Development of an Inactivated Vaccine Candidate, BBIBP-CorV, with Potent Protection against SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 µg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.

Evaluation of Varicella-zoster virus-specific cell-mediated immunity by interferon-γ Enzyme-Linked Immunosorbent Assay in adults ≥50 years of age administered a herpes zoster vaccine.

Varicella-zoster virus (VZV)-specific cell-mediated immunity (CMI) is critical for preventing and controlling the onset of herpes zoster (HZ). To assess VZV CMI, an interferon-γ (IFN-γ) enzyme-linked immunosorbent assay (ELISA) was validated by examining the influence of VZV-specific antigen content, incubation time, and interval from whole blood collection on the assay. In phase II clinical trial, VZV-specific CMI in adults ≥50 years of age administered an HZ vaccine were evaluated by IFN-γ ELISA, as determined by measuring IFN-γ production in the whole blood in response to stimulation with ultraviolet light-inactivated VZV. The VZV-specific IFN-γ levels varied among individuals from prevaccination (baseline) to 6 weeks postvaccination. In most subjects, VZV-specific CMI was increased at 6 weeks postvaccination. The HZ vaccine elicited a significant increase in the VZV-specific CMI response as measured by ELISA; the geometric mean fold-rises from baseline to 6 weeks postvaccination were 3.50, 4.22, and 5.24 in the 4.3, 4.7, and 4.9 log plaque-forming unit vaccine groups, respectively, which was significantly higher than in the placebo group (P < 0.05). These results indicate that vaccination enhances the VZV-specific CMI responses in subjects; IFN-γ ELISA is an effective method for evaluating the CMI response and may be useful for identifying individuals at a high risk of HZ infection.

Sequence Analysis and Comparison of the Key Genes of Domestic Live Attenuated Varicella Vaccine.

Objective To analyze the safety and consistency of domestic live attenuated varicella vaccines (LAVVs) at gene level.Methods The key genes (ORF38,ORF54,and ORF62) of LAVVs produced by four Chinese manufacturers were amplified by polymerase chain reaction (PCR) and sequenced.The sequencing results were compared with the sequences of Dumas,P-Oka,and V-Oka strains in GenBank and with the sequences of Varilrix (GSK) and Varivax (Merck).Results The ORF38 and ORF54 gene sequences of four domestic LAVVs were the same as each other and completely consistent with the sequences of V-Oka and Varilrix;however,it was different from Varivax (Merck) at one site.The ORF62 gene sequences of four domestic LAVVs were similar,and had individual nucleotide differences with V-Oka,Varilrix(GSK),and Varivax (Merck).Conclusions The sequences of ORF38,ORF54,and ORF62 of four domestic LAVVs are almost the same,showing good stability.They have small differences with V-Oka,Varilrix(GSK),and Varivax (Merck),without introducing new mutations.

Immunogenicity of intranasal vaccine based on SARS-CoV-2 spike protein during primary and booster immunizations in mice.

Mucosal immunity plays a crucial role in combating and controlling the spread of highly mutated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recombinant subunit vaccines have shown safety and efficacy in clinical trials, but further investigation is necessary to evaluate their feasibility as mucosal vaccines. This study developed a SARS-CoV-2 mucosal vaccine using spike (S) proteins from a prototype strain and the omicron variant, along with a cationic chitosan adjuvant, and systematically evaluated its immunogenicity after both primary and booster immunization in mice. Primary immunization through intraperitoneal and intranasal administration of the S protein elicited cross-reactive antibodies against prototype strains, as well as delta and omicron variants, with particularly strong effects observed after mucosal vaccination. In the context of booster immunization following primary immunization with inactivated vaccines, the omicron-based S protein mucosal vaccine resulted in a broader and more robust neutralizing antibody response in both serum and respiratory mucosa compared to the prototype vaccine, enhancing protection against different variants. These findings indicate that mucosal vaccination with the S protein has the potential to trigger a broader and stronger antibody response during primary and booster immunization, making it a promising strategy against respiratory pathogens.

