An overview of animal cell substrates for biological products

The issue of which cells to use as substrates for the production of biological products, and especially vaccines, has been with us in one form or another ever since the development of cell cultures in the 1950s.The major cell substrate events that occurred over the past 50 years are reviewed briefly. Although numerous conferences were held during that period, incomplete resolution of some cell substrate issues has remained. Specifically, the potential oncogenicity of cellular DNA derived from continuous cell lines, and the tests that are used to rule out the presence of adventitious agents have been recognized as areas that could benefit greatly from studies using state-of-the-art techniques.A collaborative effort involving WHO, NIAID, and IABS resulted from consensus recommendations of a 2004 conference, and the prospects for revised guidance in the near future on the characterization and use of animal cell substrates are bright.     

HPV16l1基因的克隆及其在真核细胞中的表达

克隆并表达人乳头瘤病毒16型(HPV16)晚期基因l1,以期为研制防治宫颈癌的DNA疫苗奠定基础。本实验采用PCR方法从质粒p16L1BN1中获得HPV16l1基因片段,利用基因重组技术,将其克隆至含巨细胞病毒(CMV)启动子的真核表达载体中,核酸序列鉴定HPV16l1基因真核表达质粒构建成功,再用脂质体介导基因转染7721人肝癌细胞。转化阳性细胞经SDS-PAGE显示在分子量大约为55kDa的位置出现一条特异性条带,与HPV16L1分子量大小相符。表达产物经Western blotting分析:能与HPV16L1单克隆抗体特异结合。真核表达质粒pcDNA3-HPV16L1构建成功并能在真核细胞7721中有效表达,为下一步进行动物DNA免疫实验奠定了基础。     

Conformational studies of the capsular polysaccharide produced by Neisseria meningitidis group A

The effect of different cations on the conformational and morphological properties of the capsular polysaccharide produced by Neisseria meningitidis group A was investigated. Circular dichroism studies showed that the presence of Na⁺, or Ca²⁺ ions induced different local conformations of the polysaccharide chain through interactions with the phosphodiester group bridging the saccharide residues in the polymer chain. Atomic force microscopy experiments confirmed that the morphology of the polysaccharide chains was different depending on the nature of the counterion. Ammonium ions were associated with the presence of single polymer chains in an elongated conformation, whereas sodium ions favored the folding of the chains into a globular conformation. The addition of calcium ions produced the aggregation of a limited number of globular polysaccharide chains to form a ‘toroidal-like’ structure.     

Type-1 polarized dendritic cells primed for high IL-12 production show enhanced activity as cancer vaccines

While multiple pathways of dendritic cell (DC) maturation result in transient production of IL-12, fully mature DCs show reduced ability to produce IL-12p70 upon a subsequent interaction with Ag-specific T cells, limiting their in vivo performance as vaccines. Such “DC exhaustion” can be prevented by the presence of IFNγ during the maturation of human DCs (type-1-polarization), resulting in improved induction of tumor-specific Th1 and CTL responses in vitro. Here, we show that type-1 polarization of mouse DCs strongly enhances their ability to induce CTL responses against a model tumor antigen, OVA, in vivo, promoting the induction of protective immunity against OVA-expressing EG7 lymphoma. Interestingly, in contrast to the human system, the induction of mouse DC1s requires the participation of IL-4, a nominal Th2-inducing cytokine. The current data help to explain the previously reported Th1-driving and anti-tumor activities of IL-4, and demonstrate that type-1 polarization increases in vivo activity of DC-based vaccines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Adam S. Giermasz and Julie A. Urban contributed equally to this work.     

