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Since the outbreak of COVID-19, a variety of vaccine platforms have been developed. Amongst these, inactivated vaccines have been authorized for emergency use or conditional marketing in many countries. To further enhance the protective immune responses in populations that have completed vaccination regimen, we investigated the immunogenic characteristics of different vaccine platforms and tried homologous or heterologous boost strategy post two doses of inactivated vaccines in a mouse model. Our results showed that the humoral and cellular immune responses induced by different vaccines when administered individually differ significantly. In particular, inactivated vaccines showed relatively lower level of neutralizing antibody and T cell responses, but a higher IgG2a/IgG1 ratio compared with other vaccines. Boosting with either recombinant subunit, adenovirus vectored or mRNA vaccine after two-doses of inactivated vaccine further improved both neutralizing antibody and Spike-specific Th1-type T cell responses compared to boosting with a third dose of inactivated vaccine. Our results provide new ideas for prophylactic inoculation strategy of SARS-CoV-2 vaccines.

The outbreak of COVID-19 has posed a huge threat to global health and economy. Countermeasures have revolutionized norms for working, socializing, learning, and travel. Importantly, vaccines have been considered as most effective tools to combat with COVID-19. As of the beginning of 2021, >200 COVID-19 vaccine candidates, covering nearly all existing technologies and platforms, are being research and development (R&D) by multiple manufacturers worldwide. This has posed a huge obstacle to the quality control and evaluation of those candidate vaccines, especially in China, where five vaccine platforms are deployed in parallel. To accelerate the R&D progress of COVID-19 vaccines, the guidances on R&D of COVID-19 vaccine have been issued by National Regulatory Authorities or organizations worldwide. The Center for Drug Evaluation and national quality control laboratory in China have played a leading role in launching the research on quality control and evaluation in collaboration with relevant laboratories involved in the vaccine R&D, which greatly supported the progression of vaccines R&D, and accelerated the approval for emergency use and conditional marketing of currently vaccine candidates. In this paper, the progress and experience gained in quality control and evaluation of COVID-19 vaccines developed in China are summarized, which might provide references for the R&D of current and next generation of COVID-19 vaccines worldwide.

To cope with the decline in COVID-19 vaccine-induced immunity caused by emerging SARS-CoV-2 variants, a heterologous immunization regimen using chimpanzee adenovirus vectored vaccine expressing SARS-CoV-2 spike (ChAd-S) and an inactivated vaccine (IV) was tested in mice and non-human primates (NHPs). Heterologous regimen successfully enhanced or at least maintained antibody and T cell responses and effectively protected against SARS-CoV-2 variants in mice and NHPs. An additional heterologous booster in mice further improved and prolonged the spike-specific antibody response and conferred effective neutralizing activity against the Omicron variant. Interestingly, priming with ChAd-S and boosting with IV reduced the lung injury risk caused by T cell over activation in NHPs compared to homologous ChAd-S regimen, meanwhile maintained the flexibility of antibody regulation system to react to virus invasion by upregulating or preserving antibody levels. This study demonstrated the satisfactory compatibility of ChAd-S and IV in prime-boost vaccination in animal models.

Ischemia/reperfusion (I/R) injury is a serious clinical condition characterized by high morbidity and mortality rates. Donepezil plays a neuroprotective role in I/R‑associated diseases. The aim of the present study was to investigate the role and the potential mechanism of action of donepezil in I/R‑induced myocardial microvascular endothelial cell dysfunction. An I/R model was simulated using oxygen‑glucose deprivation/reoxygenation (OGD/R) injury in human cardiac microvascular endothelial cells (CMECs). Cell viability and lactate dehydrogenase release were examined following treatment with donepezil. Commercial kits were used to evaluate cell apoptosis, cell permeability and caspase‑3 activity. The expression levels of apoptosis‑associated proteins, as well as proteins found in tight junctions or involved in the poly(ADP‑ribose) polymerase 1 (PARP1)/NF‑κB pathway, were measured using western blotting. These parameters were also examined following PARP1 overexpression. The results demonstrated that donepezil increased cell viability and reduced toxicity in OGD/R‑treated CMECs. The apoptotic rate, caspase‑3 activity and protein expression levels of Bax and cleaved caspase‑3 were significantly reduced following donepezil treatment, which was accompanied by Bcl‑2 upregulation. Moreover, cell permeability was notably reduced, coupled with a marked increase in the expression of tight junction‑associated proteins. The expression levels of proteins related to PARP1/NF‑κB signaling were significantly downregulated in CMECs following donepezil treatment. However, the protective effects of donepezil on OGD/R‑induced CMEC injury were reversed following PARP1 overexpression. In conclusion, donepezil suppressed OGD/R‑induced CMEC dysfunction via PARP1/NF‑κB signaling. This finding provided insight into the mechanism underlying myocardial I/R injury.

Although enormous success has been achieved with anti-PD-1/PD-L1 and anti-CTLA-4 monoclonal antibodies (mAbs), their unsatisfactory response rate in cancer patients has been driving the research and development of novel immune checkpoint inhibitors (ICIs). Anti-LAG-3 mAbs, as one of the most promising candidates, are now being tested for various human cancers at different stages of clinical trials. Here, we describe the development and validation of a reporter gene assay (RGA) to measure the bioactivity of anti-LAG-3 mAbs. We established the bioassay based on parental Raji cells and a Jurkat cell line stably transfected with human LAG-3 gene and luciferase reporter elements controlled by nuclear factor of activated T cell (NFAT) from the IL-2 promoter. After optimization of key parameters, the established RGA showed excellent precision, specificity, accuracy, and stability. The mechanism of action (MOA) relatedness and the excellent assay performance make the RGA suitable for the characterization, lot release, and stability test of anti-LAG-3 mAbs.

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.

CTLA-4 is an important immune checkpoint for the regulation of T cell activation, and anti-CTLA-4 monoclonal antibodies (mAbs) are being developed as mono- or combination therapy for various tumors with reliable clinical efficacy. Ipilimumab is the first approved inhibitor of immune checkpoint, and many other anti-CTLA-4 mAbs, including ipilimumab biosimilars, are in different stages of clinical trials. However, due to the immunomodulating nature of the mAbs targeting CTLA-4, mode of action (MoA) and cell-based bioassay to determine their bioactivities as the lot release or stability test has been a great challenge to quality control laboratories. In this study, we have developed and validated a reporter gene assay (RGA), in which two kinds of cell lines were engineered to measure the bioactivity of anti-CTLA-4 mAbs. Raji cells were stably transfected with the membrane-anchored anti-CD3 single chain antibody fragment (scFv) as antigen-presenting cells (APCs, Raji-CD3scFv cells), while Jurkat cells were stably transfected with CTLA-4 with Y201V mutation and NFAT controlled luciferase as the effector cells (Jurkat-CTLA-4-NFAT-luc cells). The ligation of CD80/CD86 on the APCs with CTLA-4 could reduce the luciferase expression accompanied with the activation of effector cells, while the anti-CTLA-4 mAb could reverse the reduction, which resulted in good dose response curve to determine its bioactivity. After optimizing various assay conditions, we performed full validation according to ICH-Q2 (R1), which demonstrated the excellent specificity, accuracy, precision, linearity, and the cell passage stability. The satisfied performance characteristics render the RGA a good bioassay in the bioactivity determination of anti-CTLA-4 mAbs, as applied in characterization, batch release control, stability study, and biosimilar assessment.