Immunogenicity and safety of a live-attenuated varicella vaccine in a healthy population aged 13 years and older: A randomized, double-blind, controlled study.

OBJECTIVES: Vaccines for prevention against varicella are important for adolescents and adults, who have an increased risk of severe varicella. This study aimed to evaluate the immunogenicity and safety of a two-dose immunization schedule of a live-attenuated varicella vaccine (VarV) manufactured by Sinovac (Dalian) in healthy adolescents and adults. METHODS: A randomized, double-blind, controlled clinical trial was conducted in healthy population aged ≥ 13 years old in China. Participants in block 1 were randomly assigned (1:1) to receive two doses of either the test vaccine or an active control vaccine, administered 4, 6 or 8 weeks apart. Participants in block 2 were randomly assigned (2:1) to receive two doses of test vaccine or placebo, administered 10 weeks apart. The primary immunogenicity endpoint was the seroconversion rates and GMTs of varicella zoster virus (VZV) antibodies measured by fluorescent-antibody-to-membrane-antigen (FAMA) 4 weeks post-immunization. The primary safety endpoint was the incidence of adverse reactions within 4 weeks after each dose. RESULTS: A total of 2398 participants were enrolled. The seroconversion rates of VZV antibodies were 79.55 % in the test group and 76.41 % in the active control group respectively 4 weeks after two doses of pooled schedule, with the difference of 3.14 % (95 %CI: -0.69 %, 6.97 %). The GMTs were 1:162.07 and 1:160.04 respectively, with the ratio of 1.013 (95 %CI: 0.910, 1.127). Both the seroconversion rates and GMTs reached the prespecified non-inferiority criteria. Two-dose schedule with an interval of 10 weeks could also induce high immune responses, with a seroconversion rate of 83.22 % and a GMT of 1:160.38 in the test group. Safety profiles were similar among the test group, active control group and placebo group. CONCLUSION: VarV, manufactured by Sinovac (Dalian), demonstrated higher immune response and better flexibility in the immunization schedule among heathy population aged 13 years and older, without increased safety risk.

Heterologous booster vaccination enhances antibody responses to SARS-CoV-2 by improving Tfh function and increasing B-cell clonotype SHM frequency.

Heterologous prime-boost has broken the protective immune response bottleneck of the COVID-19 vaccines. however, the underlying mechanisms have not been fully elucidated. Here, we investigated antibody responses and explored the response of germinal center (GC) to priming with inactivated vaccines and boosting with heterologous adenoviral-vectored vaccines or homologous inactivated vaccines in mice. Antibody responses were dramatically enhanced by both boosting regimens. Heterologous immunization induced more robust GC activation, characterized by increased Tfh cell populations and enhanced helper function. Additionally, increased B-cell activation and antibody production were observed in a heterologous regimen. Libra-seq was used to compare the differences of S1-, S2- and NTD-specific B cells between homologous and heterologous vaccination, respectively. S2-specific CD19+ B cells presented increased somatic hypermutations (SHMs), which were mainly enriched in plasma cells. Moreover, a heterologous booster dose promoted the clonal expansion of B cells specific to S2 and NTD regions. In conclusion, the functional role of Tfh and B cells following SARS-CoV-2 heterologous vaccination may be important for modulating antibody responses. These findings provide new insights for the development of SARS-CoV-2 vaccines that induce more robust antibody response.

The Immunogenicity of CpG, MF59-like, and Alum Adjuvant Delta Strain Inactivated SARS-CoV-2 Vaccines in Mice.