Protein profiling in the gut of Penaeus monodon gavaged with oral WSSV‐vaccines and live white spot syndrome virus

White spot syndrome virus (WSSV) is a pathogen that causes considerable mortality of the farmed shrimp, Penaeus monodon. Candidate ‘vaccines’, WSSV envelope protein VP28 and formalin‐inactivated WSSV, can provide short‐lived protection against the virus. In this study, P. monodon was orally intubated with the aforementioned vaccine candidates, and protein expression in the gut of immunised shrimps was profiled. The alterations in protein profiles in shrimps infected orally with live‐WSSV were also examined. Seventeen of the identified proteins in the vaccine and WSSV‐intubated shrimps varied significantly compared to those in the control shrimps. These proteins, classified under exoskeletal, cytoskeletal, immune‐related, intracellular organelle part, intracellular calcium‐binding or energy metabolism, are thought to directly or indirectly affect shrimp's immunity. The changes in the expression levels of crustacyanin, serine proteases, myosin light chain, and ER protein 57 observed in orally vaccinated shrimp may probably be linked to immunoprotective responses. On the other hand, altered expression of proteins linked to exoskeleton, calcium regulation and energy metabolism in WSSV‐intubated shrimps is likely to symbolise disturbances in calcium homeostasis and energy metabolism.     

基因疫苗导入技术研究进展

基因疫苗积极的临床结果证明了,基因免疫是一种有效的临床免疫模式。虽然,喷射注射法的精确作用机制还不太清楚,但临床前研究表明,在皮肤内直接打靶抗原呈递细胞可有效地增强免疫反应。另外,局部给药法和树突细胞体外加载抗原的实验结果显示,直接打靶抗原呈递细胞可放大、控制和调节预防及治疗性基因疫苗的免疫结果。尽管基因枪有许多令人鼓舞的优点,但由于价格和便利性的障碍,它是否能商业化还不能确定。利用基因法治疗和预防疾病所涉及的安全性对基因疫苗要求更严格。这要有可控的质粒导入系统和组织特异性表达系统。     

Current development of COVID-19 diagnostics,vaccines and therapeutics

A novel coronavirus, designated as SARS-CoV-2, first emerged in Wuhan City, Hubei Province, China, in late December 2019. The rapidly increasing number of cases has caused worldwide panic. In this review, we describe some currently applied diagnostic approaches, as well as therapeutics and vaccines, to prevent, treat and control further outbreaks of SARS-CoV-2 infection.     

Toxoplasma gondii: 25 years and 25 major advances for the field

This article is an attempt to identify the most significant highlights of Toxoplasma research over the last 25 years. It has been a period of enormous progress and the top 25 most significant advances, in the view of this author, are described. These range from the bench to the bedside and represent a tremendous body of work from countless investigators. And, having laid out so much that has been discovered, it is impossible not to also reflect on the challenges that lie ahead. These, too, are briefly discussed. Finally, while every effort has been made to view the field as a whole, the molecular biology background of the author almost certainly will have skewed the relative importance attached to past and future advances. Despite this, it is hoped that the reader will agree with, or at least not disagree too strongly with, most of the choices presented here.     

RNA interference technology to improve the baculovirus-insect cell expression system

The baculovirus expression vector system (BEVS) is a popular manufacturing platform for the production of recombinant proteins, antiviral vaccines, gene therapy vectors, and biopesticides. Besides its successful applications in the industrial sector, the system has also played a significant role within the academic community given its extensive use in the production of hard-to-express eukaryotic multiprotein complexes for structural characterization for example. However, as other expression platforms, BEVS has to be continually improved to overcome its limitation and adapt to the constant demand for manufacturing processes that provide recombinant products with improved quality at higher yields and lower production cost.RNA interference, or RNAi, is a relatively recent technology that has revolutionized how scientist study gene function. Originally introduced as a tool to study biological and disease-related processes it has recently been applied to improve the yield and quality of recombinant proteins produced in several expression systems. In this review, we provide a comprehensive summary of the impact that RNAi-mediated silencing of cellular or viral genes in the BEVS has on the production of recombinant products. We also propose a critical analysis of several aspects of the methodologies described in the literature for the use of RNAi technology in the BEVS with the intent to provide the reader with eventually useful guidance for designing experiments.