Enhanced Downstream Processing for a Cell-Based Avian Influenza (H5N1) Vaccine.

H5N1 highly pathogenic avian influenza virus (HPAIV) infections pose a significant threat to human health, with a mortality rate of around 50%. Limited global approval of H5N1 HPAIV vaccines, excluding China, prompted the need to address safety concerns related to MDCK cell tumorigenicity. Our objective was to improve vaccine safety by minimizing residual DNA and host cell protein (HCP). We developed a downstream processing method for the cell-based H5N1 HPAIV vaccine, employing CaptoTM Core 700, a multimodal resin, for polishing. Hydrophobic-interaction chromatography (HIC) with polypropylene glycol as a functional group facilitated the reversible binding of virus particles for capture. Following the two-step chromatographic process, virus recovery reached 68.16%. Additionally, HCP and DNA levels were reduced to 2112.60 ng/mL and 6.4 ng/mL, respectively. Western blot, high-performance liquid chromatography (HPLC), and transmission electron microscopy (TEM) confirmed the presence of the required antigen with a spherical shape and appropriate particle size. Overall, our presented two-step downstream process demonstrates potential as an efficient and cost-effective platform technology for cell-based influenza (H5N1 HPAIV) vaccines.

Safety and immunogenicity of rabies vaccine (PVRV-WIBP) in healthy Chinese aged 10-50 years old: Randomized, blinded, parallel controlled phase III clinical study.

This phase III clinical trial aimed to assess the safety and demonstrate the immunogenicity of a candidate freeze-dried purified Vero cell-based rabies vaccine (PVRV-WIBP) developed for human use. A cohort of 40 participants in stage 1 and 1956 subjects in stage 2 with an age range of 10-50 years were recruited for the phase III clinical trial. For safety analysis in stage 1, 20 participants received either 4-dose or 5-dose regimen of PVRV-WIBP. In stage 2, 1956 subjects were randomly divided into the 5-dose PVRV-WIBP, 5-dose PVRV-LNCD, and 4-dose PVRV-WIBP groups. The serum neutralizing antibody titer against rabies was determined on day 7 or 14 and day 35 or 42. Adverse reactions were recorded for more than 6 months. Most adverse reactions, which were mild and moderate in severity, occurred and resolved within 1 week after each injection in the PVRV-WIBP (4 and 5 doses) and PVRV-LNCD (5 doses) groups. All three groups achieved complete seroconversion 14 days after the initial dose and 14 days after completing the full vaccination schedule, the susceptible subjects in the PVRV-WIBP group (4-dose or 5-dose regimen) displayed higher neutralizing antibody titers against the rabies virus compared to those in the PVRV-LNCD group (5-dose regimen). PVRV-WIBP induced non-inferior immune responses versus PVRV-LNCD as assessed by seroconversion rate. PVRV-WIBP was well tolerated and non-inferior to PVRV-LNCD in healthy individuals aged 10-50 years. The results indicated that PVRV-WIBP (both 4- and 5-dose schedules) could be an alternative to rabies post-exposure prophylaxis.

Sequence-specific eDNA extraction using hydrophobic magnetic ionic liquids attached with oligonucleotide strand.

Isolation of high-purity nucleic acids, especially sequence-specific DNA, from complex samples is critical to the downstream nucleic acid analysis. In this work, an oligonucleotide strand-attached magnetic ionic liquid (OSMIL) was designed and prepared for DNA extraction. The attached oligonucleotide strand has a sequence complementary to that of a specific DNA to be extracted. The OSMIL has good hydrophobicity and magnetic response properties. At the extraction temperature, OSMIL was in a liquid state, which was favorable for maximizing the adsorption of DNA; while at the separation temperature, OSMIL was in a solid state (with an average particle size of 897 nm) and could be attracted by an external magnet in 3s, which was favorable for the separation and recovery of DNA. The sequence-specific DNA extraction process with OSMIL is simple and fast. After extraction, the DNA-enriched OSMILs were quickly attracted and separated by an external magnetic field. The extracted DNA was evaluated by a NanoDrop (wavelength detection at 260-280 nm) and the OSMIL can be recycled and reused. The enrichment factor was 0.81. Through single-factor experimental analysis, the effects of OSMIL extraction volume, thermal excitation temperature, thermal excitation time, pH, and other factors on the DNA extraction process were systematically investigated. The RSD of repeatability experiment was 1.19% (n = 3), showing the method has good repeatability. The extraction method presented here has been shown to extract DNA with specific sequences from mixtures containing DNA of different sequences and from mixtures containing proteins, respectively. In addition, the OSMIL has been applied to extract target environmental DNA with specific sequences from different water environments with high extraction efficiency. In the long run, OSMIL has great potential for identifying existing organisms in environmental samples or exploring unknown organisms.

