M2e奈米殼疫苗對雞隻禽流感廣泛保護力之評估

Abstract

家禽流行性感冒是一種易於突變的人畜共通傳染病，持續對全球家禽產業造成鉅大的經濟損失與公共衛生的威脅。在開發具廣效保護力的禽流感疫苗的過程中，流感病毒基質蛋白2的細胞外結構域 (M2e) 是一個適合的候選抗原，因為M2e的序列在多數A型流感病毒中具有高度保守性，產生之抗體可介導抗體依賴性細胞毒殺作用（ADCC），能在病毒釋放前清除受感染的細胞。然而，M2e因分子量小且表現量低，免疫原性相當有限，因此需要有效的抗原遞送系統來提升其免疫效果。本研究中，我們使用專利奈米殼來同時包覆M2e胜肽作為抗原、STING促進劑作為佐劑，評估其在無特定病原雞中的免疫原性與保護效果。結果顯示，雞隻免疫M2e 奈米殼疫苗之後，能在多種淋巴組織中偵測到顯著較多的B細胞標誌與IgG產生細胞，並在血液中測得顯著較高之M2e特異性抗體，此抗體能辨識跨7種不同亞型流感病毒感染MDCK細胞所表現的M2e蛋白質。在H6N1病毒攻毒後，皮下注射疫苗雞隻的肺與腎臟病毒量有下降的趨勢，腎臟的肉眼病變明顯減輕，組織病理分析亦顯示腎臟、肺及氣管有較低的病變指數。此外，以雞脾臟來源之自然殺手細胞進行ADCC的細胞試驗，發現免疫M2e奈米殼疫苗之雞隻血清能有效誘發MDCK細胞在流感病毒感染後的死亡。總結來說，本研究顯示M2e奈米殼疫苗在雞隻可誘發具跨病毒亞型的交叉免疫反應，並透過ADCC路徑減少病毒增殖、降低組織損傷，有潛力作為廣效保護性之禽流感疫苗，對於禽流感的防控策略具有應用價值。

Avian influenza virus (AIV) is a zoonotic pathogen that easily mutates and continues to cause economic loss in the poultry industry and poses a threat to public health. To develop a broadly protective influenza vaccine, the extracellular domain of matrix protein 2 (M2e) is a promising antigen candidate as it is highly conserved across most influenza A subtypes. M2e-induced immune protection is primarily mediated through antibody-dependent cellular cytotoxicity (ADCC), which enables the clearance of infected cells before viral release. However, the immunogenicity of M2e is limited due to its small size and low abundance on the viral surface, necessitating effective antigen delivery systems to enhance immunogenicity. In this study, we encapsulated M2e peptides as the antigen and STING agonist as an adjuvant within proprietary nanoshells and evaluated their immunogenicity and protective efficacy in specific-pathogen-free (SPF) chickens. The results showed that immunization of SPF chickens with the M2e nanoshell vaccine significantly enhanced the proliferation of B cells and IgG-producing cells in various lymphoid tissues, thereby inducing a robust and durable M2e-specific antibody response. In addition, the induced antibodies demonstrated cross-reactivity by effectively recognizing the M2e protein expressed on the surface of MDCK cells infected with seven different subtypes of influenza viruses. Following challenge with H6N1, vaccinated chickens exhibited a trend of reduced viral loads in both lung and kidney tissues and fewer gross lesions in the kidneys. Histopathological analysis revealed attenuated tissue damage in the kidney, lung, and trachea, with significantly lower pathology scores in the M2e nanoshell vaccinated group. Furthermore, to assess ADCC activity, an in vitro killing assay demonstrated that chicken splenic natural killer cells were capable of lysing influenza-infected MDCK cells in the presence of M2e nanoshells immune sera. In conclusion, this study demonstrates that the M2e nanoshell vaccine elicits durable and cross-reactive immune responses and reduces tissue damage through ADCC-mediated mechanisms. M2e nanoshell vaccine shows potential as a broadly protective avian influenza vaccine and holds value for controlling AIV outbreaks in poultry.