The emerging variants of severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) in pandemic call for the urgent development of universal corona virus disease 2019 (COVID‐19) vaccines which could be effective for both wild‐type SARS‐CoV‐2 and mutant strains. In the current study, we formulated protein subunit vaccines with AS03 adjuvant and recombinant proteins of S1 subunit of SARS‐CoV‐2 (S1‐WT) and S1 variant (K417N, E484K, N501Y, and D614G) subunit (S1‐Mut), and immunized transgenic mice that express human angiotensin‐converting enzyme 2 (hACE2). The S1 protein‐specific antibody production and the neutralization capability for SARS‐CoV‐2 and B.1.351 variant were measured after immunization in mice. The results revealed that the S1‐Mut antigens were more effective in inhibiting the receptor‐binding domain and ACE2 binding in B.1.351 variant than in wild‐type SARS‐CoV‐2. Furthermore, the development of a bivalent vaccine exhibited the ideal neutralization properties against wild‐type and B.1.351 variant, as well as other variants. Our findings may provide a rationale for the development of a bivalent recombinant vaccine targeting the S1 protein that can induce the neutralizing antibodies against both SARS‐CoV‐2 variants and wild‐type of the virus and may be of importance to explore the potential clinical use of bivalent recombinant vaccine in the future., The bivalent recombinant vaccine composed of S1 subunit of SARS‐CoV‐2 and S1 variant (K417N, E484K, N501Y, and D614G) subunit could induce the neutralizing antibodies against both SARS‐CoV‐2 variants and wild‐type of the virus.

The world has entered the second wave of the COVID-19 pandemic, and its intensity is significantly higher than that of the first wave of early 2020. Many countries or regions have been forced to start the second round of lockdowns. To respond rapidly to this global pandemic, dozens of COVID-19 vaccine candidates have been developed and many are undergoing clinical testing. Evaluating and defining effective vaccine candidates for human use is crucial for prioritizing vaccination programs against COVID-19. In this review, we have summarized and analyzed the efficacy, immunogenicity and safety data from clinical reports on different COVID-19 vaccines. We discuss the various guidelines laid out for the development of vaccines and the importance of biological standards for comparing the performance of vaccines. Lastly, we highlight the key remaining challenges, possible strategies for addressing them and the expected improvements in the next generation of COVID-19 vaccines.

Influenza vaccine potency is determined by the quantification of immunologically active hemagglutinin capable of eliciting neutralizing antibodies upon immunization. Currently, the single radial immunodiffusion (SRID) method is the standard in vitro potency assay used for lot release of seasonal inactivated influenza vaccines. Despite the proven usage of SRID, significant limitations such as the time-consuming preparation of reagents and limited dynamic range warrant the need for the development of alternative potency assays. Such alternative approaches need to discriminate and quantify relevant hemagglutinin material, provide strain identity, and be independent of strain-specific and seasonal reagents. Herein, we present a proof of concept method that combines the capture of conformationally well-folded hemagglutinin via a sialic acid binding step with the resolving power of reversed-phase high-performance liquid chromatography for strain identity and determination. Details of the protocol for the selective capture of receptor-binding hemagglutinin, its release from the receptor, and its relative determination are presented. This approach was found to provide flexibility for the reagents to be used and was adaptable to varying strain compositions of influenza vaccines. This proof of concept approach was developed as an antibody-independent methodology.

Background The high cost and insufficient supply of human papillomavirus (HPV) vaccines have slowed the pace of controlling cervical cancer. A phase III clinical trial was conducted to evaluate the efficacy, safety, and immunogenicity of a novel Escherichia coli-produced bivalent HPV-16/18 vaccine. Methods A multicenter, randomized, double-blind trial started on November 22, 2012 in China. In total, 7372 eligible women aged 18–45 years were age-stratified and randomly assigned to receive three doses of the test or control (hepatitis E) vaccine at months 0, 1, and 6. Co-primary endpoints included high-grade genital lesions and persistent infection (over 6 months) associated with HPV-16/18. The primary analysis was performed on a per-protocol susceptible population of individuals who were negative for relevant HPV type-specific neutralizing antibodies (at day 0) and DNA (at day 0 through month 7) and who received three doses of the vaccine. This report presents data from a prespecified interim analysis used for regulatory submission. Results In the per-protocol cohort, the efficacies against high-grade genital lesions and persistent infection were 100.0% (95% confidence interval = 55.6% to 100.0%, 0 of 3306 in the vaccine group vs 10 of 3296 in the control group) and 97.8% (95% confidence interval = 87.1% to 99.9%, 1 of 3240 vs 45 of 3246), respectively. The side effects were mild. No vaccine-related serious adverse events were noted. Robust antibody responses for both types were induced and persisted for at least 42 months. Conclusions The E coli-produced HPV-16/18 vaccine is well tolerated and highly efficacious against HPV-16/18–associated high-grade genital lesions and persistent infection in women.

AP25 is an anti-tumor peptide with a high affinity for integrins. It exerts its anti-tumor activity by inhibiting angiogenesis and by directly inhibiting the growth of tumor cells. Its half-life time in vivo is only about 50 minutes, which limits its clinical application. In order to prolong the half-life time of AP25 while preserving its anti-tumor activity, several fusion proteins of AP25 and IgG4 Fc were designed and expressed; their anti-tumor activity and pharmacokinetics properties were evaluated. Firstly, four AP25-Fc fusion protein sequences were designed, and the corresponding proteins were expressed and purified. Based on the results of HUVEC migration inhibition assay, HUVEC and tumor cell proliferation inhibition assay and yields of expression by HEK293 cells, the fusion protein designated PSG4R was selected for further evaluation. The anti-tumor effect of PSG4R was then evaluated in vivo on HCT-116 nude mice xenograft model. And the pharmacokinetics properties of PSG4R were investigated in rats. The results showed that PSG4R could inhibit the growth of xenografts of human colon cancer cell line HCT-116 in nude mice by intravenous administration of 40 mg/kg once every two days. The half-life time of PSG4R was 56.270 ± 15.398 h. This study showed that the construction of AP25-Fc fusion protein could significantly prolong the half-life of AP25 while retaining its anti-tumor activity, which provides a new direction for new drug development of AP25.

We studied Phellinus lonicerinus to determine the cytotoxic effect and the dual estrogenic activities of methyl-hispolon and their relation to estrogen signals in vivo and in vitro. The Glide scores of methyl-hispolon-estrogen receptor α (ERα) and methyl-hispolon-ERβ docked complexes were -7.29 kcal/mol and -6.68 kcal/mol in docking simulations. Methyl-hispolon had a significant antiproliferative effect for estrogen-sensitive ER(+) MCF-7 cells in the absence of estrogen, and it exhibited dual estrogen activities. Methyl-hispolon increased the serum E2 in rats with premature ovarian failure and fulfilled the estrogenic function in the uterus and ovary. Methyl-hispolon significantly inhibited the expression of Ras, API, ERα, C-myc, and cyclinDl, as well as their gene transcription in RL95-2 cells. The phosphorylation of ERK1/2 was inhibited by methyl-hispolon. Thus, methyl-hispolon has potential use in treating estrogen deficiency-related diseases, with good antitumor effects and estrogenic activity.

