. Rates of local reactions and unsolicited AEs differed among groups (p = .045 and 0.011). Immunogenicity analyses showed higher antibody levels, seroconversion rates, and pre-immune seroconversion rates in the 4-dose experimental group versus the 5-dose group (p < .001) on day 7 post-first dose, with similar GMCs (geometric mean concentrations) by day 14. Pre-immune negative subjects had comparable antibody levels in the 4-dose and 5-dose experimental groups, outperforming the 5-dose control group (p = .039). The rate difference (95% CI) for first-dose seroconversion at 14 days post-vaccination among pre-immune antibody-negative subjects indicated non-inferiority between the 5-dose experimental and control groups. Overall, the Shandong Yeedo rabies vaccine demonstrated satisfactory safety and efficacy, offering a new option for rabies prevention.

The efficacy of the G-protein is influenced by N-linked glycosylation, which serves as the sole immunogen of the rabies virus vaccine. However, achieving satisfactory immune-protection efficacy remains challenging, owing to the heterogeneous glycosylation of G-proteins. Within molecular dynamics, examining the impact of N-glycan heterogeneity on the structural characteristics of G-proteins provides insights into the relationship between antigens and the efficacy of rabies virus vaccines. Glycosylation is regulated by host cells. In rabies virus cultured in Vero cells (VRV), all N-glycosylation sites of the G-protein underwent modification. In contrast, rabies virus G-protein cultured in KMB17 cells (human diploid cell vaccine [HDCV]) was only modified by N-glycans at amino acid positions 247 and 319. Furthermore, treatment of VRV with de-glycosylation significantly improved its immune-protective efficacy, whereas de-glycosylation did not alter the immune-protective efficacy of HDCV. To support the impact of glycosylation on VRV efficacy, the structures and dynamics of G-proteins were analyzed using GROMACS. Specifically, the hydrophobicity, flexibility, and radius of gyration of the G-protein trimer in VRV were significantly altered by excessive hydrogen bonds formed by the three-branched hybrid glycan at the aa 319 site. These changes increase the instability of the G-protein trimer and may lead to a decrease in vaccine protective efficacy. Ultimately, we determined that N-glycan heterogeneity affects the immune-protection effect of antigen proteins by altering their dynamic characteristics, enhancing our understanding of the correlation between antigen structural characteristics and efficacy. IMPORTANCE: N-glycosylation of rabies virus glycoprotein dynamically regulates protein folding, stability, and antigenicity. Therefore, regulation of N-glycan modification is key to improving vaccine stability and protective efficacy. How the type and modification sites of N-glycans affect the protective efficacy of rabies vaccines remains unclear. Our research indicates that there are differences in the protective efficacy of rabies virus G-proteins modified with different N-glycans. Moreover, the modification of the three-branched hybrid glycan at the aa 319 site of G-protein significantly altered the hydrophobicity, flexibility, and radius, and increased its trimeric antigen instability through molecular dynamics demonstrations. These findings update the current understanding of the impact of glycans on vaccine antigenicity and develop a system to evaluate the stability of antigen glycoproteins based on molecular dynamics.