Sączyńska Violetta, Romanik Agnieszka, Florys Katarzyna, Cecuda-Adamczewska Violetta, Kęsik-Brodacka Małgorzata, Śmietanka Krzysztof, Olszewska Monika, Domańska-Blicharz Katarzyna, Minta Zenon, Szewczyk Bogusław, Płucienniczak Grażyna, Płucienniczak Andrzej
Institute of Biotechnology and Antibiotics, Warsaw, Poland.
Department of Poultry Diseases, National Veterinary Research Institute, Puławy, Poland.
PLoS One. 2017 Feb 17;12(2):e0172008. doi: 10.1371/journal.pone.0172008. eCollection 2017.
The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design. The HA protein with the sequence of the H5N1 HPAIV strain was efficiently expressed in Escherichia coli, recovered in the form of inclusion bodies and refolded by dilution between two chromatographic purification steps. Antigenicity studies showed that the resulting antigen, referred to as rH5-E. coli, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses in vitro. The proper conformation of this protein and its ability to form functional oligomers were confirmed by a hemagglutination test. Consistent with the biochemical characteristics, prime-boost immunizations with adjuvanted rH5-E. coli protected 100% and 70% of specific pathogen-free, layer-type chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively. The observed protection was related to the positivity in the FluAC H5 test (IDVet) but not to hemagglutination-inhibiting antibody titers. Due to full protection, the effective contact transmission of the homologous challenge virus did not occur. Survivors from both challenges did not or only transiently shed the viruses, as established by viral RNA detection in oropharyngeal and cloacal swabs. Our results demonstrate that vaccination with rH5-E. coli could confer control of H5N1 HPAIV infection and transmission rates in chicken flocks, accompanied by reduced virus shedding. Moreover, the role of H5 subtype-specific neutralizing antibodies in anti-influenza immunity and a novel correlate of protection are indicated.
高致病性（HP）H5N1禽流感病毒（AIV）在感染的鸡中致死率高达100%，并构成持续的大流行威胁。研发针对H5N1高致病性禽流感病毒的有效疫苗的努力主要集中在血凝素（HA）上，它是一种能引发中和抗体的免疫显性病毒抗原。绝大多数疫苗项目是使用真核表达系统进行的。相比之下，我们根据自己的设计使用细菌表达系统来生产疫苗HA蛋白（细菌HA）。具有H5N1高致病性禽流感病毒株序列的HA蛋白在大肠杆菌中高效表达，以包涵体形式回收，并在两个色谱纯化步骤之间通过稀释进行复性。抗原性研究表明，所得抗原称为rH5-大肠杆菌，保留了H5亚型HA特异性抗体靶向的构象表位，在体外抑制血凝和/或中和流感病毒。血凝试验证实了该蛋白的正确构象及其形成功能性寡聚体的能力。与生化特性一致，用佐剂化的rH5-大肠杆菌进行的初免-加强免疫分别使100%和70%的无特定病原体蛋鸡型鸡免受同源和异源H5N1高致病性禽流感病毒的攻击。观察到的保护作用与FluAC H5检测（IDVet）呈阳性有关，而与血凝抑制抗体滴度无关。由于获得了完全保护，同源攻击病毒未发生有效的接触传播。两次攻击的存活鸡未排出病毒或仅短暂排出病毒，这通过口咽和泄殖腔拭子中的病毒RNA检测得以证实。我们的结果表明，用rH5-大肠杆菌疫苗接种可控制鸡群中H5N1高致病性禽流感病毒的感染和传播率，同时减少病毒排出。此外，还表明了H5亚型特异性中和抗体在抗流感免疫中的作用以及一种新的保护相关指标。