A rapid shrinkage of Daihai Lake was found in recent decades. The present study analyzed the characteristics of Daihai Lake shrinkage and quantified the contribution of climate and human activities. The results of Mann-Kendall- Sneyers test and moving t-test showed that there was an obvious mutation point of lake level in 2006 and the descending speed of Daihai Lake level post-2006 (-0.46m/a) was 3.22 times that of pre-2006 (-0.14m/a). The centroid of Daihai Lake moved 1365.18 m from southwest to northeast during 1989 ~ 2018 with an average speed of 47.08 m/a. The results of Mann-Kendall trend test revealed that the annual evaporation showed a significant downward trend with a rate of approximately -5.33 mm/a, while no significant trend was found in precipitation. Daihai lake water level showed a very weak relationship with evaporation (r = 0.078, p < 0.01) and precipitation (p>0.05) respectively. Daihai Lake was influenced by human activities mainly from land use/ land cover, building reservoirs, pumping groundwater and directly consuming Daihai Lake water by Daihai power plant (DHPP). It was thought-provoking that DHPP began to consume Daihai lake water in 2006, which was consistent with abrupt change of Daihai lake level. The proportion of human impact was fluctuating upward. Human factors were the main factor of lake water reduction in last 10 years and the 5-year average contribution of human activities to Daihai Lake shrinkage was more than 61.99%. More attention and economic support should be given to prevent the continuous shrinkage of Daihai Lake.

Calcitonin gene-related peptide (CGRP) is critical for the pathophysiology of migraine, and four therapeutic antibodies targeting CGRP and its corresponding receptors have been approved by the Food and Drug Administration (FDA), while many others are in the different stages of clinical trials. Bioactivity determination is essential for the quality control and clinical application of therapeutic monoclonal antibodies (mAbs). However, no bioassay has been reported to date. In this study, we developed a reporter gene assay (RGA) based on SK-N-MC cells stably expressing firefly luciferase driven by cAMP response element (CRE). The key assay parameters were optimized according to signal-to-noise (SNR), the response value, and the fitted dose-response curve. Validation of the RGA in accordance with ICH-Q2 guidelines showed that the method had good specificity, accuracy, linearity, and precision. The established RGA can be utilized as a reference method for release testing and stability studies of relevant antibodies.

Background Exploring herbicide resistance mechanisms in weeds is regarded as an important part of designing resistance management strategies and rationalizing the use of herbicides. Beckmannia syzigachne is one of the most important agricultural weeds in China. Long-term use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has led to evolution of herbicide resistance in B. syzigachne. ACCase-inhibiting herbicides consist of three chemical families, including the aryloxyphenoxypropionates (APPs), cyclohexanediones (CHDs) and phenylpyraxoline (DENs). Results Based on whole-plant dose-response experiments, a B. syzigachne population (BS-R) was confirmed to be 20- and 12-fold resistant to the APP herbicides clodinafop-propargyl and quizalofop-P-ethyl; 2.2-, 2.8-, and 2.8-fold resistant to fenoxaprop-P-ethyl, the CHD herbicide sethoxydim and PPZ herbicide pinoxaden, respectively, compared to the susceptible counterpart (BS-S). Resistance to clodinafop-propargyl in the BS-R population could not be reversed by the known cytochrome P450 (CYP450) inhibitor malathion and glutathione S-transferase (GST) inhibitor 4-chloro-7-nitrobenzoxadiazole. In addition, no difference in CYP450 and GST activity was confirmed in BS-R and BS-S population. ACCase gene sequencing revealed an Ile-2041-Val mutation in the BS-R population. A derived cleaved amplified polymorphic sequence (dCAPS) marker was developed for rapid detection of the specific Ile-2041-Val mutation. Correlation quantification of resistance in homo- and hetro-resistant versus WT plants showed that the resistance to clodinafop-propargyl in this population is conferred by the Ile-2041-Val mutation. Conclusion Unlike the previous reports on the unique cross-resistance pattern by the 2041 mutation, this study demonstrates that the BS-R population confers resistance to certain ACCase-inhibiting APP, CHD and PPZ herbicides in B. syzigachne. This article is protected by copyright. All rights reserved.

The tumor necrosis factor alpha (TNF-α)/nuclear factor-kappa B (NF-κB) signaling pathway plays a crucial role in the pathogenesis of inflammatory diseases. Several therapeutic monoclonal antibodies (mAbs) and biosimilars against TNF-α have been developed to treat patients who suffer from inflammatory diseases caused by disordered expression of TNF-α. Hence, quality control of biopharmaceuticals is crucial during research and development. The high-order structure of these complex molecules cannot be entirely identified by physiochemical attributes; however, they can be inferred by observing biological activities. Thus, we developed a U937-based bioassay to determine the biological activities of mAbs and biosimilars against TNF-α using a low-basal NF-κB-inducible lentiviral reporter gene. The reporter gene assay (RGA) can be induced with a high signal-to-noise ratio (SNR) in a short time by TNF-α. Validation of the RGA showed accuracy (% relative standard deviation [RSD] = 4.64%), linearity (r2 = 0.9856), and precision (Interday RSD = 4.6%, between analysts RSD = 3.51%) as well as reasonable specificity and robustness. The measured potency values of a biosimilar to adalimumab were between 90% and 110%. Results showed our RGA is suitable for mAb quality control and lot release, and for evaluation of the biological activity similarity of the biosimilar.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.

Type 2 inflammatory cytokines, including IL-4, IL-5 and IL-13, contribute considerably to the pathogenesis of asthma. Anti-IL-4R monoclonal antibody (mAb) has been approved for the therapeutic treatment of asthma, and many mAbs with the same target are in the different stages of R&D and clinical trials. Bioactivity determination is required to ensure the quality control of mAbs. However, current ELISA and SPR assays or cell-based anti-proliferation assays for IL-4R mAbs are either not mechanism-of-action (MOA) representative or tedious and time consuming. Therefore, we developed a reporter gene assay (RGA) based on the HEK-293 cell line that stably expressed signal transducer and activator of transcription 6 (STAT6) and the luciferase reporter controlled by STAT6 binding elements. Anti-4R mAb could bind to IL-4R, and block the interaction between IL-4 and IL-4R, resulting in the reduction of IL-4 induced STAT6 controlled luciferase expression. After careful optimization of the experiment parameters, the RGA method demonstrated optimal dose-response curve between anti-IL-4R mAb concentration and luciferase expression level. Validation according ICH-Q2 proved the excellent assay performance characteristics of the established RGA, including specificity, accuracy, precision, linearity and range. The established transgenic cell line was stable for the bioactivity determination of anti-IL-4R mAb up to 46 generations, and the RGA was also suitable for the bioactivity determination of anti-IL-4 mAbs, and potentially of anti-IL-13 mAbs. The established RGA could be adopted to determine the bioactivity during the development, characterization, lot release, stability, and comparability studies of anti-IL-4R mAbs.

