Development and characterization of an enterovirus 71 (EV71) virus-like particles (VLPs) vaccine produced in Pichia pastoris • https://doi.org/10.1080/21645515.2019.1649554 • Abstract • Enterovirus 71 (EV71) is one of the major causative agents for hand, foot and mouth disease (HFMD) in children. Although there are three inactivated virus-based HFMD vaccines licensed in China, alternative approaches have been taken to produce an effective and safer vaccine that is easier to manufacture in large scale. Among these, a virus-like particles (VLPs) based EV71 vaccine is under active development. For this purpose, an efficient methodology for the production of EV71-VLPs by recombinant technology is needed. We here report the construction and expression of the P1 and 3C genes of EV71 in Pichia pastoris for producing VLP-based EV71 vaccine antigen with a high yield and simple manufacturing process. Based on codon-optimized P1 and 3C genes, EV71-VLPs were efficiently expressed in Pichia pastoris system, and the expression level reached 270 mg/L. Biochemical and biophysical analyses showed that the produced EV71-VLPs consisted of processed VP0, VP1, and VP3 present as ~35nm spherical particles. The immune response as a function of EV71-VLPs and adjuvant dose ratio was investigated for vaccine development. Immunization with EV71-VLPs of 1-5 μg/dose and adjuvant of 225 μg/dose induced robust neutralizing antibody responses in mice and provided effective protection against lethal challenge in both maternally transferred antibody and passive transfer protection mouse models. Therefore, the yeast produced EV71-VLPs antigen is a promising candidate for the development of vaccine against HFMD. • Keywords: Enterovirus 71, hand foot and mouth disease, vaccine, immunogenicity, Pichia pastoris, virus-like particles E print link : https://www.tandfonline.com/eprint/HG25RAZNRRDBYFIV9JXU/full?target=10.1080/21645515.2019.1649554
Parainfluenza virus 5 expressing the g protein of rabies virusprotects mice after rabies virus infection(表达狂犬病病毒G蛋白的副流感病毒5型可保护已感染狂犬病毒的小鼠)。 ( J Virol. 2015 Mar 15;89(6):3427-9. doi: 10.1128/JVI.03656-14. Epub 2014 Dec 31.) 狂犬病仍然是对全球公共卫生的主要威胁之一。狂犬病是一种死亡率最高(几乎达 100%)的传染病。而且 狂犬病只能预防,不能治疗 :狂犬病一旦出现症状,目前尚无能防止死亡的有效治疗方法。 至今国际主流学术界从未正式承认过任何一例狂犬病治疗成功的病例。 人被患病动物咬伤后,只有及时预防性注射合格的狂犬病疫苗才可能挽救生命。若错过了最佳的处治时间,狂犬病毒会沿外周神经系统逐渐转移到中枢神经系统,最终到达大脑。一旦大脑受感染并引发神经性症状,即狂犬病一旦发病,则受感染者通常必死无疑,此时任何治疗措施都无法挽救生命。 由于狂犬病的潜伏期通常较长,被疯动物咬伤后及时预防性接种疫苗的效果相当好。但狂犬病的潜伏期变化很大,从一周到数月(极个别情况下甚至可能超过一年),所以被疯动物咬伤后接种疫苗有效的时间间隔—— 窗口期 的长短是不同的。 窗口期 的长短取决于伤口与大脑的距离、伤口的严重程度、伤口处置情况、病毒毒株的特性、受伤者的免疫状态等。所以一般而言,被疯动物咬伤后接种狂犬病疫苗越早,效果就越好。对于人类来说,成功预防接种的 窗口期 可能在 6天之内;但由于狂犬病的潜伏期变化很大,因此接种疫苗有效的 窗口期 也有可能长达一个月甚至更久。 最近, 美国佐治亚大学华裔科学家 傅振芳 教授等 开发出了一种新型狂犬病疫苗,在小鼠实验中证明有望延长狂犬病疫苗预防接种的 窗口期 ,使延时的接种仍能达到成功治疗狂犬病的目标。 在该项研究中,所使用小鼠在实验室狂犬病病毒株感染后疫苗接种有效的 窗口期 是 6天。研究人员使用的病毒毒株到达试验小鼠大脑的时间在3天之内,神经系统症状通常在第6天出现,这些症状的出现就意味着病毒感染已经进展到无药可救的程度。然而作者发现,新疫苗PIV5-G在第6天,即在小鼠已经开始出现症状时使用,仍能挽救50%小鼠的生命。而如果此时使用其他现有的疫苗,结果通常是白费功夫。 所以,该新型重组疫苗能治愈晚期,即感染已波及大脑的狂犬病。 在本项研究中,作者用 副流感病毒 5型(parainfluenza virus 5,简称 PIV5 ) 表达狂犬病糖蛋白( G),并将该重组病毒( PIV5-G )用于治疗狂犬病病毒感染。 PIV5是上呼吸道病毒,通常会感染狗,但是对人类无害。PIV5可用作狂犬病蛋白质的理想递送系统,从而使免疫系统产生抗体来对抗狂犬病毒的感染。 PIV5-G 是一种可能用于预防和治疗狂犬病毒感染的很有希望的疫苗。 这项研究已于去年 12月31日由国际著名专业杂志《病毒学杂志》(Journal of Virology)在线发表,并将于今年3月15日正式发表在该杂志上 ( J Virol. 2015 Mar 15;89(6):3427-9. doi:10.1128/JVI.03656-14. Epub 2014 Dec 31.) 。 相关论文的题目是: Parainfluenza virus 5 expressing the g protein of rabiesvirus protects mice after rabies virus infection(表达狂犬病病毒G蛋白的副流感病毒5型可保护已感染狂犬病毒的小鼠)。论文的作者为 Huang Y , Chen Z , Huang J , Fu Z , He B . 研究报告的作者傅振芳教授说,“目前在世界上的许多地方,迫切需要有更好的狂犬病治疗方法。我们认为这种技术可以作为一个很好的平台,我们希望最终能挽救更多的生命。” 该论文的另一位作者,佐治亚大学华裔科学家 He B 教授说,“这是我们在科学文献中所看到的报导中最有效的治疗……,如果我们能改进这些结果,让治疗效果进一步提高并能用于人类,我们可能找到了针对晚期狂犬病感染的第一种真正有效的治疗方法。” He B 教授表示,“这些初步实验结果非常让人兴奋,我们相信可以把新疫苗与其他疗法结合起来以提高生存率,让已出现症状的动物也能存活下来。”
