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Severe acute respiratory syndrome (SARS)-coronavirus (CoV), Middle Eastern respiratory syndrome (MERS)-CoV, and SARS-CoV-2 have seriously threatened human life in the 21st century. Emerging and re-emerging β-coronaviruses after the coronavirus disease 2019 (COVID-19) epidemic remain possible highly pathogenic agents that can endanger human health. Thus, pan-β-coronavirus vaccine strategies to combat the upcoming dangers are urgently needed. In this study, four LNP-mRNA vaccines, named O, D, S, and M, targeting the spike protein of SARS-CoV-2 Omicron, Delta, SARS-CoV, and MERS-CoV, respectively, were synthesized and characterized for purity and integrity. All four LNP-mRNAs induced effective cellular and humoral immune responses against the corresponding spike protein antigens in mice. Furthermore, LNP-mRNA S and D induced neutralizing antibodies against SARS-CoV and SARS-CoV-2, which failed to cross-react with MERS-CoV. Subsequent evaluation of sequential and cocktail immunizations with LNP-mRNA O, D, S, and M effectively elicited broad immunity against SARS-CoV-2 variants, SARS-CoV, and MERS-CoV. A direct comparison of the sequential with cocktail regimens indicated that the cocktail vaccination strategy induced more potent neutralizing antibodies and T-cell responses against heterotypic viruses as well as broader antibody activity against pan-β-coronaviruses. Overall, these results present a potential pan-β-coronavirus vaccine strategy for improved preparedness prior to future coronavirus threats.

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost. Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40-44 h. Much higher cell density (8 × 10 6 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process. Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic. The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 10 6 cells/mL., (Copyright © 2019. Published by Elsevier Ltd.)

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by severe fever with thrombocytopenia syndrome virus (SFTSV). SFTS is mainly characterized by severe fever with thrombocytopenia and has a high mortality rate. The virus has been found in China, South Korea, and Japan. Effective antiviral drugs or vaccines still have been unavailable. Now, two vaccine manufacturers in China are actively engaged in the development of the vaccine. To promote the development of SFTS vaccines and ensure their effective quality control, we developed national antigen and antibody references. We collaborative calibrated the standards; evaluated the homogeneity and stability of the national SFTS standards. The national SFTS vaccine antigen and antibody references met the Chinese national standards and can be used to standardize quality control for the manufacture of SFTS vaccines. And also can be used into the study the dose-response relationship of SFTS vaccines, determine clinical doses, and evaluate vaccine immunogenicity., (Copyright © 2019. Published by Elsevier Ltd.)