Environmental disturbances may result in dysregulation of interleukin-23 (IL-23), which is a crucial modulator of immunity. Several therapeutic monoclonal antibodies (mAbs) have been developed for treating IL-23-related autoimmune inflammation, such as ustekinumab, guselkumab, tildrakizumab, and risankizumab. Accurate bioactivity determination of therapeutic mAbs is essential for their quality control and clinical application. However, the current methods are tedious and complicated. In the present study, we employed low-background lentivirus carrying sis-inducible element (SIE)-driven firefly luciferase to generate a stable DB-SIE-Luc cell line that expresses endogenous IL-23 receptors and developed a sensitive and straightforward reporter gene assay (RGA) based on DB-SIE-Luc cells. After the optimization of various assay parameters, we set up a bioassay with the best fit of a four-parameter model and an appropriate signal-to-noise ratio (SNR) for bioactivity determination of guselkumab. We further verified the excellent assay performance characteristics of our RGA, including specificity, linearity, accuracy, precision, and stability, according to ICH-Q2. Taken together, we established a reliable and robust cell-based RGA, which potentially serves as a valubale alternative bioactivity determination assay for the release control and stability study of anti-IL-23 mAbs.

To overcome the lack of availability of fresh human whole blood for pyrogen detection, we explored the feasibility of utilizing cryopreserved pooled human blood to detect the responses of the pro-inflammatory cytokines IL-6 and IL-1β to LPS. Whole blood was obtained from five donors and incubated with LPS. The quantities of pro-inflammatory cytokines were measured using ELISA, and the results were compared among the samples. After the blood was cryopreserved with Dimethyl sulfoxide (DMSO) (10% v/v) and stored for 4 mo at –196℃, the detection limits of the IL-6/IL-1β responses to LPS were 0.2/0.4 endotoxin units (EU)/ml, respectively, and IL-6/IL-1β release increased in response to LPS in a dose-dependent manner. When these experiments were performed in three separate laboratories, the within-laboratory reproducibility of the IL-6/IL-1β responses was 100%/86.7%, 93.3%/100%, and 86.7%/80%, and the inter-laboratory reproducibility was 92.9%/85.7%, 64.3%/63.6%, and 57.1%/66.7%, respectively. The sensitivity (the probability of correctly classifying positive samples) and specificity (the probability of correctly classifying negative samples) of the IL-6/IL-1β tests were 81.7%/82.5% and 100%/100%, respectively. The results of this study suggest that cryopreserved pooled blood is a convenient and viable alternative for evaluating in vitro pyrogenicity. Additionally, maintaining cryopreserved pooled blood promotes safety for the user because it is released only after pretesting for infection parameters and has lower variation than fresh donations from a variety of donors.

The management of hand-foot-and-mouth disease(HFMD) epidemic is difficult due to the frequent emergence of non-EV71 and non-CVA16 enteroviruses and some cases testing negative for HFMD-associated causative agents. To clarify the virus spectrum of mild and severe HFMD, a comprehensive virome analysis of 238 samples was performed using next-generation sequencing (NGS). The data revealed total thirteen mammalian- and plant- virus families and diverse viral populations including enteroviruses, common respiratory viruses, diarrhea-related viruses, plant viruses and anelloviruses. A total of 18 viruses from 7 virus families were identified in severe cases, versus 37 viruses from 12 virus families in mild cases. Moreover, complicated mixed-infections of enteroviruses with common respiratory viruses were mainly found in severe cases(P = 0.013), while diarrhea-related viruses were mainly found in mild cases(P 0.001). This study provides the preliminary understanding of viromes both in mild and severe cases, which may benefit the detection of etiologic agents and prevention of HFMD.

Eosinophilic asthma is characterized by the eosinophilic inflammation with the allergen independent activation of Th2 lymphocytes. Since IL-5 plays an important role in the maturation, survival and migration of eosinophils, hence the pathogenesis of eosinophilic asthma, biotherapeutics targeting IL-5/IL-5Rα have been developed and/or marketed, including Mepolizumab, Reslizumab, and Benralizumab. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current mode of action (MOA) based method used in the quality control and stability tests for anti-IL-5 mAbs is anti-proliferation assay, which is tedious with long duration and high variation. We describe here the development and validation of a reporter gene assay (RGA), based on an IL-5-dependent TF-1 cell line variant we established that stably expresses the luciferase reporter under the control of STAT5 response elements. After careful optimization, we demonstrate the excellent specificity, precision, accuracy and linearity of the established RGA. Our study also proves that the assay is superior on precision, sensitivity and assay simplicity to the anti-proliferation assay. The established RGA is also applicable to another anti-IL-5Rα mAb. These results show for the first time that this novel RGA, based on the IL-5-IL-5R-STAT5 pathway, can be a valuable supplement to the anti-proliferation assay and employed in the bioactivity determination of anti-IL-5/anti-IL-5Rα biotherapeutics.

More than 100 monoclonal antibodies (mAbs) have been approved by FDA. The mechanism of action (MoA) involves in neutralization of a specific target via the Fab region and Fc effector functions through Fc region, while the latter include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). ADCP has been recognized one of the most important MoAs, especially for anti-cancer mAbs in recent years. However, traditional bioassays measuring ADCP always introduced primary macrophages and flow cytometry, which are difficult to handle and highly variable. In this study, we engineered a monoclonal Jurkat/NFAT/CD32a-FceRIγ effector cell line that stably expresses CD32a-FceRIγ chimeric receptor and NFAT-controlled luciferase. The corresponding mAb could bind with the membrane antigens on the target cells with its Fab fragment and CD32a-FceRIγ on the effector cells with its Fc fragment, leading to the crosslinking of CD32a-FceRIγ and the resultant expression of subsequent NFAT-controlled luciferase, which represents the bioactivity of ADCP based on the MoA of the mAb. With rituximab as the model mAb, Raji cells as the target cells, and Jurkat/NFAT/CD32a-FceRIγ cells as the effector cells, we adopted the strategy of Design of Experiment (DoE) to optimize the bioassay. Then we fully validated the established bioassay according to ICH-Q2(R1), which proved the good assay performance characteristics of the bioassay, including specificity, accuracy, precision, linearity, stability and robustness. This RGA can be applied to evaluate the -ADCP bioactivity for anti-CD20 mAbs in lot release, stability testing as well as biosimilar comparability. The engineered cells may also potentially be used to evaluate the ADCP bioactivity of mAbs with other targets.

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.

Phytoestrogen has previously been proposed as an alternative to hormone replacement therapy. Hispolon has been found to have phytoestrogenic properties. However, the possible effects of hispolon on estrogen receptors and other related molecules remain to be determined. This study was performed mainly to confirm the estrogenic function of hispolon as it relates to estrogen receptors, aromatase, and cyclooxygenase 2 (COX-2). Hispolon was shown to increase the serum 17β-estradiol in vivo. Immunohistochemical staining methods showed that hispolon exhibited a biphasic effect on ERα/β and aromatase expression in MCF-7 cells. Hispolon could also significantly inhibit aromatase activity, assessed using the enzyme-linked immunosorbent assay. Western blotting showed that COX-2 and aromatase could be inhibited by hispolon. These results further prove the phytoestrogenic features of hispolon and explore some pharmacological mechanisms that suggest that hispolon could be useful in the treatment of breast cancers or other gynecologic diseases.

