Intranasal Immunization with Pressure Inactivated Avian Influenza Elicits Cellular and Humoral Responses in Mice

Influenza viruses pose a serious global health threat, particularly in light of newly emerging strains, such as the avian influenza H5N1 and H7N9 viruses. Vaccination remains the primary method for preventing acquiring influenza or for avoiding developing serious complications related to the disease. Vaccinations based on inactivated split virus vaccines or on chemically inactivated whole virus have some important drawbacks, including changes in the immunogenic properties of the virus. To induce a greater mucosal immune response, intranasally administered vaccines are highly desired as they not only prevent disease but can also block the infection at its primary site. To avoid these drawbacks, hydrostatic pressure has been used as a potential method for viral inactivation and vaccine production. In this study, we show that hydrostatic pressure inactivates the avian influenza A H3N8 virus, while still maintaining hemagglutinin and neuraminidase functionalities. Challenged vaccinated animals showed no disease signs (ruffled fur, lethargy, weight loss, and huddling). Similarly, these animals showed less Evans Blue dye leakage and lower cell counts in their bronchoalveolar lavage fluid compared with the challenged non-vaccinated group. We found that the whole inactivated particles were capable of generating a neutralizing antibody response in serum, and IgA was also found in nasal mucosa and feces. After the vaccination and challenge we observed Th1/Th2 cytokine secretion with a prevalence of IFN-γ. Our data indicate that the animals present a satisfactory immune response after vaccination and are protected against infection. Our results may pave the way for the development of a novel pressure-based vaccine against influenza virus.流感病毒对全球公共卫生构成严重威胁，尤其在新型毒株不断涌现的背景下，如禽流感H5N1与H7N9病毒。接种疫苗仍是预防流感感染、避免发生该疾病相关严重并发症的核心防控手段。当前主流的灭活裂解疫苗与化学灭活全病毒疫苗存在若干显著缺陷，其中包括病毒免疫原性特性发生改变。为诱导更强的黏膜免疫应答，鼻内接种疫苗备受青睐——其不仅可预防疾病，还能在感染原发部位阻断病毒侵染。为规避上述缺陷，静水压已被用作病毒灭活与疫苗生产的潜在技术手段。本研究证实，静水压可灭活甲型禽流感H3N8病毒，同时保留血凝素（hemagglutinin）与神经氨酸酶（neuraminidase）的生物活性。免疫接种后的攻毒动物未表现出任何疾病相关症状（如被毛蓬乱、精神萎靡、体重下降以及扎堆蜷缩）。同样，与攻毒未免疫组相比，免疫组动物的伊文思蓝染料渗漏程度更低，支气管肺泡灌洗液中的细胞计数也显著更少。研究发现，灭活全病毒颗粒可诱导血清产生中和抗体应答，且在鼻黏膜与粪便中也能检测到免疫球蛋白A（immunoglobulin A，IgA）。免疫接种与攻毒实验后，我们观察到Th1/Th2型细胞因子分泌，且以干扰素-γ（IFN-γ）的表达占优。本研究数据表明，免疫接种后的动物可产生良好的免疫应答，且能有效抵御病毒感染。本研究结果可为新型静水压灭活流感疫苗的研发开辟全新路径。