近日,，國際知名雜志《病毒學(xué)期刊》（Journal of Virology）在線發(fā)表了中國科學(xué)院上海巴斯德研究所周保羅研究組關(guān)于艾滋病病毒I型（HIV-1）疫苗的最新成果：一種基于果蠅S2細(xì)胞的新型HIV-1 病毒樣顆粒（VLP）表達(dá)系統(tǒng),。這是世界上首次報(bào)道采用果蠅S2細(xì)胞表達(dá)系統(tǒng)制備HIV-1 VLP作為艾滋病疫苗組分。
2009年在泰國完成的RV144 HIV疫苗實(shí)驗(yàn)雖然獲得了31.2%的保護(hù)率,，但是仍然不能用于人群接種,，該疫苗采用痘病毒載體初免，GP120蛋白加強(qiáng),。一個(gè)成功的HIV-1疫苗應(yīng)既能夠誘導(dǎo)出HIV-1胞膜蛋白特異性的廣譜中和抗體,，又能夠誘導(dǎo)出多種蛋白特異性的T細(xì)胞免疫反應(yīng)。在眾多的HIV-1免疫原設(shè)計(jì)中,，VLP由于能夠?qū)⑻烊粯?gòu)象的胞膜蛋白刺突展示于其表面,，并且能夠同時(shí)誘導(dǎo)體液和細(xì)胞免疫反應(yīng)而被認(rèn)為優(yōu)于GP120蛋白。但是,，目前的HIV-1 VLP表達(dá)系統(tǒng)在某些方面還存在缺陷,。
為了克服這些缺陷，博士研究生楊立飛,、宋宇峰等在周保羅研究員的指導(dǎo)下,，開發(fā)出一種基于果蠅S2細(xì)胞的新型HIV-1 VLP表達(dá)系統(tǒng)。首先,，他們構(gòu)建了表達(dá)HIV-1 VLP的S2細(xì)胞穩(wěn)轉(zhuǎn)克?。蝗缓笱芯苛嗽撓到y(tǒng)表達(dá)的VLP胞膜蛋白的表達(dá),、剪切,、組裝以及糖基化等理化特征，并且采用冷凍電鏡（Croy-EM）和冷凍電子斷層成像（Croy Electron Tomography）對VLP的形態(tài)學(xué)及其表面刺突的數(shù)量進(jìn)行了仔細(xì)分析,；最后研究了VLP的抗原性和免疫原性,。
研究表明，采用這一新型VLP表達(dá)系統(tǒng)制備的HIV-1 VLP其表面膜蛋白能夠正確剪切,、糖基化并且和gag蛋白一起組裝成病毒樣顆粒,；采用該系統(tǒng)制備的HIV-1 VLP產(chǎn)量與桿狀病毒表達(dá)系統(tǒng)的產(chǎn)量相當(dāng)，甚至更高,。Cryo-EM和Tomography研究發(fā)現(xiàn),，純化后的VLP表面平均包含17個(gè)膜蛋白刺突，并且保留了多個(gè)已知的廣譜中和抗體表位,。研究人員采用DNA初免-VLP加強(qiáng),，并伴以CpG佐劑的接種策略，可以在小鼠體內(nèi)誘導(dǎo)出胞膜蛋白特異性的抗體反應(yīng)：包括ELISA結(jié)合抗體,、中和抗體,、ADCC和ADCVI特異性抗體反應(yīng),，以及胞膜蛋白和gag特異性的CD8 T細(xì)胞免疫反應(yīng)等，具有更好的免疫保護(hù)效果,。綜上所述,，這一新型VLP表達(dá)系統(tǒng)制備的HIV-1 VLP具有很多優(yōu)良的特性，可以開發(fā)成為HIV-1疫苗的有效組分,。
該研究是與中科院生物物理研究所朱平研究組合作完成的,，得到了國家科技重大專項(xiàng)的資助,。（生物谷Bioon.com）
doi:10.1128/?JVI.07164-11
PMC:
PMID:
HIV-1 Virus-Like Particles Produced by Stably Transfected Drosophila S2 Cells: a Desirable Vaccine Component
Lifei Yanga, Yufeng Songa, Xiaomin Lib, Xiaoxing Huangb, Jingjing Liua, Heng Dinga, Ping Zhub and Paul Zhoua
The development of a successful vaccine against human immunodeficiency virus type 1 (HIV-1) likely requires immunogens that elicit both broadly neutralizing antibodies against envelope spikes and T cell responses that recognize multiple viral proteins. HIV-1 virus-like particles (VLP), because they display authentic envelope spikes on the particle surface, may be developed into such immunogens. However, in one way or the other current systems for HIV-1 VLP production have many limitations. To overcome these, in the present study we developed a novel strategy to produce HIV-1 VLP using stably transfected Drosophila S2 cells. We cotransfected S2 cells with plasmids encoding HIV-1 envelope, Gag, and Rev proteins and a selection marker. After stably transfected S2 clones were established, HIV-1 VLP and their immunogenicity in mice were carefully evaluated. Here, we report that HIV-1 envelope proteins are properly cleaved, glycosylated, and incorporated into VLP with Gag. The amount of VLP released into culture supernatants is comparable to those produced by insect cells infected with recombinant baculoviruses. Moreover, cryo-electron microscopy tomography revealed average 17 spikes per purified VLP, and antigenic epitopes on the spikes were recognized by the broadly neutralizing antibodies 2G12, b12, VRC01, and 4E10 but not by PG16. Finally, mice primed with DNA and boosted with VLP in the presence of CpG exhibited anti-envelope antibody responses, including ELISA-binding, neutralizing, antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated viral inhibition, as well as envelope and Gag-specific CD8 T cell responses. Thus, we conclude that HIV-1 VLP produced by the S2 expression system has many desirable features to be developed into a vaccine component against HIV-1.
