Donayre-Torres Alberto J, Esquivel-Soto Ernesto, Gutiérrez-Xicoténcatl María de Lourdes, Esquivel-Guadarrama Fernando R, Gómez-Lim Miguel A
Centro de Investigación y de Estudios Avanzados (CINVESTAV), Unidad Irapuato, Km 9.6 Libramiento Norte, 36500 Carretera Irapuato-León, Irapuato, Guanajuato, México.
Virol J. 2009 Feb 6;6:17. doi: 10.1186/1743-422X-6-17.
Gag protein from HIV-1 is a polyprotein of 55 kDa, which, during viral maturation, is cleaved to release matrix p17, core p24 and nucleocapsid proteins. The p24 antigen contains epitopes that prime helper CD4 T-cells, which have been demonstrated to be protective and it can elicit lymphocyte proliferation. Thus, p24 is likely to be an integral part of any multicomponent HIV vaccine. The availability of an optimal adjuvant and carrier to enhance antiviral responses may accelerate the development of a vaccine candidate against HIV. The aim of this study was to investigate the adjuvant-carrier properties of the B ricin subunit (RTB) when fused to p24.
A fusion between ricin toxin B subunit and p24 HIV (RTB/p24) was expressed in E. coli. Affinity chromatography was used for purification of p24 alone and RTB/p24 from cytosolic fractions. Biological activity of RTB/p24 was determined by ELISA and affinity chromatography using the artificial receptor glycoprotein asialofetuin. Both assays have demonstrated that RTB/p24 is able to interact with complex sugars, suggesting that the chimeric protein retains lectin activity. Also, RTB/p24 was demonstrated to be immunologically active in mice. Two weeks after intraperitoneal inoculation with RTB/p24 without an adjuvant, a strong anti-p24 immune response was detected. The levels of the antibodies were comparable to those found in mice immunized with p24 alone in the presence of Freund adjuvant. RTB/p24 inoculated intranasally in mice, also elicited significant immune responses to p24, although the response was not as strong as that obtained in mice immunized with p24 in the presence of the mucosal adjuvant cholera toxin.
In this work, we report the expression in E. coli of HIV-1 p24 fused to the subunit B of ricin toxin. The high levels of antibodies obtained after intranasal and intraperitoneal immunization of mice demonstrate the adjuvant-carrier properties of RTB when conjugated to an HIV structural protein. This is the first report in which a eukaryotic toxin produced in E. coli is employed as an adjuvant to elicit immune responses to p24 HIV core antigen.
HIV-1的Gag蛋白是一种55 kDa的多聚蛋白，在病毒成熟过程中被切割，释放出基质p17、核心p24和核衣壳蛋白。p24抗原包含能启动辅助性CD4 T细胞的表位，已证明这些表位具有保护性，并且它能引发淋巴细胞增殖。因此，p24很可能是任何多组分HIV疫苗的一个组成部分。获得一种能增强抗病毒反应的最佳佐剂和载体可能会加速抗HIV疫苗候选物的开发。本研究的目的是研究与p24融合时蓖麻毒素B亚基（RTB）的佐剂-载体特性。
蓖麻毒素B亚基与HIV p24（RTB/p24）的融合蛋白在大肠杆菌中表达。采用亲和层析法从胞质组分中纯化单独的p24和RTB/p24。通过ELISA和使用人工受体糖蛋白去唾液酸胎球蛋白的亲和层析法测定RTB/p24的生物学活性。两种测定方法均表明RTB/p24能够与复合糖相互作用，这表明嵌合蛋白保留了凝集素活性。此外，RTB/p24在小鼠体内被证明具有免疫活性。在无佐剂的情况下腹腔接种RTB/p24两周后，检测到强烈的抗p24免疫反应。抗体水平与在弗氏佐剂存在下单独用p24免疫的小鼠中发现的水平相当。在小鼠中鼻内接种RTB/p24，也引发了对p24的显著免疫反应，尽管该反应不如在粘膜佐剂霍乱毒素存在下用p24免疫的小鼠中获得的反应强烈。
在本研究中，我们报道了与蓖麻毒素亚基B融合的HIV-1 p24在大肠杆菌中的表达。小鼠经鼻内和腹腔内免疫后获得的高水平抗体证明了RTB与HIV结构蛋白偶联时的佐剂-载体特性。这是首次报道在大肠杆菌中产生的真核毒素被用作佐剂以引发对HIV p24核心抗原的免疫反应。