To investigate the pharmacokinetics of PEGylated recombinant human endostatin (M2ES) in rhesus monkey. M2ES was administered to rhesus monkeys by intravenous bolus injection, and serum M2ES concentration was determined by a self-developed ELISA assay. Pharmacokinetic parameters were calculated using a non-compartmental model of WinNonlin V2.1A software. The standard curve of self-developed ELISA assay was fitted by four-parameter method. The limit of detection (LOD) and LOQ were 0.3050 ng/mL and 0.9140 ng/mL, respectively. Following IV infusions of M2ES at 0.3, 1, and 3 mg/kg in rhesus monkeys, the serum M2ES concentration-time curve was fitted with a non-compartment model. The pharmacokinetic parameters were evaluated as follows: Terminal elimination half-life (T1/2) of M2ES were 3.30 ± 0.70, 29.50 ± 18.80 and 24.60 ± 8.90 h. Systemic clearance (CLsys) of M2ES were 339.60 ± 66.30, 161.40 ± 18.20 and 260.10 ± 43.70 mL/h/kg. AUC(0-∞) values of M2ES were 909.30 ± 199.60, 6251.00 ± 739.60 and 11758.00 ± 2010.10 ng∙h/mL. The dosage was positively correlated with AUC, and the correlation coefficient r2 = 0.9327. Self-developed ELISA assay could meet the requirements of serum M2ES concentration detection. PEGylation modification substantially expands the circulation time of recombinant human endostatin and effectively improves its pharmacokinetic behavior.

Trametenolic acid B (TAB) was a lanostane-type triterpenoid isolated from the trametes lactinea (Berk.) Pat. We have previously reported that extract from trametes lactinea (Berk.) Pat and TAB could efficiently improve learning and memory ability of the cerebral ischemia injury rats and suppress mitochondrial-mediated apoptosis in hydrogen peroxide damaged SH-SY5Y cells. However, the potential mechanisms have not been fully understood yet. The current study was to further investigate the protective effect of TAB on oxygen glucose deprivation/reoxygenation (OGD/R)-damaged SH-SY5Y cells and cerebral ischemia/reperfusion (I/R) injury rats, as well as its mechanisms involved. Cell experiments demonstrated that TAB (10, 20 and 40 μg/mL) protected OGD/R-induced SH-SY5Y cell injury by promoting cell proliferation and suppressing LDH leakage; Meanwhile, the results in vivo showed that TAB (20, 40 and 80 mg/kg) might significantly ameliorate the neurological deficit score, cerebral edema, neuronal cell loss and apoptosis, suppress cerebral infarction volume of the cerebral I/R injury rats. Further studies in vitro and in vivo indicated TAB could efficiently reduce OGD/R-damaged SH-SY5Y cell and cerebral I/R rat serum ROS, LDH and MDA levels, elevate SOD, GSH-Px and CAT activities, downregulate miR-10a mRNA and Bax, cytochrome C, cleaved-caspase-3 and cleaved-caspase-9 protein expressions, upregulate p-PIK3CA, p-Akt, p-mTOR, Bcl-2, pro-caspase-9 and pro-caspase-3 protein expressions and p-PIK3CA/PIK3CA, p-Akt/Akt, p-mTOR/mTOR ratios (P

During the past two decades, recombinant human interleukin-12 (rhIL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models and clinical studies. Purity is a critical quality attribute (CQA) in the quality control system of rhIL-12. In our study, rhIL-12 bulks from manufacturer B showed a different pattern in non-reduced SDS-PAGE compared with size-exclusion chromatography (SEC)-HPLC. A small fragment was only detected in non-reduced SDS-PAGE but not in SEC-HPLC. The results of UPLC/MS and N-terminal sequencing confirmed that the small fragment was a 261&ndash, 306 amino acid sequence of a p40 subunit of IL-12. The cleavage occurs between Lys260 and Arg261, a basic rich region. With the presence of 0.2% SDS, the small fragment appeared in both native PAGE and in SEC-HPLC, suggesting that it is bound to the remaining part of the IL-12 non-covalently, and is dissociated in a denatured environment. The results of a bioassay showed that the fractured rhIL-12 proteins had deficient biological activity. These findings provide an important reference for the quality control of the production process and the final products of rhIL-12.

The long-acting growth hormone (LAGH) is a promising alternative biopharmaceutical to treat growth hormone (GH) deficiency in children, and it was developed using a variety of technologies by several pharmaceutical companies. Most LAGH preparations, such as Fc fusion protein, are currently undergoing preclinical study and clinical trials. Accurate determination of bioactivity is critical for the efficacy of quality control systems of LAGH. The current in vivo rat weight gain assays used to determine the bioactivity of recombinant human GH (rhGH) in pharmacopoeias are time-consuming, expensive, and imprecise, and there are no recommended bioassays for LAGH bioactivity in pharmacopoeias. Therefore, we developed a cell-based bioassay for bioactivity determination of therapeutic long-acting Fc-fusion recombinant human growth hormone (rhGH-Fc) based on the luciferase reporter gene system, which is involved in the full-length human GH receptor (hGHR) and the SG (SIE and GAS) response element. The established bioassay was comprehensively validated according to the International Council for Harmonization (ICH) Q2 (R1) guidelines and the Chinese Pharmacopoeia, and is highly precise, time-saving, simple, and robust. The validated bioassay could be qualified for bioactivity determination during the research, development, and manufacture of rhGH-Fc, and other LAGH formulations.

The therapeutic recombinant human keratinocyte growth factor 1 (rhKGF-1) was approved by the FDA for oral mucositis resulting from hematopoietic stem cell transplantation for hematological malignancies in 2004. However, no recommended bioassay for rhKGF-1 bioactivity has been recorded in the U.S. Pharmacopoeia. In this study, we developed an rhKGF-1-dependent bioassay for determining rhKGF-1 bioactivity based on HEK293 and HaCat cell lines that stably expressed the luciferase reporter driven by the serum response element (SRE) and human fibroblast growth factor receptor (FGFR2) IIIb. A good responsiveness to rhKGF-1 and rhKGF-2 shared by target HEK293/HaCat cell lines was demonstrated. Our stringent validation was completely focused on specificity, linearity, accuracy, precision, and robustness according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, AAPS/FDA Bioanalytical Workshop and the Chinese Pharmacopoeia. We confirmed the reliability of the method in determining rhKGF bioactivity. The validated method is highly timesaving, sensitive, and simple, and is especially valuable for providing information for quality control during the manufacture, research, and development of therapeutic rhKGF.

