Barreto Aline Silva, de Franca Mariana Nobre Farias, Dos Reis Tatiana Leão Dos Santos, Silva Joao Antonio Barbosa Martins, Dos Santos Priscila Lima, de Oliveira Fabrícia Alvisi, da Silva Angela Maria, Magalhaes Lucas Sousa, Secco Danielle Angst, Andrade Maria Aiza Fontes, Porto Luís Cristóvão, Rosa Daniela Santoro, Cavalcante Rafael Ciro Marques, Corrêa Cristiane Bani, Sidney John, Sette Alessandro, de Almeida Roque Pacheco, Palatnik-de-Sousa Clarisa Beatriz
Molecular Biology Laboratory, University Hospital, Department of Medicine Federal University of Sergipe, Aracaju, Sergipe, Brazil.
Postgraduate Program in Health Science, Federal University of Sergipe, Aracaju, SE, Brazil.
Front Immunol. 2025 Mar 31;16:1540537. doi: 10.3389/fimmu.2025.1540537. eCollection 2025.
INTRODUCTION: No vaccine is currently licensed against human visceral leishmaniasis (VL), a fatal CD4+ T cell immunosupressive disease against which chemotherapy is reduced to a few toxic drugs. The NH36 nucleoside hydrolase is a DNA metabolism vital enzyme present in all species. A vaccine based on such a conserved antigen could protect against both VL and cutaneous leishmaniasis, whose epidemics geographically overlap. Increased frequencies of NH36-specific IL-2+TNF-α+IFN-γ+-producing CD4+ T cells were associated with VL immune protection. METHODS: the sequences of HLA-Class I and Class II T cell epitopes were predicted in the NH36 peptide sequence using the Tepitope, Propred, IEDB and NetMHCpan EL 4.1 immune informatic tools. The epitopes were synthetized and used to study their reactivity with sera samples, and to stimulate the response of PBMC of human patients cured from VL, asymptomatic individuals and healthy blood donors of a non-endemic area. Cytokine production was studied intracellularly by flow cytometry (ICS) and cytokine secretion was measured in PBMC supernatants. The HLA typing of DNA patients and the analysis of epitope conservancy in the genus were obtained. Two recombinant multiepitope proteins were designed, cloned in , expressed, purified and used for stimulation of PBMC of VL cured and asymptomatic patients. RESULTS: We identified fifteen NH36 conserved epitopes that correspond to promiscuous binders of HLA-DR, -DQ, -DP class II molecules, as well as HLA-A, B and C class I molecules. Collectively, these epitopes provide high worldwide population coverage of both class I and II alleles, and bound to alleles associated with VL susceptibility and resistance. VL asymptomatic individuals showed maximal frequencies of CD4+ and CD8+ multifunctional IL-2+TNF-α+IFN-γ+-producing T lymphocytes in response to these epitopes, with secretion of TNF-α, IL-1β and IL-6. Two recombinant multiepitope vaccines were designed using these epitopes linked by AAA or GPGPG spacers. Both proteins promoted CD4+ and CD8+ T cell responses in PBMC of VL cured and asymptomatic individuals. DISCUSSION: Both MultiAAA and MultiGPGPG proteins could be potentially used for universal human vaccination against leishmaniasis.
引言：目前尚无针对人类内脏利什曼病（VL）的许可疫苗，这是一种致命的CD4 + T细胞免疫抑制疾病，针对该病的化疗药物减少到了几种有毒药物。NH36核苷水解酶是所有物种中存在的一种对DNA代谢至关重要的酶。基于这种保守抗原的疫苗可以预防VL和皮肤利什曼病，这两种疾病的流行在地理上有重叠。产生NH36特异性IL-2 + TNF-α+ IFN-γ+的CD4 + T细胞频率增加与VL免疫保护相关。 方法：使用Tepitope、Propred、IEDB和NetMHCpan EL 4.1免疫信息学工具在NH36肽序列中预测HLA-I类和II类T细胞表位的序列。合成这些表位并用于研究它们与血清样本的反应性，并刺激来自VL治愈患者、无症状个体和非流行地区健康献血者的外周血单核细胞（PBMC）的反应。通过流式细胞术（ICS）在细胞内研究细胞因子的产生，并在PBMC上清液中测量细胞因子的分泌。获得了患者的DNA的HLA分型以及该属中表位保守性的分析。设计了两种重组多表位蛋白，克隆到……中，表达、纯化并用于刺激VL治愈患者和无症状患者的PBMC。 结果：我们鉴定出15个NH36保守表位，它们对应于HLA-DR、-DQ、-DP II类分子以及HLA-A、B和C I类分子的混杂结合物。总体而言，这些表位在全球范围内对I类和II类等位基因提供了高覆盖率，并与与VL易感性和抗性相关的等位基因结合。VL无症状个体对这些表位的反应显示，产生多功能IL-2 + TNF-α+ IFN-γ+的CD4 +和CD8 + T淋巴细胞频率最高，并分泌TNF-α、IL-1β和IL-6。使用通过AAA或GPGPG间隔物连接的这些表位设计了两种重组多表位疫苗。两种蛋白均在VL治愈个体和无症状个体的PBMC中促进了CD4 +和CD8 + T细胞反应。 讨论：MultiAAA和MultiGPGPG蛋白都有可能用于人类针对利什曼病的通用疫苗接种。
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