Quantitation of Enterovirus A71 Empty and Full Particles by Sedimentation Velocity Analytical Ultracentrifugation.

The enterovirus A71 (EV71) inactivated vaccine is an effective intervention to control the spread of the virus and prevent EV71-associated hand, foot, and mouth disease (HFMD). It is widely administered to infants and children in China. The empty particles (EPs) and full particles (FPs) generated during production have different antigenic and immunogenic properties. However, the antigen detection methods currently used were established without considering the differences in antigenicity between EPs and FPs. There is also a lack of other effective analytical methods for detecting the different particle forms, which hinders the consistency between batches of products. In this study, we analyzed the application of sedimentation velocity analytical ultracentrifugation (SV-AUC) in characterizing the EPs and FPs of EV71. Our results showed that the proportions of the two forms could be quantified simultaneously by SV-AUC. We also determined the repeatability and accuracy of this method and found that both parameters were satisfactory. We assessed SV-AUC for bulk vaccine quality control, and our findings indicated that SV-AUC can be used effectively to analyze the percentage of EPs and FPs and monitor the consistency of the process to ensure the quality of the vaccine.

Immunogenicity and safety of an enterovirus 71 vaccine in children aged 36-71 months: A double-blind, randomised, similar vaccine-controlled, non-inferiority phase III trial.

Background: The enterovirus 71 (EV71) vaccine produced by Wuhan Institute of Biological Products Co., Ltd. (WIBP) (B-EV71) has been given to children aged 6-35 months, and it has shown good safety, immunogenicity and efficacy. However, the administration of EV71 vaccine in children aged 36-71 months, which is another target population, needs further exploration. Methods: We conducted a double-blind, randomised, controlled, non-inferiority phase III clinical trial in children aged 36-71 months, with a further comparison group of children aged 6-35 months in China. Children aged 6-71 months with no history of hand, foot and mouth disease or prior-vaccination of EV71 vaccine were eligible and recruited. Eligible participants aged 36-71 months were randomly assigned (1:1) to receive two doses of the B-EV71 vaccine (Older-B group) or the control EV71 vaccine (C-EV71 vaccine, produced by Institute of Medical Biology, Chinese Academy of Medical Sciences) (Older-C group), administered at a 30-day interval. Eligible participants aged 6-35 months were enrolled consecutively to receive two doses of the B-EV71 vaccine (Younger-B group) at a 30-day interval. Participants, investigators and those assessing outcomes were masked to the vaccine received. Non-inferiority analyses were conducted to compare the immunogenicity of EV71 vaccine in the Older-B group with that in the Older-C and Younger-B groups. Non-inferiority margins were 10% for seroconversion rate differences and 0.5 for geometric mean titre (GMT) ratios. The primary endpoints were the GMT level and seroconversion rate of anti-EV71 neutralising antibody 30 days after the second dose of vaccination. The primary analysis was performed in the per-protocol population. Safety analyses were conducted amongst participants receiving at least one dose of vaccine. This trial was registered at Chinadrugtrials.org.cn (#CTR20192345). Findings: Between June 3 and June 30, 2020, 1600 participants were enrolled and assigned, including 625 participants in the Older-B group, 625 participants in the Older-C group and 350 participants in the Younger-B group. The seroconversion rate of anti-EV71 neutralising antibody in the Older-B group (99.66%; 95% CI: 99.18%-100.00%) was non-inferior to that of the Older-C (99.32%; 95% CI: 98.65%-99.98%) and Younger-B groups (100.00%; 95% CI: 100.00%-100.00%). The differences in seroconversion rates in the Older-B group to those in the Older-C and Younger-B groups were 0.34% (95%CI: -2.17%-2.86%) and -0.34% (95%CI: -2.78%-2.09%). The GMT of the anti-EV71 neutralising antibody in the Older-B group (693.87) was also non-inferior to that in the Older-C (289.37) and Younger-B groups (634.80). The ratios of GMTs in the Older-B group to those in the Older-C and Younger-B groups were 2.67 (95%CI: 2.00-3.00) and 1.00 (95%CI: 0.75-1.00), respectively. The incidence of any adverse event (AE) related to vaccination was similar amongst the three groups (34/625 [5.44%] in the Older-B group, 32/623 [5.14%] in the Older-C group, and 26/349 [7.45%] in the Younger-B group), with only 2 (0.57%) participants having grade 3 AEs in the Younger-B group. Fifteen (0.94%) participants from these three groups had reported serious AEs (SAEs), all of which were unrelated to vaccines. Interpretation: EV71 vaccine produced by WIBP could extend to be administered to children aged 36-71 months against EV71 infection. However, the persistence of vaccine-induced immunities needs to be further investigated. Funding: Hubei Province's young medical talent program (20191229), Hubei Province's young talent program (2021), Hubei Province's young public health talent program (2021); and the Wuhan Institute of Biological Products Co., Ltd.

Clinical evaluation of the lot-to-lot consistency of an enterovirus 71 vaccine in a commercial-scale phase IV clinical trial.

