Laboratory of Biotechnology and Physiology of Viral Infections, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil.
Laboratory of Immunopharmacology, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil.
Front Immunol. 2019 Jul 3;10:1522. doi: 10.3389/fimmu.2019.01522. eCollection 2019.
The importance of the cellular immune response against DENV has been increasingly highlighted in the past few years, in particular for vaccine development. We have previously constructed two plasmids, pE1D2, and pcTPANS1, encoding the envelope (E) ectodomain (domains I, II, and III) and the non-structural 1 (NS1) protein of dengue virus serotype 2 (DENV2), respectively. In the present work, we analyzed the induction of the cellular response in mice immunized with these DNA vaccines and identified the immunogenic peptides. Vaccinated BALB/c mice became protected against a lethal challenge of DENV2. Depletion of CD4 cells in vaccinated animals almost completely abolished protection elicited by both vaccines. In contrast, a significant number of pE1D2- and pcTPANS1-immunized mice survived virus challenge after depletion of CD8 cells, although some animals presented morbidity. To identify immunogenic peptides recognized by T cells, we stimulated splenocytes with overlapping peptide libraries covering the E and NS1 proteins and evaluated the production of IFN-γ by ELISPOT. We detected two and three immunodominant epitopes in the E and NS1 proteins, respectively, and four additional NS1-derived peptides after virus challenge. Characterization by intracellular cytokine staining (ICS) revealed that both CD4 and CD8 T cells were involved in IFN-γ and TNF-α production. The IFN-γ ICS confirmed reaction of almost all E-derived peptides before challenge and identified other epitopes after infection. All NS1-derived peptides were able to elicit IFN-γ production in CD4 cells, while only a few peptides induced expression of this cytokine in CD8 T lymphocytes. Interestingly, we observed an increase in the frequency of either CD4 or CD8 T cells producing TNF-α after immunization with the pE1D2 and challenge with DENV2, while lymphocytes from pcTPANS1-vaccinated animals maintained ordinary TNF-α production after virus infection. We also assessed the recognition of E and NS1 immunogenic peptides in C57BL/6 mice due to the difference in MHC haplotype expression. Two NS1-derived epitopes featured prominently in the IFN-γ response with cells from both animal strains. Overall, our results emphasize the importance of the T cell response involved in protection against dengue induced by E and NS1 based DNA vaccines.
细胞免疫反应对 DENV 的重要性在过去几年中越来越受到重视，特别是在疫苗开发方面。我们之前构建了两个质粒，pE1D2 和 pcTPANS1，分别编码登革热病毒血清型 2（DENV2）的包膜（E）外显子（I、II 和 III 结构域）和非结构蛋白 1（NS1）。在本工作中，我们分析了用这些 DNA 疫苗免疫的小鼠中细胞反应的诱导，并鉴定了免疫原性肽。接种 BALB/c 小鼠可免受 DENV2 的致死性攻击。用抗 CD4 细胞的抗体耗尽接种动物中的 CD4 细胞几乎完全消除了两种疫苗引起的保护作用。相比之下，在耗尽 CD8 细胞后，大量 pE1D2 和 pcTPANS1 免疫的小鼠在病毒攻击后存活下来，尽管有些动物出现了发病。为了鉴定 T 细胞识别的免疫原性肽，我们用覆盖 E 和 NS1 蛋白的重叠肽文库刺激脾细胞，并通过 ELISPOT 评估 IFN-γ 的产生。我们分别在 E 和 NS1 蛋白中检测到两个和三个免疫优势表位，以及病毒攻击后另外四个 NS1 衍生的肽。通过细胞内细胞因子染色（ICS）进行的表征表明，CD4 和 CD8 T 细胞均参与 IFN-γ 和 TNF-α 的产生。ICS 证实了 IFN-γ 之前在挑战前 E 衍生肽的反应，并鉴定了感染后的其他表位。所有 NS1 衍生的肽都能够在 CD4 细胞中诱导 IFN-γ 的产生，而只有少数肽在 CD8 T 淋巴细胞中诱导该细胞因子的表达。有趣的是，我们观察到在用 pE1D2 免疫和用 DENV2 攻击后，CD4 或 CD8 T 细胞产生 TNF-α的频率增加，而来自 pcTPANS1 疫苗接种动物的淋巴细胞在病毒感染后仍保持普通 TNF-α的产生。我们还评估了由于 MHC 单倍型表达的差异，在 C57BL/6 小鼠中对 E 和 NS1 免疫原性肽的识别。两个 NS1 衍生的表位在来自两种动物品系的细胞中均在 IFN-γ 反应中突出。总之，我们的结果强调了基于 E 和 NS1 的 DNA 疫苗诱导的登革热保护中涉及的 T 细胞反应的重要性。