The continuous evolution and mutation of SARS-CoV-2 have highlighted the need for more effective vaccines. In this study, CpG, MF59-like, and Alum adjuvant Delta strain inactivated SARS-CoV-2 vaccines were prepared, and the immunogenicity of these vaccines in mice was evaluated. The Delta + MF59-like vaccine group produced the highest levels of S- and RBD-binding antibodies and live Delta virus neutralization levels after one shot of immunization, while mice in the Delta + Alum vaccine group had the highest levels of these antibodies after two doses, and the Delta + MF59-like and Delta + Alum vaccine groups produced high levels of cross-neutralization antibodies against prototype, Beta, and Gamma strain SARS-CoV-2 viruses. There was no significant decrease in neutralizing antibody levels in any vaccine group during the observation period. CpG, MF59-like, and Alum adjuvant Delta strain inactivated SARS-CoV-2 vaccines excited different antibody subtypes compared with unadjuvanted vaccines; the Delta + CpG vaccine group had a higher proportion of IgG2b antibodies, indicating bias towards Th1 immunity. The proportions of IgG1 and IgG2b in the Delta + MF59-like vaccine group were similar to those of the unadjuvanted vaccine. However, the Delta + Alum vaccine group had a higher proportion of IgG1 antibodies, indicating bias towards Th2 immunity. Antigen-specific cytokine secretion CD4/8+ T cells were analyzed. In conclusion, the results of this study show differences in the immune efficacy of CpG, MF59-like, and Alum adjuvant Delta strain inactivated SARS-CoV-2 vaccines in mice, which have significant implications for the selection strategy for vaccine adjuvants.

The Quantification of Spike Proteins in the Inactivated SARS-CoV-2 Vaccines of the Prototype, Delta, and Omicron Variants by LC-MS.

Developing variant vaccines or multivalent vaccines is a feasible way to address the epidemic as the SARS-CoV-2 variants of concern (VOCs) posed an increased risk to global public health. The spike protein of the SARS-CoV-2 virus was usually used as the main antigen in many types of vaccines to produce neutralizing antibodies against the virus. However, the spike (S) proteins of different variants were only differentiated by a few amino acids, making it difficult to obtain specific antibodies that can distinguish different VOCs, thereby challenging the accurate distinction and quantification of the variants using immunological methods such as ELISA. Here, we established a method based on LC-MS to quantify the S proteins in inactivated monovalent vaccines or trivalent vaccines (prototype, Delta, and Omicron strains). By analyzing the S protein sequences of the prototype, Delta, and Omicron strains, we identified peptides that were different and specific among the three strains and synthesized them as references. The synthetic peptides were isotopically labeled as internal targets. Quantitative analysis was performed by calculating the ratio between the reference and internal target. The verification results have shown that the method we established had good specificity, accuracy, and precision. This method can not only accurately quantify the inactivated monovalent vaccine but also could be applied to each strain in inactivated trivalent SARS-CoV-2 vaccines. Hence, the LC-MS method established in this study can be applied to the quality control of monovalent and multivalent SARS-CoV-2 variation vaccines. By enabling more accurate quantification, it will help to improve the protection of the vaccine to some extent.

Safety, immunogenicity, and lot-to-lot consistency of live attenuated varicella vaccine in 1-3 years old children: a double-blind, randomized phase III trial.

The study was to evaluate the safety, immunogenicity and lot-to-lot consistency of live attenuated varicella vaccine in Chinese population aged 1-3 years. The double-blind, randomized phase III trial was conducted in Henan Province, China. In total, 1197 subjects were included in this study. Subjects were randomly assigned into four groups in a 2:2:2:1 ratio to receive one of the three lots of commercial scale (CS) vaccine or the licensed pilot scale (LPS) vaccine. Seroconversion rate and neutralizing antibody titers (NATb) were assessed at day 0 pre-vaccination and at day 30 post-vaccination. Safety data were recorded for 30 days post-vaccination. After vaccination, the geometric mean titers (GMTs) of the three CS groups were 25.04 (95% confidence interval [CI], 22.85 to 27.44), 24.47 (95% CI, 22.35 to 26.78) and 25.88 (95% CI, 23.61 to 28.36), respectively (P= 0.6928). The ratio of GMTs adjusted for covariates of each pair of lots were all between 0.67 to 1.50 in susceptible subjects. The difference of seroconversion rate between pooled CS group and LPS group was 3.82 (95% CI, 0.55 to 8.81). Meanwhile, the percentage of solicited local, systemic and unsolicited adverse reactions showed no difference across the four groups, and most of the adverse reactions were mild or moderate in intensity. The CS group was comparable to the LPS group in safety and immunogenicity. The consistency of three consecutive CS lots was reliable. Moreover, the CS group was non-inferior to the LPS group.

