利用腺相關病毒載體表現對新冠病毒突變株有廣泛保護效果的抗人類ACE2黏膜抗體

Abstract

2019年底出現的新冠病毒因病毒持續突變產生突變株令疫情至今仍無法趨緩。現有的SARS-CoV-2疫苗和單株抗體藥物是以病毒的棘蛋白為目標，防止病毒和血管收縮素轉換酶II（hACE2）結合進入人體感染。但是被WHO列為高度關注的突變株，包含alpha、beta、gamma、delta和omicron等，在棘蛋白上都有不少突變位點。研究顯示這些突變位點除了增加病毒和hACE2的結合能力外，更大幅降低現有疫苗和抗體的保護效果。因此，我們迫切需要研發能夠廣泛預防和治療COVID-19的方法。先前我們實驗室利用融合瘤技術分離出一株和hACE2高度結合的單株抗體，命名為2H2，並將其可變區接到human IgG骨架產生嵌合抗體ch2H2-IgG。 於本篇論文中我們首先利用HDX-MS、Cryo-EM與Biolayer Interferometry實驗分析，證實ch2H2-IgG和hACE2的結合位點與SARS-CoV-2和hACE2的結合位點有所重疊，且抗體與受體間的親和力大於病毒與受體間的親和力，因此能夠阻止病毒與hACE2的結合，達到預防SARS-CoV-2感染的效果。接著我們以腺相關病毒（AAV）作為載體在K18轉基因小鼠中表現ch2H2-IgG，攻毒結果顯示ch2H2-IgG可以保護小鼠對抗omicron BA.5感染，降低肺臟中病毒感染量及肺部組織損傷程度。為了更進一步增加ch2H2抗體在呼吸道黏膜預防SARS-CoV-2感染的效果，我們將ch2H2抗體的可變區接到human IgA1的骨架上，並使用peptide M 來純化CHO cell產生的ch2H2-IgA抗體。之後先以西方墨點法確認抗體在輕鏈、重鏈以及J chain的表現符合預期，再以偽病毒中和試驗證實ch2H2-IgA對不同突變株皆具有中和效果。但在動物實驗我們以AAV6表現ch2H2-IgA，無論是氣管注射（i.t.）、鼻腔注射（i.n.）或靜脈注射（i.v.）都無法在血清、肺部沖洗液或鼻腔沖洗液以ELISA測得ch2H2-IgA的表現。 本篇論文證明會辨認hACE2的ch2H2-IgG與ch2H2-IgA抗體確實可以中和SARS-CoV-2的不同突變株，小鼠攻毒試驗則證明以AAV6作為載體遞送抗體可以有效保護小鼠免於病毒感染。目前正在研究ch2H2-IgA無法在小鼠體內被正常表現的原因，但本研究成果仍對SARS-CoV-2突變株以及未來新興病毒提供另一種預防策略。

Since the emergence of the novel coronavirus in late 2019, it has sparked a global pandemic. Current interventions against SARS-CoV-2 include vaccines and monoclonal antibody therapies targeting the viral spike protein, which prevents the virus from binding to the angiotensin-converting enzyme II (hACE2) and infecting human cells. However, variants of concern, such as alpha, beta, gamma, delta, and omicron, harboring numerous mutations in the spike protein have enhanced the binding affinity between the virus and hACE2 and diminished the protective efficacy of existing vaccines and antibodies. Consequently, there is an urgent need to develop new interventions that can provide broad protectivity to limit the transmission of continuously emerging SARS-CoV-2 variants in the future. In our previous study, we isolated a high-affinity anti-hACE2 monoclonal antibody, named 2H2, using hybridoma technology, and further engineered a chimeric antibody, ch2H2-IgG, by grafting its variable regions onto the human IgG framework. In this study, we investigated the binding mechanism of ch2H2-IgG and its efficacy in preventing SARS-CoV-2 infection. The HDX-MS and Cryo-EM results revealed that ch2H2-IgG binds to a site on hACE2 that overlaps with the SARS-CoV-2 RBD binding site, thus blocking the virus from interacting with hACE2. We further utilized adeno-associated virus (AAV) as a vector to express ch2H2-IgG in K18-hACE2 transgenic mice and demonstrated its protective effect against omicron BA.5 infections. To further enhance the efficacy of the ch2H2 antibody in preventing SARS-CoV-2 infection in the respiratory mucosa, we generated ch2H2-IgA by grafting the variable regions of 2H2 onto the human IgA1 framework and purified the antibody produced by CHO cells using peptide M. Subsequently, we confirmed the expression of the antibody through Western blot analysis. Pseudovirus neutralization assays confirmed the neutralizing activity of ch2H2-IgA against different SARS-CoV-2 variants. In animal experiments, however, we found that the expression of ch2H2-IgA could not be detected in serum, lung lavage fluid, or nasal lavage fluid by ELISA, regardless of whether it was administered via intratracheal (i.t.), intranasal (i.n.), or intravenous (i.v.) routes. Currently, we are investigating the underlying reasons why ch2H2-IgA fails to express normally in mice. This study demonstrated that both ch2H2-IgG and ch2H2-IgA antibodies, which recognized hACE2, effectively neutralize different variants of SARS-CoV-2 and that rAAV-mediated antibody delivery protects mice from viral infection. In conclusion, our study provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and other hACE2-dependent pathogens that may emerge in the future.