Transcription factor AP-2α activates RNA polymerase III-directed transcription and tumor cell proliferation by controlling expression of c-MYC and p53.

Deregulation of transcription factor AP2 alpha (TFAP2A) and RNA polymerase III (Pol III) products is associated with tumorigenesis. However, the mechanism underlying this event is not fully understood and the connection between TFAP2A and Pol III-directed transcription has not been investigated. Here, we report that TFAP2A functions as a positive factor in the regulation of Pol III-directed transcription and cell proliferation. We found TFAP2A is also required for the activation of Pol III transcription induced by the silencing of filamin A, a well-known cytoskeletal protein and an inhibitor in Pol III-dependent transcription identified previously. Using a chromatin immunoprecipitation technique, we showed TFAP2A positively modulates the assembly of Pol III transcription machinery factors at Pol III-transcribed gene loci. We found TFAP2A can activate the expression of Pol III transcription-related factors, including BRF1, GTF3C2, and c-MYC. Furthermore, we demonstrate TFAP2A enhances expression of MDM2, a negative regulator of tumor suppressor p53, and also inhibits p53 expression. Finally, we found MDM2 overexpression can rescue the inhibition of Pol III-directed transcription and cell proliferation caused by TFAP2A silencing. In summary, we identified that TFAP2A can activate Pol III-directed transcription by controlling multiple pathways, including general transcription factors, c-MYC and MDM2/p53. The findings from this study provide novel insights into the regulatory mechanisms of Pol III-dependent transcription and cancer cell proliferation.

Phenylpropyl Guanidinium Magnetic Ionic Liquid for Green and Selective Extraction of RNA.

A phenylpropyl guanidinium magnetic ionic liquid (PGMIL) was designed and prepared to extract RNA from complex samples. The properties of PGMIL were characterized by a vibrating sample magnetometer, Fourier transform infrared spectrometer, thermogravimetric analyzer, transmission electron microscope, and scanning electron microscope. Through single-factor analysis, the factors affecting the RNA extraction process, such as PGMIL volume, temperature, extraction time, and pH, were systematically investigated. The ability of PGMIL to selectively extract RNA was investigated by a NanoDrop. Under the optimized conditions, the extraction efficiency of RNA can reach 81.9 ± 1.9%. The proposed extraction method has been demonstrated with the extraction of RNA from a series of complex sample matrices, including a metal ion mixture and medicinal yeast. After extraction, the retained RNA could be readily recovered by simply using Tris-HCl buffer, with a recovery rate of 68.11 ± 2.45%. Regeneration studies have shown that the extraction efficiency of PGMIL did not change significantly after using 4 times. This study provides a green, rapid, and environmental friendly extraction method for the selective extraction of RNA.

Preclinical immunogenicity assessment of a cell-based inactivated whole-virion H5N1 influenza vaccine.

In influenza vaccine development, Madin-Darby canine kidney (MDCK) cells provide multiple advantages, including large-scale production and egg independence. Several cell-based influenza vaccines have been approved worldwide. We cultured H5N1 virus in a serum-free MDCK cell suspension. The harvested virus was manufactured into vaccines after inactivation and purification. The vaccine effectiveness was assessed in the Wuhan Institute of Biological Products BSL2 facility. The pre- and postvaccination mouse serum titers were determined using the microneutralization and hemagglutination inhibition tests. The immunological responses induced by vaccine were investigated using immunological cell classification, cytokine expression quantification, and immunoglobulin G (IgG) subtype classification. The protective effect of the vaccine in mice was evaluated using challenge test. Antibodies against H5N1 in rats lasted up to 8 months after the first dose. Compared with those of the placebo group, the serum titer of vaccinated mice increased significantly, Th1 and Th2 cells were activated, and CD8+ T cells were activated in two dose groups. Furthermore, the challenge test showed that vaccination reduced the clinical symptoms and virus titer in the lungs of mice after challenge, indicating a superior immunological response. Notably, early after vaccination, considerably increased interferon-inducible protein-10 (IP-10) levels were found, indicating improved vaccine-induced innate immunity. However, IP-10 is an adverse event marker, which is a cause for concern. Overall, in the case of an outbreak, the whole-virion H5N1 vaccine should provide protection.