Neutralizing antibodies (NTAbs) is a major criterion for evaluation the immunogenicity of many vaccines, for example, poliovirus and EV71 vaccine. Here, we firstly discovered that polyclonal antibodies induced by inactivated CVA16 vaccine and lived CVA16 virus have poor ability to neutralize circulating CVA16 strains in vitro. However, the passive transfer of poorly neutralizing polyclonal antibodies can protect suckling mice from lethally challenged with circulating strains in vivo. In addition, the obvious dose response was found between the titer of antibodies and the survival rate. Interestingly, poorly neutralizing polyclonal antibodies against circulating CVA16 strains, have good ability to neutralize prototype strain G10 in vitro. Between G10 and circulating CVA16 strains, there are total 47 variant sites in capsid, which are near the interface of VP1, VP2, and VP3, and close to 2-fold axis. Based on the structure of CVA16, the obvious structural changes were observed in residue 213 of VP1 GH loop, residue 139 of VP2 EF loop, and residues 59, 182 and 183 of VP3 GH loop. What we found may provide a new sight for the development of CVA16 vaccine.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by a phlebovirus of the Bunyaviridae family, which is designated as SFTS virus (SFTSV). To our knowledge, no efficient SFTSV vaccine exists. Here, we report the identification of a standard virus strain for the eight major SFTSV strains circulating in China for use in evaluating the SFTSV vaccine. Rabbits were immunized with the SFTSV strains and the cross-neutralization capacities of SFTSV anti-sera were determined in microculture cytopathic effect (CPE)-inhibition assays. The mean cross-neutralization capacity of the eight SFTSV anti-sera ranged from 62.4 to 142.6%, compared to autologous strains. The HB29 strain demonstrated strong cross-reactivity with heterologous antibodies, and 33 serum samples from SFTS patients efficiently neutralized HB29, suggesting its broad cross-reactivity. In addition, HB29 demonstrated good replication in Vero and MRC-5 cells (8.0 and 6.0 lg 50% cell culture-infectious dose/mL, respectively) and significant CPE, which satisfied the requirements for a standard virus strain. The HB29 isolate was proven identical to the reported HB29 strain by DNA sequencing, and showed high homology in the S segments with other SFTSV strains (94.8-99.7%). Our results suggest that HB29 may be the best candidate standard strain for use in SFTS vaccine development in China., (Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Background:  This study evaluated the 2-year efficacy, immunogenicity, and safety of the Vigoo enterovirus 71 (EV71) vaccine., Method:  In an initial phase 3 study, we randomly assigned healthy infants and children aged 6-35 months (ratio, 1:1) to receive 2 doses of either EV71 vaccine (5120 participants) or placebo (5125 participants) at days 0 and 28, and followed them for 12 months after vaccination. In this extended follow-up study, we continued to evaluate the efficacy, immunogenicity, and safety of the EV71 vaccine for up to 2 years., Results:  Overall efficacy was 94.84% (95% confidence interval [CI], 83.53%-98.38%) during the 2-year follow-up period (P < .0001), and the vaccine efficacy during the second year was 100.00% (95% CI, 84.15%-100.00%) against EV71-associated hand-foot-and-mouth disease (HFMD; P < .0001). Geometric mean titers of neutralizing antibody in participants remained high during the 2-year follow-up period, and no vaccine-related serious adverse events were recorded., Conclusions:  Two doses of Vigoo EV71 vaccine could provide sustained protection against EV71-associated HFMD in healthy Chinese children., Clinical Trials Registration:  NCT01508247., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background: Enterovirus 71 (EV71) is the etiologic agent of hand-foot-and-mouth disease (HFMD) in the Asia-Pacific region, Many strategies have been applied to develop EV71 vaccines but no vaccines are currently available. Mucosal immunization of the VP1, a major immunogenic capsid protein of EV71, may be an alternative way to prevent EV71 infection., Results: In this study, mucosal immunogenicity and protect function of recombinant VP1 protein (rVP1) in formulation with chitosan were tested and assessed in female ICR mouse model. The results showed that the oral immunization with rVP1 induced VP1-specific IgA antibodies in intestine, feces, vagina, and the respiratory tract and serum-specific IgG and neutralization antibodies in vaccinated mice. Splenocytes from rVP1-immunized mice induced high levels of Th1 (cytokine IFN-γ), Th2 (cytokine IL-4) and Th3 (cytokine TGF-β) type immune responses after stimulation. Moreover, rVP1-immunized mother mice conferred protection (survival rate up to 30%) on neonatal mice against a lethal challenge of 103 plaque-forming units (PFU) EV71., Conclusions: These data indicated that oral immunization with rVP1 in formulation with chitosan was effective in inducing broad-spectrum immune responses and might be a promising subunit vaccine candidate for preventing EV71 infection.