Etiological analysis of clinical cases of live attenuated varicella vaccine / 中国生物制品学杂志

@#Objective To analyze the etiology of clinical cases of live attenuated varicella vaccine.Methods 64 samples of varicella vesicle fluid from 49 patients clinically diagnosed as varicella cases in phase Ⅲ clinical trial of live attenuated varicella vaccine in enterprises(the test site was Henan Province) were collected,extracted for DNA,and distinguished for wild-type strains and vaccine strains by PCR and restriction fragment length polymorphism(PCR-RFLP).Genotype was analyzed using the genotyping method recommended at the international(varicella-zoster virus,VZV) nomenclature meeting2008.Results 64 vesicle fluids were all wild-type strains(Pst Ⅰ~+Sma Ⅰ~-BssH Ⅱ~-Nae Ⅰ~-),and no vaccine-related cases occurred.All 49 isolates belonged to Clade 2.Additionally,compared with Clade 2,a synonymous mutation(T→C) in SNP18 082 was detected in all 49 isolates,and a mutation(C→A) in SNP790 was detected in one isolate.Conclusion In the clinical trial of live attenuated varicella vaccine in Henan Province,all the clinical cases were caused by infection of wild-type strain which belonged to Clade 2 genetic branch.

Bactericidal activity of strong acidic hypochlorous water against Escherichia coli O157:H7 and Listeria monocytogenes in biofilms attached to stainless steel.

This study aims to investigate the bactericidal activity of strong acidic hypochlorous water (SAHW) against Escherichia coli O157:H7 and L. monocytogenes in bacterial biofilms. The bactericidal activity of SAHW against both bacteria in colony biofilm increased with the elevation of the available chlorine concentration (ACC) and extension of the treatment time. The survived cell counts of E. coli O157:H7 and L. monocytogenes in the biofilms were significantly (p < 0.05) decreased compare to tap water at more than 30 mg/L of ACC in SAHW and 15 s of treatment time. E. coli O157:H7 and L. monocytogenes in the biofilms reduced to less than the detection limit by treatment of 50 mg/L of ACC in SAHW for 300 and 600 s, respectively. SAHW may be a potential disinfecting agent for removing bacterial biofilms from food processing equipment and other facilities.

Trehalase regulates neuroepithelial stem cell maintenance and differentiation in the Drosophila optic lobe.

As one of the major hydrolases in Drosophila, trehalase (Treh) catalyzes the hydrolysis of trehalose into glucose providing energy for flight muscle activity. Treh is highly conserved from bacteria to humans, but little is known about its function during animal development. Here, we analyze the function of Treh in Drosophila optic lobe development. In the optic lobe, neuroepithelial cells (NEs) first divide symmetrically to expand the stem cell pool and then differentiate into neuroblasts, which divide asymmetrically to generate medulla neurons. We find that the knockdown of Treh leads to a loss of the lamina and a smaller medulla. Analyses of Treh RNAi-expressing clones and loss-of-function mutants indicate that the lamina and medulla phenotypes result from neuroepithelial disintegration and premature differentiation into medulla neuroblasts. Although the principal role of Treh is to generate glucose, the Treh loss-of-function phenotype cannot be rescued by exogenous glucose. Thus, our results indicate that in addition to being a hydrolase, Treh plays a role in neuroepithelial stem cell maintenance and differentiation during Drosophila optic lobe development.

Evaluation of bactericidal activity of weakly acidic electrolyzed water (WAEW) against Vibrio vulnificus and Vibrio parahaemolyticus.

