Bissa Massimiliano, Forlani Greta, Zanotto Carlo, Tosi Giovanna, De Giuli Morghen Carlo, Accolla Roberto S, Radaelli Antonia
Department of Pharmacological and Biomolecular Sciences, University of Milan, via Balzaretti 9, Milan, Italy.
Department of Experimental Medicine, University of Insubria, Via O. Rossi 9, Varese, Italy.
PLoS One. 2018 Jan 31;13(1):e0190869. doi: 10.1371/journal.pone.0190869. eCollection 2018.
A complete eradication of an HIV infection has never been achieved by vaccination and the search for new immunogens that can induce long-lasting protective responses is ongoing. Avipoxvirus recombinants are host-restricted for replication to avian species and they do not have the undesired side effects induced by vaccinia recombinants. In particular, Fowlpox (FP) recombinants can express transgenes over long periods and can induce protective immunity in mammals, mainly due to CD4-dependent CD8+ T cells. In this context, the class II transactivator (CIITA) has a pivotal role in triggering the adaptive immune response through induction of the expression of class-II major histocompatibility complex molecule (MHC-II), that can present antigens to CD4+ T helper cells. Here, we report on construction of novel FPgp and FPenv recombinants that express the highly immunogenic SIV Gag-pro and Env structural antigens. Several FP-based recombinants, with single or dual genes, were also developed that express CIITA, driven from H6 or SP promoters. These recombinants were used to infect CEF and Vero cells in vitro and determine transgene expression, which was evaluated by real-time PCR and Western blotting. Subcellular localisation of the different proteins was evaluated by confocal microscopy, whereas HLA-DR or MHC-II expression was measured by flow cytometry. Fowlpox recombinants were also used to infect syngeneic T/SA tumour cells, then injected into Balb/c mice to elicit MHC-II immune response and define the presentation of the SIV transgene products in the presence or absence of FPCIITA. Antibodies to Env were measured by ELISA. Our data show that the H6 promoter was more efficient than SP to drive CIITA expression and that CIITA can enhance the levels of the gag/pro and env gene products only when infection is performed by FP single recombinants. Also, CIITA expression is higher when carried by FP single recombinants than when combined with FPgp or FPenv constructs and can induce HLA-DR cell surface expression. However, in-vivo experiments did not show any significant increase in the humoral response. As CIITA already proved to elicit immunogenicity by improving antigen presentation, further in-vivo experiments should be performed to increase the immune responses. The use of prime/boost immunisation protocols and the oral administration route of the recombinants may enhance the immunogenicity of Env peptides presented by MHC-II and provide CD4+ T-cell stimulation.
通过疫苗接种从未实现过对HIV感染的完全根除，目前仍在寻找能够诱导持久保护性反应的新免疫原。禽痘病毒重组体在复制上宿主限制于禽类物种，并且它们没有痘苗重组体诱导的不良副作用。特别是，鸡痘（FP）重组体可以长期表达转基因，并且可以在哺乳动物中诱导保护性免疫，这主要归因于CD4依赖性CD8 + T细胞。在这种情况下，II类反式激活因子（CIITA）在通过诱导II类主要组织相容性复合体分子（MHC-II）的表达来触发适应性免疫反应中起关键作用，MHC-II可以将抗原呈递给CD4 +辅助性T细胞。在此，我们报告了表达高度免疫原性的SIV Gag-pro和Env结构抗原的新型FPgp和FPenv重组体的构建。还开发了几种基于FP的单基因或双基因重组体，它们表达由H6或SP启动子驱动的CIITA。这些重组体用于体外感染CEF和Vero细胞并确定转基因表达，通过实时PCR和蛋白质印迹进行评估。通过共聚焦显微镜评估不同蛋白质的亚细胞定位，而通过流式细胞术测量HLA-DR或MHC-II表达。鸡痘重组体也用于感染同基因T/SA肿瘤细胞，然后注射到Balb/c小鼠中以引发MHC-II免疫反应，并确定在存在或不存在FPCIITA的情况下SIV转基因产物的呈递。通过ELISA测量针对Env的抗体。我们的数据表明，H6启动子在驱动CIITA表达方面比SP更有效，并且仅当通过FP单重组体进行感染时，CIITA才能提高gag/pro和env基因产物的水平。此外，当由FP单重组体携带时，CIITA表达高于与FPgp或FPenv构建体组合时，并且可以诱导HLA-DR细胞表面表达。然而，体内实验并未显示体液反应有任何显著增加。由于CIITA已被证明通过改善抗原呈递引发免疫原性，因此应进行进一步的体内实验以增强免疫反应。使用初免/加强免疫方案和重组体的口服给药途径可能会增强MHC-II呈递的Env肽的免疫原性并提供CD4 + T细胞刺激。