Despite significant advances in health care, outbreaks of infections by enteroviruses (EVs) continue to plague the Asia-Pacific region every year. Enterovirus 71 (EV71) causes hand-foot-and-mouth disease (HFMD), for which there are currently no therapeutics. Here, we report two new antibodies, A9 and D6, that potently neutralize EV71. A9 exhibited a 50% neutralizing concentration (neut50) value of 0.1 nM against EV71, which was 10-fold lower than that observed for D6. Investigation into the mechanisms of neutralization revealed that binding of A9 to EV71 blocks receptor binding but also destabilizes and damages the virus capsid structure. In contrast, D6 destabilizes the capsid only slightly but interferes more potently with the attachment of the virus to the host cells. Cryo-electron microscopy (cryo-EM) structures of A9 and D6 bound with EV71 shed light on the locations and nature of the epitopes recognized by the two antibodies. Although some regions of the epitopes recognized by the two antibodies overlap, there are differences that give rise to dissimilarities in potency as well as in the mechanisms of neutralization. Interestingly, the overlapping regions of the epitopes encompass the site that the virus uses to bind SCARB2, explaining the reason for the observed blocking of the virus-receptor interaction by the two antibodies. We also identified structural elements that might play roles in modulating the stability of the EV71 particles, including particle integrity. The molecular features of the A9 and D6 epitopes unveiled in this study open up new avenues for rationally designing antiviral drugs., IMPORTANCE During the course of viral infections, the human body produces neutralizing antibodies which play a defining role in clearing the virus. From this study, we report two new, highly potent neutralizing antibodies, A9 and D6, against enterovirus 71 (EV71), the causative agent of HFMD. Both antibodies prevent the virus from entering the host cell, a step that is important for establishing a successful infection. A9 destabilizes and damages the virus capsid that forms an outer protective covering around the genome of the virus, while also interfering with virus attachment to the host cells. In contrast, D6 only prevents binding of the virus to its receptor(s). The mechanism of neutralization of A9 is unique and has not been observed before for neutralizing antibodies targeting EVs. The two antibodies that we are reporting in this study have potential to be developed into much-needed therapeutic interventions for treatment of HFMD, outbreaks of which are reported every year in the Asia-Pacific region.

The 2014 Ebola outbreak in West Africa brought great threat to public health worldwide. There was no approved antiviral therapy or vaccine available to control the disease at that time. Several kinds of Ebola vaccines were urgently under development across the world. Among these, the novel recombinant adenovirus type 5 vector-based Ebola vaccine (Ad5-EBOV)-the first Ebola vaccine based on the 2014 Zaire Guinea epidemic strain-was developed in China, and its safety and immunogenicity were demonstrated in China and Sierra Leone. The license to market the drug was approved on October 19, 2017, by the Chinese Food and Drug Administration. In order to standardize the test on the Ad5-EBOV virus titer, China's national standard substance for the virus titer of Ad5-EBOV was established according to the recommendations for the preparation, characterization, and establishment of international and other biological reference standards from the World Health Organization and Chinese Pharmacopoeia (third edition). The standard for the Ad5-EBOV virus titer was prepared with a volume of 0.5 mL per ampoule in lyophilized form. The samples of the standard, designated as A, B, C, D with different aims, were blinded and distributed to five laboratories to be collaboratively calibrated. The virus titer for this standard was determined with the antibody staining method according to the instructions in the Adeno-X™ Rapid Titer Kit. The homogeneity and stability of the standard substance were also satisfied. The virus titer standard value was 8.54 lg infectious units (IFU)/mL, and the 95% confidence interval was between 7.94 lg IFU/mL and 9.14 lg IFU/mL. This standard was approved by the Chinese national committee and is available on the National Institutes for Food and Drug Control Web site ( www.nifdc.org.cn ; lot no. 250019-201501).

Bioassay of recombinant protein products is important tests to ensure protein effectiveness. Some recombinant protein products have no cells used in their bioassay but instead use animal models, while others have no suitable method. Here, we developed a method to obtain responsive cells used in bioassay of proteins. After screening of a CRISPR/Cas9 library, we obtained a responsive cell line that grew faster in the presence of rhEGF (recombinant human epidermal growth factor) than that of control cells. We used this cell line for bioassay of rhEGF. This cell line, compared with the control cells, had a 2 day shorter operation time and had lower interference. The responsive cell line is more suitable for use in bioassay of rhEGF.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that causes substantial morbidity and mortality in livestock and humans. To date, there are no licensed human vaccines or therapeutics available. Here, we report the isolation of monoclonal antibodies from a convalescent patient, targeting the RVFV envelope proteins Gn and Gc. The Gn-specific monoclonal antibodies exhibited much higher neutralizing activities in vitro and protection efficacies in mice against RVFV infection, compared to the Gc-specific monoclonal antibodies. The Gn monoclonal antibodies were found to interfere with soluble Gn binding to cells and prevent infection by blocking the attachment of virions to host cells. Structural analysis of Gn complexed with four Gn-specific monoclonal antibodies resulted in the definition of three antigenic patches (A, B and C) on Gn domain I. Both patches A and B are major neutralizing epitopes. Our results highlight the potential of antibody-based therapeutics and provide a structure-based rationale for designing vaccines against RVFV.

Development of anti-VEGF based biologic agents has been a focus in cancer treatment for the past decades, and several anti-VEGF pharmaceuticals have been already approved for treatment of various medical indications especially in cancer. The first anti-angiogenic agent approved by FDA was bevacizumab (BVZ, trade name Avastin, Genentech/Roche), a humanized anti-VEGF monoclonal antibody. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current method widely used in the lot release and stability test for clinical trial batches of BVZ is anti-proliferation assay using primary human umbilical vein endothelial cells (HUVEC), which is tedious with high assay variations. We describe here the development and preliminary validation of a reporter gene assay (RGA) that is based on an HEK293 cell line stably expressing vascular endothelial growth factor receptor 2 (VEGFR-2), and a luciferase reporter under the control of nuclear factor activated T cell (NFAT) response elements. Our study shows this assay not only to be superior on precision, sensitivity and assay simplicity compared with HUVEC assay, but also applicable to other VEGF-targeted biotherapeutics. These results show for the first time that this new reporter assay, based on the VEGF-VEGFR-NFAT pathway, can be a viable supplement to the HUVEC assay and employed in potency determination of BVZ and other kinds of anti-VEGF antibody-based biotherapeutics.

Soluble glycoprotein 130 kDa (sgp130)-Fc fusion protein, an innovative therapeutic bio-macromolecular drug specifically targeting IL-6 trans-signaling, proved to have good potential for application in the treatment of chronic inflammatory diseases. A simple and quick bioassay for sgp130-Fc was developed in this study. First, a stable reporter cell line was obtained by transfecting CHO-K1 cells with a sis-inducible element (SIE)-driving luciferase reporter gene (CHO/SIE-Luc). Sgp130-Fc could inhibit the expression of luciferase induced by IL-6/sIL-6R&alpha, complex, and the dose&ndash, response curve fitted the four-parameter logistic model, with 50% inhibitive concentration (IC50) being about 500 ng/mL and detection range between 40 and 5000 ng/mL. Both the intra-assay and inter-assay coefficient of variation (CV) were below 10.0%, and the accuracy estimates ranged from 94.1% to 106.2%. The assay indicated a good linearity (R&sup2, = 0.99) in the range of 50% to 150% of optimized initial concentration. No significant difference was found between the test results of new assay and BAF3/gp130 proliferation assay (unpaired t test, p = 0.4960, n = 6). The dose-response effect and copy number of the luciferase gene was basically unchanged after long-term culture (up to passage 60), demonstrating the stability of CHO/SIE-Luc cells. These results suggested that the new reporter assay was suited to routine potency determination of therapeutic sgp130-Fc.