Development of Magnetic Deep Eutectic Solvent-Based Liquid-Liquid Extraction for the Selective Extraction and Separation of RNA.

Four kinds of hydrophobic magnetic deep eutectic solvents (HMDESs) were prepared and applied to RNA extraction. Based on the HMDESs, a mechanical shaking-assisted liquid-liquid extraction (MSLLE) was developed for the extraction of RNA. Factors that influence the extraction, including the extraction time, temperature, volume of HMDES, buffer types, and pH, were evaluated. After the optimization of all conditions, the RNA extraction efficiency was 82.31 ± 0.02%. RNA can be extracted from complex samples and medicinal yeast by the method proposed in this work and can be recovered from the HMDESs after being extracted.

Hydrophobic magnetic deep eutectic solvent: Synthesis, properties, and application in DNA separation.

Isolating high-purity nucleic acids from complex biological samples is critical to nucleic acid analysis. In the current work, four hydrophobic magnetic deep eutectic solvents (HMDESs) were firstly designed and prepared for the extraction of DNA. The conformations of the HMDESs were simulated and H-bonding interactions in the HMDESs were investigated by density functional theory (DFT) calculation. Characterization of HMDESs' physical (magnetism, density, viscosity and hydrophobicity), and thermal (melting point and decomposition temperature) properties were conducted. Single stranded DNA (ssDNA), double stranded DNA (dsDNA) and DNA sodium salts (stDNA) that were extracted by HMDESs could be quickly collected by an external magnet. Three auxiliary extraction methods, including vortex auxiliary extraction, mechanical shaking auxiliary extraction and ultrasonic auxiliary extraction, were introduced to extract DNA with HMDESs and the extraction efficiencies were evaluated using NanoDrop. Factors that could impact the DNA extraction process, such as HMDESs volume, temperature, time, and pH, were systematically investigated via single-factor experimental analysis. The proposed extraction method can successfully extract DNA from complex matrices and E. coli cell lysate. The DNA extracted by using HMDESs are well suitable for PCR amplifications. The interaction and corresponding binding sites between HMDESs and DNA were investigated by FT-IR and DFT calculation. The extraction mechanisms were discussed: hydrophobic interaction and electrostatic interaction are two main forces driving DNA extraction by HMDESs.

Synthesis of low-viscosity hydrophobic magnetic deep eutectic solvent: Selective extraction of DNA.

Fast extraction of high-purity nucleic acid from complex biological sample is the key to downstream nucleic acid analysis. In this work, two low-viscosity hydrophobic magnetic deep eutectic solvents (HMDESs) were synthesized for the selective extraction of DNA. The conformation of the HMDES was simulated by density functional theory (DFT) calculation. Characterization of HMDESs' physical (magnetism, density, viscosity, and hydrophobicity) properties and thermal (melting point and decomposition temperature) properties were conducted. Based on the HMDESs, a vortex-assisted liquid-liquid micro-extraction (VALLME) DNA method was developed. Single stranded DNA that was extracted by HMDESs could be quickly collected by an external magnet. Factors that could impact the DNA extraction process, such as HMDESs volume, temperature, extraction time, and pH were systematically investigated via single-factor experimental analysis. Under the optimized condition, the proposed extraction method has been demonstrated with the extraction of DNA from a series of complex sample matrices, including metal mixture, protein mixture and E. coli cell lysate. The DNA extracted by using HMDES-based VALLME method was well suitable for PCR amplifications. After extraction, the retained DNAs could be readily recovered by simply using Britton-Robison (BR) buffer. In addition, the interaction and corresponding binding sites between HMDESs and DNA were investigated by FT-IR and DFT calculation. This work provides a new green magnetic solvent and a rapid and environmental-friendly extraction method for the enrichment of DNA and other biological macromolecules.

The transcription factor Sp1 modulates RNA polymerase III gene transcription by controlling BRF1 and GTF3C2 expression in human cells.