Background: Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD). Three inactivated EV71 whole-virus vaccines of different strains developed by different manufacturers in mainland China have recently entered clinical trials. Although several studies on these vaccines have been published, a study directly comparing the immunogenicity and protective effects among them has not been carried out, which makes evaluating their relative effectiveness difficult. Thus, properly comparing newly developed vaccines has become a priority, especially in China., Methods and Findings: This comparative immunogenicity study was carried out on vaccine strains (both live and inactivated), final container products (FCPs) without adjuvant, and corresponding FCPs containing adjuvant (FCP-As) produced by three manufacturers. These vaccines were evaluated by neutralizing antibody (NAb) responses induced by the same or different dosages at one or multiple time points post-immunization. The protective efficacy of the three vaccines was also determined in one-day-old ICR mice born to immunized female mice. Survival rates were observed in these suckling mice after challenge with 20 LD(50) of EV71/048M3C2. Three FCP-As, in a dose of 200 U, generated nearly 100% NAb positivity rates and similar geometric mean titers (GMTs), especially at 14-21 days post-inoculation. However, the dynamic NAb responses were different among three vaccine strains or three FCPs. The FCP-As at the lowest dose used in clinical trials (162 U) showed good protective effects in suckling mice against lethal challenge (90-100% survival), while the ED(50) of NAb responses and protective effects varied among three FCP-As., Conclusions: These studies establish a standard method for measuring the immunogenicity of EV71 vaccines in mice. The data generated from our mouse model study indicated a clear dose-response relationship, which is important for vaccine quality control and assessment, especially for predicting protective efficacy in humans when combined with future clinical trial results.

Enterovirus 71 (EV71) is a highly infectious agent that causes hand-foot-mouth disease (HFMD) in humans. Effective vaccination against EV71 infection is critically important, given the recent outbreak of HFMD in the Asia-Pacific region, where it has shown significant mortality and morbidity. There is currently no approved anti-viral therapy available to treat the disease. While several vaccine manufacturers are actively developing EV71 vaccines, there are no international reference standards available to conduct quality control on EV71 vaccines or to assess the effectiveness of EV71 vaccines in immunized populations. In the current report, antigen reference standard based on the C4 subtype of the EV71 vaccine strain was developed. In addition, neutralizing antibody (NTAb) reference panels were analyzed and standards with various neutralizing titers were selected. These reference antigens were used to calibrate vaccine samples from several producers and found that five EV71 antigens and the national reference standards showed good linearity and parallelism. Moreover, mice immunized with various vaccines at doses standardized by these national references showed comparable NTAb responses. Finally, the national NTAb reference panels were found to effectively reduce assay discrepancy between different labs. Taken together, these national reference standards are highly valuable for the standardization and evaluation of EV71 vaccines., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Enterovirus 71 (EV71) has led to recent outbreaks of hand, foot and mouth disease (HFMD) in China, resulting in high mortality. In this study, several monoclonal antibodies were generated by immunizing mice with two synthetic peptides, SP55 and SP70, containing amino acids 163-177 and 208-222 of VP1. The specificities of the anti-EV71 peptide monoclonal antibodies were confirmed by Western blot analysis and immunocytochemistry against EV71 virus. Most importantly, we have identified a monoclonal antibody, clone 22A12, which shows strong neutralizing activity against EV71 in an in vitro neutralization assay. Because there is no vaccine available and treatment is very limited, mouse anti-EV71 monoclonal antibody, clone 22A12, could be a promising candidate to be humanized and used for treatment of EV71 infection.

A liquid chromatography–tandem mass spectrometry method was developed to determine nine types of capsid proteins simultaneously in nine‐valent human papillomavirus vaccines. Signature peptides were optimized in terms of specificity, repeatability, determination accuracy and sensitivity. As a result, three signature peptides per capsid protein were obtained. The linear calibration curves were achieved in the range of 11.6–373.6 nmol/L (R2 > 0.998). Compared to our previous liquid chromatography–tandem mass spectrometry method, the current method was more sensitive (3.18‐fold) and it can be used for quality evaluation of nine‐valent human papillomavirus vaccines, unlike the previous method, which could only be used for bivalent human papillomavirus vaccines. Then, they were utilized to determine nine types of capsid proteins in nine‐valent human papillomavirus vaccines from four different manufactures. Intraday and interday precision values for the determination of capsid proteins in nine‐valent human papillomavirus vaccines were less than 6.8 and 9.1%, respectively. Recovery rates of all capsid proteins investigated were in the range of 80–120%. In addition, the current assay was used for determination of free capsid protein in nine‐valent human papilloma virus vaccines, and the results were used to evaluate the adsorption rate of the adjuvant. [ABSTRACT FROM AUTHOR]