Outbreaks of the highly pathogenic avian influenza H5N1 and H7N9 viruses have spurred an unprecedented research effort to develop antivirals and vaccines against influenza. Standardized methods for vaccine evaluation are critical for facilitating vaccine development. Compared with hemagglutination inhibition assays, mounting evidence suggest that microneutralization tests (MNTs) is a better choice for the evaluation of candidate pandemic influenza vaccines because they measure neutralizing antibody activity in cell cultures and are more sensitive in detecting H5 and H7. Here, we report a MNT measuring neuraminidase activity as the read-out (NA-MNT) for quantitative analysis of neutralizing antibodies against avian influenza viruses. Compared to the conventional microneutralization assay (ELISA-MNT), the NA-MNT is faster with lower intra- and inter-assay variations, while no difference in geometric mean titers was found between these two assays for the evaluation of H5N1 and H7N9 vaccines. These results suggest that NA-MNT is a reliable and high throughput method which could facilitate the development of candidate pandemic influenza vaccine.

IL-6 has an important role in the pathogenesis of autoimmunity and chronic inflammation. Several mAbs that target IL-6 or the IL-6 receptor (IL-6R) have been established and approved for the treatment of various diseases such as multicentric Castleman's disease and rheumatoid arthritis. Quality control of therapeutic antibodies requires accurate determination of bioactivity. However, current cell-based anti-proliferation assays are tedious, time consuming, and result in high variation. We therefore developed a reporter gene assay (RGA) based on an IL-6-dependent DS-1 cell line that stably expressed the reporter luciferase controlled by the serum-induced element (SIE) response element, which was a key element located downstream of the IL-6 signaling pathway. The RGA method demonstrated good performance characteristics after careful optimization, including high specificity, stability, accuracy, precision, and robustness. It also had superior precision and sensitivity. The assay is simple compared with the traditional anti-proliferation assay. This novel RGA based on the IL-6-IL-6R-STAT3 pathway can be useful, in conjunction with the anti-proliferation bioassay, to determine the bioactivity of anti-IL-6/anti-IL-6R therapeutic mAbs. Graphical abstract The mechanism sketch of the reporter gene assay for the bioactivity determination of anti-IL-6/anti-IL-6Rα mAbs.

MicroRNAs (miRNAs) have been reported to be associated with heart valve disease, which can be caused by inflammation. This study aimed to investigate the functional impacts of miR-27a on TNF-α-induced inflammatory injury in human mitral valve interstitial cells (hMVICs). hMVICs were subjected to 40 ng/mL TNF-α for 48 h, before which the expressions of miR-27a and NELL-1 in hMVICs were altered by stable transfection. Trypan blue staining, BrdU incorporation assay, flow cytometry detection, ELISA, and western blot assay were performed to detect cell proliferation, apoptosis, and the release of proinflammatory cytokines. We found that miR-27a was lowly expressed in response to TNF-α exposure in hMVICs. Overexpression of miR-27a rescued hMVICs from TNF-α-induced inflammatory injury, as cell viability and BrdU incorporation were increased, apoptotic cell rate was decreased, Bcl-2 was up-regulated, Bax and cleaved caspase-3/9 were down-regulated, and the release of IL-1β, IL-6, and MMP-9 were reduced. NELL-1 was positively regulated by miR-27a, and NELL-1 up-regulation exhibited protective functions during TNF-α-induced cell damage. Furthermore, miR-27a blocked JNK and Wnt/β-catenin signaling pathways, and the blockage was abolished when NELL-1 was silenced. This study demonstrated that miR-27a overexpression protected hMVICs from TNF-α-induced cell damage, which might be via up-regulation of NELL-1 and thus modulation of JNK and Wnt/β-catenin signaling pathways.

Both influenza viral hemagglutinin and neuraminidase can induce protective immune responses in humans. Although the viral hemagglutinin antigens have been quantified in influenza vaccines, the amounts of neuraminidase remain undetermined. Using comprehensive bioinformatics analyses of all neuraminidase sequences, we identified highly conserved and subtype-specific peptide epitopes within each of N1, N2 and type B neuraminidase groups. Mono-specific antibodies generated against these peptides bound to their respective subtype/type only while demonstrating remarkable specificity against the viral neuraminidase sequences without any cross-reactivity with allantoic and cellular proteins. Moreover, the subtype/type-specific antibodies were found not to interfere with one another when a mixture of vaccine samples was analysed. Importantly, immunoassay based on these antibodies can quantitatively determine neuraminidase in commercial trivalent vaccine samples. Analyses of vaccines from eight manufacturers using the same vaccine seeds revealed significant differences in neuraminidase levels. Specifically, while the ratio between neuraminidase and hemagglutinin in some products are found to be close 1/5, other products have a ratio of approximately 1/100, a level which is far below the theoretical ratio between neuraminidase and hemagglutinin in a virus. The antibody-based assays reported here could be of great value for better quality control of both monovalent and trivalent vaccines.

MRC-5 represents the most frequent human diploid cells (HDCs)-type cell substrate in the production of human viral vaccines. However, early-passage MRC-5 is diminishing and, due to both technical and ethical issues, it is extremely difficult to derive novel HDCs from fetal lung tissues, which are the common sources of HDCs. Our previous studies suggested that human umbilical cord may represent an alternative but convenient source of new HDCs. Here, we established a three-tiered cell banking system of a hUC-MSC line, designated previously as Cell Collection and Research Center-1 (CCRC-1). The full characterization indicated that the banked CCRC-1 cells were free from adventitious agents and remained non-tumorigenic. The CCRC-1 cells sustained its rapid proliferation even at passage 30 and were susceptible to the infection of a wide spectrum of viruses. Interestingly, the CCRC-1 cells showed much higher production of EV71 or Rubella viruses than MRC-5 and Vero cells when growing in serum-free medium. More importantly, the EV71 vaccine produced from CCRC-1 cells induced immunogenicity while eliciting no detectable toxicities in the tested mice. Collectively, these studies further supported that CCRC-1, and likely other hUC-MSCs as well, may serve as novel, safe and high-yielding HDCs for the production of human viral vaccines.

The present study aimed to develop a symbiotic selection-marker-free plasmid and host system that would allow successful plasmid maintenance and amplification for use in gene therapy. Initially, the chromosomal aspartate‑semialdehyde dehydrogenase (asd) gene was disrupted in DH10B Escherichia coli using Red recombinase‑mediated homologous recombination. This method required the use of linear DNA fragments carrying kan‑kil genes, and/or homologous extensions to the targeted locus. The resultant auxotrophic cell wall‑deficient strain (DH10BΔasd) was evaluated as a symbiotic host for amplification of the marker‑free plasmid, allowing it to supply ASD activity. In order to construct the plasmid, an asd expression cassette was inserted, under the control of the nirB promoter, into a eukaryotic expression vector, and its kanamycin resistance gene was subsequently removed. The symbiotic plasmid and host system was assessed for numerous plasmid production and stability parameters, including structure, yield, plasmid‑retention rate, and bacterial storability, under various conditions. The presence of the plasmid was subsequently confirmed by growth test, restriction enzyme mapping, and sequencing. The plasmid yield and copy number produced in the symbiotic cells, in the absence of antibiotic selection, were shown to be similar to those produced under kanamycin selection, in the cells containing the precursor plasmid and kanamycin resistance gene. Furthermore, the results of the present study demonstrated that when inoculated with1% inoculant volume,98% of the cells in the culture retained the plasmid regardless of the number of passages. The strain was stable when stored at ‑70˚C, with negligible viability loss over 12 months. The constructed plasmid is stable and has potential in future gene therapy, while much work is still required.