OBJECTIVE: To evaluate the immunogenicity, safety and lot-to-lot consistency of an inactivated enterovirus 71 (EV71) vaccine cultured in bioreactors with different specifications after full immunization. METHODS: A randomized, double-blind trial was performed in 3,000 children aged 6 ~ 35 months with six vaccine batches, which were prepared in 40 L and 150 L bioreactors for three consecutive batches respectively. Children were immunized on day 0 and 28, serum samples were collected on day 0 and 56, and neutralizing antibody titers were determined by the microcytopathic method. Immediate reactions were recorded within 30 min, local and systemic symptoms were recorded within 0 ~ 28 days, and serious adverse events were recorded within 6 months. RESULTS: After immunization with two doses of the inactivated EV71 vaccine, the neutralizing antibody GMT was 825.52 ± 4.09, and the positive conversion rate was 96.18%, with no significant difference. The 95% CI of the serum neutralizing antibody GMT ratio between the two groups after immunization with the three vaccine batches produced in the 150 L and 40 L bioreactors ranged from .67 ~ 1.5. The overall incidence of adverse reactions, mainly grade 1 reactions, for all 6 batches from 0 to 28 days after vaccination was 49.62%, with no significant difference (p = .8736). The incidence of systemic adverse reactions, primarily fever and diarrhea, was 45.14%; the incidence of local adverse reactions, primarily erythema and tenderness, was 9.43%. CONCLUSION: The EV71 vaccine was highly immunogenic and safe in children aged 6-35 months, and 6 consecutive batches produced by the two bioreactors with different specifications were consistent.

Bladder cancer-associated protein is suppressed in human cervical tumors.

Bladder cancer-associated protein (BLCAP) is downregulated in bladder cancer and has been identified as a prognostic biomarker for human bladder cancer. We previously reported that BLCAP mRNA is decreased in cervical cancer tissues, and overexpression of BLCAP was found to inhibit cell growth and induce apoptosis in the human cervical cancer HeLa cell line To investigate the BLCAP protein expression in cervical cancer and its potential clinical indications, we developed a polyclonal antibody against human BLCAP to assess the BLCAP protein expression in 30 cervical cancer tissues and 30 non-tumor cervical tissues from patients. Western blotting data showed that a single band of recombinant protein was probed by antiserum of BLCAP and no band was probed by pre-immune serum. BLCAP expression was significantly downregulated in cervical carcinoma tissues compared with its expression in the non-tumor cervical tissues. Moreover, cervical carcinoma tissues from patients with stage III-IV had significantly lower BLCAP expression percentage compared with stage I-II. Similarly, a significantly lower BLCAP expression percentage was observed in moderately/poorly differentiated tumor tissues and in the tumor tissues from patients with lymphatic metastasis (LM) compared with well-differentiated tumor tissues and non-LM patients, respectively. Our results suggest that decreased BLCAP protein expression is associated with poor prognosis and it could be a potential bio-index to predict cervical tumor patient outcome.

[Identification and sequential analysis on rabies virus isolated from a donkey].

OBJECTIVE: To identify and analyze the genetic characteristics of nucleoprotein (N) and glycoprotein (G) genes of rabies virus (RABV) isolated from a donkey in Wuhan. N gene and G gene of the virus were compared with other representative street strains isolated around Hubei areas as well as the vaccine strains used in China and abroad. METHODS: RABV in brain tissue of a donkey was detected by direct immunofluorescent method and then inoculated in suckling mice to observe the incidence of rabies. Brain samples of the donkey and infected suckling mice were detected by ELISA. The N gene and G gene fragment of the isolated RABV were amplified by RT-PCR and cloned into pMD18-T vector for sequencing and genetic analysis. RESULTS: RABVs were detected in both donkey brain and suckling mice brain samples. The N gene and G gene nucleotide homology of RABV isolated from the donkey with other representative street strains found around Hubei areas as well as vaccine strains used in China and abroad were 85.7% - 99.1% and 82.2% - 99.7%, and the deduced amino acid identity were 95.6% - 99.8% and 87.8% - 99.4%, respectively. CONCLUSION: Novel RABV was successfully identified and isolated from a donkey and showed close relationship to the representative street strains found around Hubei areas as well as vaccine strains used in China through genetic analysis.

Molecular characterization of the complete genome of a street rabies virus WH11 isolated from donkey in China.

The complete genomic sequence of a rabies virus isolate WH11, isolated from brain tissue of a rabid donkey in China, was determined and compared with other rabies viruses. This is the first Chinese street strain which was isolated from donkey and the entire length and organization of the virus was similar to that of other rabies viruses. Multiple alignments of amino acid sequences of the nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and large protein of WH11 with those of other rabies viruses were undertaken to examine the conservative degree of functional regions. Phylogenetic analysis using the complete genomic sequence of WH11 determined that this isolate is most closely related with rabies viruses previously isolated in China and the attenuated Chinese vaccine strain CTN181.