Vibrio parahaemolyticus and Vibriovulnificus cause severe foodborne illness in humans; thus, to reduce outbreaks of disease, it is clearly important to reduce food contamination by these pathogens. Although electrolyzed oxidizing (EO) water has been reported to exhibit strong bactericidal activities against many pathogens, it has never been tested against V. vulnificus and V. parahaemolyticus. The purpose of this study was to evaluate the bactericidal activity of weakly acidic electrolyzed water (WAEW), a type of EO water, against V. vulnificus and V. parahaemolyticus. Cell suspensions and cell cultures of both pathogens were treated for 30s with sodium hypochlorite solution containing 35mg/L available chlorine concentration (ACC) or WAEW containing 35mg/L ACC. After an initial inoculum of 5.7logCFU/mL, the number of viable V. vulnificus cells was reduced by 2.2 logs after treatment for 60s with sodium hypochlorite solution containing 35mg/L ACC, while no cells survived treatment with WAEW for 30s. Similar results were obtained for V. parahaemolyticus. Under open storage conditions, WAEW maintained bactericidal activities against cell suspensions of both strains after 5weeks but disappeared against cell cultures of the two strains after 5weeks. Under closed storage conditions, however, WAEW maintained bactericidal activities against both cell suspensions and cell cultures of each strain after 5weeks. No cells were detected in the cell suspensions and cultures when the ACC of WAEW was more than 20mg/L and treatment time was greater than 15s. Bactericidal activity of WAEW against V. vulnificus cell culture was reduced when the ACC of WAEW was less than 15mg/L but was maintained in the V. vulnificus cell suspension when the ACC of WAEW was 0.5mg/L. Thus, the bactericidal activity of WAEW was primarily affected by ACC rather than treatment time. Similar results were obtained for V. parahaemolyticus, indicating that WAEW kills these microorganisms more quickly than a chemical product such as sodium hypochlorite (NaClO), even at equivalent ACCs.

Novel alpha-conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity.

Conotoxins (CTX) from the venom of marine cone snails (genus Conus) represent large families of proteins, which show a similar precursor organization with surprisingly conserved signal sequence of the precursor peptides, but highly diverse pharmacological activities. By using the conserved sequences found within the genes that encode the alpha-conotoxin precursors, a technique based on RT-PCR was used to identify, respectively, two novel peptides (LiC22, LeD2) from the two worm-hunting Conus species Conus lividus, and Conus litteratus, and one novel peptide (TeA21) from the snail-hunting Conus species Conus textile, all native to Hainan in China. The three peptides share an alpha4/7 subfamily alpha-conotoxins common cysteine pattern (CCX(4)CX(7)C, two disulfide bonds), which are competitive antagonists of nicotinic acetylcholine receptor (nAChRs). The cDNA of LiC22N encodes a precursor of 40 residues, including a propeptide of 19 residues and a mature peptide of 21 residues. The cDNA of LeD2N encodes a precursor of 41 residues, including a propeptide of 21 residues and a mature peptide of 16 residues with three additional Gly residues. The cDNA of TeA21N encodes a precursor of 38 residues, including a propeptide of 20 residues and a mature peptide of 17 residues with an additional residue Gly. The additional residue Gly of LeD2N and TeA21N is a prerequisite for the amidation of the preceding C-terminal Cys. All three sequences are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The properties of the alpha4/7 conotoxins known so far were discussed in detail. Phylogenetic analysis of the new conotoxins in the present study and the published homologue of alpha4/7 conotoxins from the other Conus species were performed systematically. Patterns of sequence divergence for the three regions of signal, proregion, and mature peptides, both nucleotide acids and residue substitutions in DNA and peptide levels, as well as Cys codon usage were analyzed, which suggest how these separate branches originated. Percent identities of the DNA and amino acid sequences of the signal region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between inter- and intra-species. Notably, the diversity of the proregion was also high, with an intermediate percentage of divergence between that observed in the signal and in the toxin regions. The data presented are new and are of importance, and should attract the interest of researchers in this field. The elucidated cDNAs of these toxins will facilitate a better understanding of the relationship of their structure and function, as well as the process of their evolutionary relationships.