Up to now, all tested Ebola virus vaccines have been based on the virus strain from the Zaire outbreak in 1976. We aimed to assess the safety and immunogenicity of a novel recombinant adenovirus type-5 vector-based Ebola vaccine expressing the glycoprotein of the 2014 epidemic strain.We did this randomised, double-blind, placebo-controlled, phase 1 clinical trial at one site in Taizhou County, Jiangsu Province, China. Healthy adults (aged 18-60 years) were sequentially enrolled and randomly assigned (2:1), by computer-generated block randomisation (block size of six), to receive placebo, low-dose adenovirus type-5 vector-based Ebola vaccine, or high-dose vaccine. Randomisation was pre-stratified by dose group. All participants, investigators, and laboratory staff were masked to treatment allocation. The primary safety endpoint was occurrence of solicited adverse reactions within 7 days of vaccination. The primary immunogenicity endpoints were glycoprotein-specific antibody titres and T-cell responses at day 28 after the vaccination. Analysis was by intention to treat. The study is registered with ClinicalTrials.gov, number NCT02326194.Between Dec 28, 2014, and Jan 9, 2015, 120 participants were enrolled and randomly assigned to receive placebo (n=40), low-dose vaccine (n=40), or high-dose vaccine. Participants were followed up for 28 days. Overall, 82 (68%) participants reported at least one solicited adverse reaction within 7 days of vaccination (n=19 in the placebo group vs n=27 in the low-dose group vs n=36 in the high-dose group; p=0·0002). The most common reaction was mild pain at the injection site, which was reported in eight (20%) participants in the placebo group, 14 (35%) participants in the low-dose group, and 29 (73%) participants in the high-dose vaccine group (p0·0001). We recorded no statistical differences in other adverse reactions and laboratory tests across groups. Glycoprotein-specific antibody titres were significantly increased in participants in the low-dose and high-dose vaccine groups at both day 14 (geometric mean titre 421·4 [95% CI 249·7-711·3] and 820·5 [598·9-1124·0], respectively; p0·0001) and day 28 (682·7 [424·3-1098·5] and 1305·7 [970·1-1757·2], respectively; p0·0001). T-cell responses peaked at day 14 at a median of 465·0 spot-forming cells (IQR 180·0-1202·5) in participants in the low-dose group and 765·0 cells (400·0-1460·0) in those in the high-dose group. 21 (18%) participants had mild fever (n=9 in the placebo group, n=6 in the low-dose group, and n=6 in the high-dose group). No serious adverse events were recorded.Our findings show that the high-dose vaccine is safe and robustly immunogenic. One shot of the high-dose vaccine could mount glycoprotein-specific humoral and T-cell response against Ebola virus in 14 days.China National Science and Technology, Beijing Institute of Biotechnology, and Tianjin CanSino Biotechnology.

The outbreak of human infections of a novel avian influenza virus A (H7N9) prompted the development of the vaccines against this virus. Like all types of influenza vaccines, H7N9 vaccine must be tested for its potency prior to being used in humans. However, the unavailability of international reference reagents for the potency determination of H7N9 vaccines substantially hinders the progress in vaccine development. To facilitate clinical development, we enlisted 5 participants in a collaborative study to develop critical reagents used in Single Radial Immunodiffusion (SRID), the currently acceptable assay for potency determination of influenza vaccine. Specifically, the hemagglutinin (HA) content of one vaccine bulk for influenza A (H7N9), herein designated as Primary Liquid Standard (PLS), was determined by SDS-PAGE. In addition, the freeze-dried antigen references derived from PLS were prepared to enhance the stability for long term storage. The final HA content of lyophilized antigen references were calibrated against PLS by SRID assay in a collaborative study. Importantly, application of these national reference standards to potency analyses greatly facilitated the development of H7N9 vaccines in China.

Quality control of vaccine strains is directly associated with the safety and efficacy of inactivated whole bacterial vaccines. The assessment of genetic stability is one of the essential elements to guarantee the quality of vaccine strains. The multiple-valence inactivated leptospiral vaccine, comprising the main circulating serogroups, has played an important role in the control of Leptospira infection in China. In the present study, to assess the genetic stability of vaccine strains and develop novel quality control tests that enhance and extend the existing procedures, 7 Chinese leptospiral vaccine strains were characterized during in vivo and in vitro passages by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) analysis. The seven vaccine strains were found to have distinct sequence types (STs) and PFGE profiles. Further analysis showed that the ST and PFGE pattern of each vaccine strain, after in vivo or serial in vitro passages (up to 20 passages), were identical to those of the initial strain, demonstrating that these strains were genetically stable and homogeneous. Taken together, PFGE and MLST provide a reproducible and reliable means for confirming the identity and genetic stability of vaccine seeds, suggesting that these approaches can be used to evaluate the quality of leptospiral vaccine strains.

Through the continuous efforts of several generations, China has become one of the few countries in the world that is capable of independently addressing all the requirements by the Expanded Program on Immunization. Regulatory science is applied to continuously improve the vaccine regulatory system. Passing the prequalification by WHO has allowed Chinese vaccine products to go global. Chinese vaccine products not only secure disease prevention and control domestically but also serve the needs for international public health. This article describes the history of Chinese vaccine development, the current situation of Chinese vaccine industry and its contribution to the prevention and control of infectious diseases. We also share our experience of national quality control and vaccine regulation during the past decades. China's experience in vaccine development and quality control can benefit other countries and regions worldwide, including the developing countries.

Hypoxia‑inducible factor‑1alpha(HIF‑1alpha) is key in tumor progression and aggressiveness as it regulates a series of genes involved in angiogenesis and anaerobic metabolism. Previous studies have shown that the transcriptional levels of HIF‑1alpha may be downregulated by endostatin. However, the molecular mechanism by which endostatin represses HIF‑1alpha expression remains unknown. The current study investigated the mechanism by which nuclear‑translocated endostatin suppresses HIF‑1alpha activation by disrupting Zn(II) homeostasis. Endostatin was observed to downregulate HIF‑1alpha expression at mRNA and protein levels. Blockage of endostatin nuclear translocation by RNA interference of importinalpha1/beta1 or ectopic expression of NLS‑deficient mutant nucleolin in human umbilical vein endothelial cells co‑transfected with small interfering (si)‑nucleolin siRNA compromises endostatin‑reduced HIF‑1alpha expression. Nuclear‑translocated apo‑endostatin, but not holo‑endostatin, significantly disrupts the interaction between CBP/p300 and HIF‑1alpha by disturbing Zn(II) homeostasis, which leads to the transcriptional inactivation of HIF‑1alpha. The results reveal mechanistic insights into the method by which nuclear‑translocated endostatin downregulates HIF‑1alpha activation and provides a novel way to investigate the function of endostatin in endothelial cells.