植物热休克蛋白 90 是一种新型佐剂，能诱导针对弓形虫 SAG1 肽的 B 细胞和 T 细胞表位的强烈体液和细胞免疫应答。

Plant Hsp90 is a novel adjuvant that elicits a strong humoral and cellular immune response against B- and T-cell epitopes of a Toxoplasma gondii SAG1 peptide.

机构信息

Unidad de Biotecnología 6-UB6, IIB-INTECH, CONICET-UNSAM, Intendente Marino Km 8.2, B7130IWA, Chascomús, Buenos Aires Province, Argentina.

出版信息

Parasit Vectors. 2019 Mar 25;12(1):140. doi: 10.1186/s13071-019-3362-6.

DOI:10.1186/s13071-019-3362-6

PMID:30909938

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6434815/

Abstract

BACKGROUND

The 90-kDa heat-shock protein (Hsp90) from Nicotiana benthamiana (NbHsp90.3) is a promising adjuvant, especially for those vaccines that require a T cell-mediated immune response. Toxoplasma gondii SAG1 is considered one of the most important antigens for the development of effective subunit vaccines. Some epitopes located in the SAG1 C-terminus region have showed a strong humoral and cellular immune response. In the present study, we aimed to assess the efficacy of NbHsp90.3 as carrier/adjuvant of SAG1-derived peptide (SAG1) in a T. gondii infection murine model.

METHODS

In the present study, C57BL/6 mice were intraperitoneal immunized with the NbHsp90.3-SAG1 fusion protein (NbHsp90.3-SAG1 group), mature SAG1 (SAG1m group), NbHsp90.3 (NbHsp90.3 group) or PBS buffer 1× (PBS group). The levels of IgG antibodies and the cytokine profile were determined by ELISA. Two weeks after the last immunization, all mice were orally challenged with 20 cysts of T. gondii Me49 strain and the number of brain cysts was determined. In addition, both humoral and cellular immune responses were also evaluated during the acute and chronic phase of T. gondii infection by ELISA.

RESULTS

The characterization of the immune response generated after vaccination with NbHsp90.3 as an adjuvant showed that NbHsp90.3-SAG1-immunized mice produced antibodies that were able to recognize not only rSAG1m but also the native SAG1 present in the total lysate antigen extract (SAG1) from T. gondii tachyzoites, while control groups did not. Furthermore, anti-rSAG1m IgG2a/2b antibodies were significantly induced. In addition, only the spleen cell cultures from NbHsp90.3-SAG1-immunized mice showed a significantly increased production of IFN-γ. During the chronic phase of T. gondii infection, the antibodies generated by the infection were unable to detect the recombinant protein, but they did react with TLA extract. In addition, splenocytes from all groups showed a high production of IFN-γ when stimulated with rGRA4, but only those from NbHsp90.3-SAG1 group stimulated with rSAG1m showed high production of IFN-γ. Finally, NbHsp90.3-SAG1-immunized mice exhibited a significant reduction in the cyst load (56%) against T. gondii infection.

CONCLUSIONS

We demonstrated that NbHsp90.3 enhances the humoral and cell-mediated immune response through a Th1 type cytokine production. Mice vaccinated with NbHsp90.3-SAG1 exhibited a partial protection against T. gondii infection and it was correlated with the induction of memory immune response. We developed and validated a vaccine formulation which, to our knowledge, for the first time includes the NbHsp90.3 protein covalently fused to a peptide from T. gondii SAG1 protein that contains T- and B-cell epitopes.

摘要

背景

来自 Nicotiana benthamiana（NbHsp90.3）的 90kDa 热休克蛋白（Hsp90）是一种很有前途的佐剂，特别是对于那些需要 T 细胞介导的免疫反应的疫苗。刚地弓形虫 SAG1 被认为是开发有效亚单位疫苗的最重要抗原之一。位于 SAG1 C 末端区域的一些表位表现出强烈的体液和细胞免疫反应。在本研究中，我们旨在评估 NbHsp90.3 作为刚地弓形虫感染鼠模型中 SAG1 衍生肽（SAG1m）的载体/佐剂的功效。

方法

本研究中，C57BL/6 小鼠经腹腔免疫 NbHsp90.3-SAG1 融合蛋白（NbHsp90.3-SAG1 组）、成熟 SAG1（SAG1m 组）、NbHsp90.3（NbHsp90.3 组）或 PBS 缓冲液 1×（PBS 组）。通过 ELISA 测定 IgG 抗体水平和细胞因子谱。末次免疫后 2 周，所有小鼠均经口感染 20 个刚地弓形虫 Me49 株囊包，测定脑囊包数。此外，还通过 ELISA 在刚地弓形虫感染的急性和慢性阶段评估体液和细胞免疫反应。

结果

用 NbHsp90.3 作为佐剂免疫后产生的免疫反应特征表明，NbHsp90.3-SAG1 免疫的小鼠产生的抗体不仅能够识别 rSAG1m，还能够识别来自刚地弓形虫速殖子总裂解物抗原提取物（SAG1）中的天然 SAG1，而对照组则不能。此外，还诱导了抗 rSAG1m IgG2a/2b 抗体。此外，只有 NbHsp90.3-SAG1 免疫的脾细胞培养物显示 IFN-γ 的产生显著增加。在刚地弓形虫感染的慢性阶段，感染产生的抗体无法检测到重组蛋白，但它们与 TLA 提取物反应。此外，所有组的脾细胞在刺激 rGRA4 时均表现出 IFN-γ 的高产生，但仅 NbHsp90.3-SAG1 组刺激 rSAG1m 时 IFN-γ 的产生较高。最后，NbHsp90.3-SAG1 免疫的小鼠对刚地弓形虫感染的囊包负荷（56%）显著降低。

结论

我们证明 NbHsp90.3 通过 Th1 型细胞因子产生增强体液和细胞介导的免疫反应。用 NbHsp90.3-SAG1 免疫的小鼠对刚地弓形虫感染表现出部分保护作用，这与诱导记忆免疫反应有关。我们开发并验证了一种疫苗制剂，据我们所知，该制剂首次将 NbHsp90.3 蛋白与含有 T 和 B 细胞表位的刚地弓形虫 SAG1 蛋白肽共价连接。