Specificity protein 1 (Sp1) is an important transcription factor implicated in numerous cellular processes. However, whether Sp1 is involved in the regulation of RNA polymerase III (Pol III)-directed gene transcription in human cells remains unknown. Here, we first show that filamin A (FLNA) represses Sp1 expression as well as expression of TFIIB-related factor 1 (BRF1) and general transcription factor III C subunit 2 (GTF3C2) in HeLa, 293T, and SaOS2 cell lines stably expressing FLNA-silencing shRNAs. Both BRF1 promoter 4 (BRF1P4) and GTF3C2 promoter 2 (GTF3C2P2) contain putative Sp1-binding sites, suggesting that Sp1 affects Pol III gene transcription by regulating BRF1 and GTF3C2 expression. We demonstrate that Sp1 knockdown inhibits Pol III gene transcription, BRF1 and GTF3C2 expression, and the proliferation of 293T and HeLa cells, whereas Sp1 overexpression enhances these activities. We obtained a comparable result in a cell line in which both FLNA and Sp1 were depleted. These results indicate that Sp1 is involved in the regulation of Pol III gene transcription independently of FLNA expression. Reporter gene assays showed that alteration of Sp1 expression affects BRF1P4 and GTF3C2P2 activation, suggesting that Sp1 modulates Pol III-mediated gene transcription by controlling BRF1 and GTF3C2 gene expression. Further analysis revealed that Sp1 interacts with and thereby promotes the occupancies of TATA box-binding protein, TFIIAα, and p300 at both BRF1P4 and GTF3C2P2. These findings indicate that Sp1 controls Pol III-directed transcription and shed light on how Sp1 regulates cancer cell proliferation.

A novel method to investigate the effects of gene mutations at the cellular level using a dual expression lentiviral vector.

One of the conventional methods to study the effects of gene mutations is that gene mutants are transfected into mammalian cells, and the dominant effects of gene mutants in the cells are examined. However, the result obtained using this method is not always satisfactory due to the interference of endogenous expression. Whether there is a better method to investigate the effects of gene mutations in cells remains to be examined. In the present study, a novel dual expression lentiviral vector was constructed using a shRNA-expressing lentiviral vector and combined techniques. Using this dual expression system, the vectors expressing both transcription factor IIA γ (TFIIAγ) shRNA and HA-TFIIAγ or its mutants were generated, and the effects of TFIIAγ gene mutations on transcription and protein-DNA interaction were investigated. We show that the transfection of the vector expressing TFIIAγ shRNA and HA-TFIIAγ fusion gene was able to silence the expression of endogenous TFIIAγ gene but not affect that of exogenous HA-TFIIAγ fusion gene in either transiently transfected cells or stable cell lines. Mutations in the conservative domain between AA62 and AA69 in TFIIAγ inhibit the activities of promoters and endogenous gene expression, and reduce TFIIAγ binding to AdML core promoter compared with wild-type (WT) TFIIAγ. ChIP-qPCR data suggest that the TFIIAγ N63A mutant inhibits insulin-like growth factor 2 (IGF2) transcription by reducing the recruitments of TFIIAγ, polymerase II (Pol II), TATA box-binding protein (TBP), and TBP associated factor 1 (250 kDa) (TAF1) at its promoter. Our study provides a novel method that is used to investigate the effects of gene mutations at the cellular level.

Rapid Regeneration and Reuse of Silica Columns from PCR Purification and Gel Extraction Kits.

Silica columns from PCR purification and gel extraction kits are widely used in laboratories worldwide to assist in gene cloning. However, the use of these columns can generate plastic waste that has an environmental impact due to their one-off design and massive consumption. Thus, it is important to develop a novel method that can reduce the utilization of silica columns but not affect research efficiency. In this study, various chemical and nonchemical reagents were used to eliminate residual DNA within used columns from PCR purification and gel extraction kits. We show that phosphoric acid is the most effective reagent among those tested to remove DNA contamination from used columns. Columns regenerated using 1 M phosphoric acid have a DNA purification capability that is comparable to that of fresh columns. We demonstrate that silica columns can be regenerated and reused a minimum of five times. The lab-made buffers are compatible with the regenerated columns for DNA purification, and DNA that is prepared with the regenerated columns can be used for gene cloning without affecting the gene cloning efficiency. Thus, the use of this novel method greatly reduces the production of laboratory waste and benefits numerous laboratories worldwide.