Finding a match-How do homologous sequences get together for recombination Barzel A, Kupiec M. Finding a match: how do homologous sequences get together for recombination? Nat Rev Genet. 2008 Jan; 9 (1): 27-37. Decades of research into homologous recombination have unravelled many of the details concerning the transfer of information between two homologous sequences. By contrast, the processes by which the interacting molecules initially colocalize are largely unknown. How can two homologous needles find each other in the genomic haystack? Is homologous pairing the result of a damage-induced homology search, or is it an enduring and general feature of the genomic architecture that facilitates homologous recombination whenever and wherever damage occurs? This Review presents the homologous-pairing enigma, delineates our current understanding of the process and offers guidelines for future research. Recent advances in plant recombination Li J, Hsia AP, Schnable PS. Recent advances in plant recombination. Curr Opin Plant Biol. 2007 Apr; 10 (2): 131-5. Epub 2007 Feb 8. Recombination is an essential cellular process and a source of genetic diversity. Recent studies have demonstrated the effects of various factors (e.g. DNA sequence similarity and activation of transposons) on rates of recombination and the distribution of recombination breakpoints in plants. These studies have also provided detailed characterizations of interchromatid and interhomolog recombination events. New approaches offer the promise of achieving the long-awaited goal of gene targeting in plants. Plant genome modification by homologous recombination Hanin M, Paszkowski J. Plant genome modification by homologous recombination. Curr Opin Plant Biol. 2003 Apr; 6 (2): 157-62. The mechanisms and frequencies of various types of homologous recombination (HR) have been studied in plants for several years. However, the application of techniques involving HR for precise genome modification is still not routine. The low frequency of HR remains the major obstacle but recent progress in gene targeting in Arabidopsis and rice, as well as accumulating knowledge on the regulation of recombination levels, is an encouraging sign of the further development of HR-based approaches for genome engineering in plants. Recombination-an underappreciated factor in evolution of plant genomes Gaut BS, Wright SI, Rizzon C, Dvorak J, Anderson LK. Recombination: an underappreciated factor in the evolution of plant genomes. Nat Rev Genet. 2007 Jan; 8 (1): 77-84. Our knowledge of recombination rates and patterns in plants is far from being comprehensive. However, compelling evidence indicates a central role for recombination, through its influences on mutation and selection, in the evolution of plant genomes. Furthermore, recombination seems to be generally higher and more variable in plants than in animals, which could be one of the primary reasons for differences in genome lability between these two kingdoms. Much additional study of recombination in plants is needed to investigate these ideas further. Genetics: paramutable possibilities Soloway PD. Genetics: paramutable possibilities. Nature. 2006 May 25; 441 (7092): 413-4 Paramutation: an encounter leaving a lasting impression Stam M, Mittelsten Scheid O. Paramutation: an encounter leaving a lasting impression. Trends Plant Sci. 2005 Jun; 10 (6): 283-90. Paramutation is the result of heritable changes in gene expression that occur upon interaction between alleles. Whereas Mendelian rules, together with the concept of genetic transmission via the DNA sequence, can account for most inheritance in sexually propagating organisms, paramutation-like phenomena challenge the exclusiveness of Mendelian inheritance. Most paramutation-like phenomena have been observed in plants but there is increasing evidence for its occurrence in other organisms, including mammals. Our knowledge of the underlying mechanisms, which might involve RNA silencing, physical pairing of homologous chromosomal regions or both, is still limited. Here, we discuss the characteristics of different paramutation-like interactions in the light of arguments supporting each of these alternative mechanisms. Paramutation and transgene silencing: A common responsive to invasive DNA? Finding a match-How do homologous sequences get together for recombination Recent advances in plant recombination Plant genome modification by homologous recombination Recombination-an underappreciated factor in evolution of plant genomes Genetics-paramutable possibilities Paramutation-an encounter leaving a lasting impression Paramutation and transgene silencing-A common responsive to invasive DNA From centiMorgans to base-pairs: homologous recombination in plants From centiMorgans to base-pairs: homologous recombination in plants