Noisumdaeng Pirom, Roytrakul Thaneeya, Prasertsopon Jarunee, Pooruk Phisanu, Lerdsamran Hatairat, Assanasen Susan, Kitphati Rungrueng, Auewarakul Prasert, Puthavathana Pilaipan
Faculty of Public Health, Thammasat University, Khlong Luang, Pathum Thani, Thailand.
Thammasat University Research Unit in Modern Microbiology and Public Health Genomics, Thammasat University, Khlong Luang, Pathum Thani, Thailand.
PeerJ. 2021 Mar 10;9:e11021. doi: 10.7717/peerj.11021. eCollection 2021.
Protection against the influenza virus by a specific antibody is relatively strain specific; meanwhile broader immunity may be conferred by cell-mediated immune response to the conserved epitopes across influenza virus subtypes. A universal broad-spectrum influenza vaccine which confronts not only seasonal influenza virus, but also avian influenza H5N1 virus is promising.
This study determined the specific and cross-reactive T cell responses against the highly pathogenic avian influenza A (H5N1) virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals. Ex vivo IFN- ELISpot assay using overlapping peptides spanning the entire nucleoprotein (NP), matrix (M) and hemagglutinin (HA) derived from A/Thailand/1(KAN-1)/2004 (H5N1) virus was employed in adjunct with flow cytometry for determining T cell functions. Microneutralization (microNT) assay was performed to determine the status of previous H5N1 virus infection.
IFN- ELISpot assay demonstrated that survivors nos. 1 and 2 had markedly higher T cell responses against H5N1 NP, M and HA epitopes than survivors nos. 3 and 4; and the magnitude of T cell responses against NP were higher than that of M and HA. Durability of the immunoreactivity persisted for as long as four years after disease onset. Upon stimulation by NP in IFN- ELISpot assay, 60% of H3N2 patients and 39% of healthy subjects exhibited a cross-reactive T cell response. The higher frequency and magnitude of responses in H3N2 patients may be due to blood collection at the convalescent phase of the patients. In H5N1 survivors, the effector peptide-specific T cells generated from bulk culture PBMCs by in vitro stimulation displayed a polyfunction by simultaneously producing IFN- and TNF-, together with upregulation of CD107a in recognition of the target cells pulsed with peptide or infected with rVac-NP virus as investigated by flow cytometry.
This study provides an insight into the better understanding on the homosubtypic and heterosubtypic T cell-mediated immune responses in H5N1 survivors and non-H5N1 subjects. NP is an immunodominant target of cross-recognition owing to its high conservancy. Therefore, the development of vaccine targeting the conserved NP may be a novel strategy for influenza vaccine design.
特定抗体对流感病毒的保护作用具有相对的毒株特异性；同时，针对流感病毒各亚型保守表位的细胞介导免疫应答可能赋予更广泛的免疫力。一种不仅能应对季节性流感病毒，还能应对禽流感H5N1病毒的通用广谱流感疫苗很有前景。
本研究测定了4名H5N1幸存者以及33名非H5N1受试者（包括10名H3N2患者和23名健康个体）针对高致病性甲型禽流感（H5N1）病毒的特异性和交叉反应性T细胞应答。采用体外干扰素-ELISpot检测法，使用覆盖源自A/泰国/1（KAN-1）/2004（H5N1）病毒的整个核蛋白（NP）、基质蛋白（M）和血凝素（HA）的重叠肽，并结合流式细胞术来测定T细胞功能。进行微量中和（microNT）检测以确定既往H5N1病毒感染状况。
干扰素-ELISpot检测表明，1号和2号幸存者针对H5N1的NP、M和HA表位的T细胞应答明显高于3号和4号幸存者；针对NP的T细胞应答强度高于M和HA。免疫反应性的持久性在疾病发作后长达四年。在干扰素-ELISpot检测中，经NP刺激后，60%的H3N2患者和39%的健康受试者表现出交叉反应性T细胞应答。H3N2患者中较高的应答频率和强度可能是由于在患者恢复期采集血液所致。在H5N1幸存者中，通过体外刺激从大量培养的外周血单个核细胞（PBMC）中产生的效应肽特异性T细胞通过同时产生干扰素和肿瘤坏死因子以及上调CD107a表现出多功能性，这是通过流式细胞术在识别用肽脉冲或感染rVac-NP病毒的靶细胞时进行研究的。
本研究有助于更深入了解H5N1幸存者和非H5N1受试者中同亚型和异亚型T细胞介导的免疫应答。由于NP具有高度保守性，它是交叉识别的免疫显性靶点。因此，开发针对保守NP的疫苗可能是流感疫苗设计的一种新策略。
