單株抗體於茲卡病毒及新型冠狀病毒之研究與應用

Abstract

茲卡病毒 (Zika virus；ZIKV) 感染可能造成嚴重神經疾病，然而目前並無FDA 核准的茲卡病毒疫苗及抗病毒藥物可以預防或治療該疾病。此外，茲卡病毒感染之症狀與其他Flavivirus 類似，因此不易診斷。茲卡病毒利用表面套膜蛋白 (envelope protein；E蛋白) 結合宿主細胞並引發膜融合，為重要之中和性抗體標的。本研究以融合瘤細胞技術開發出抗E 蛋白之單株抗體A1、B1、C1 及9E-1，雖然這四株抗體皆無法中和茲卡病毒，但是抗體B1 及9E-1 可以結合茲卡病毒，因此利用這兩株抗體開發出茲卡病毒之快篩檢測試片。 新型冠狀病毒 (Severe Acute Respiratory Syndrome Coronavirus 2；SARS-CoV-2) 所引起的新型冠狀病毒肺炎 (Coronavirus-induced disease；COVID-19) 在全球造成嚴重疫情，雖然目前已有疫苗與藥物可以預防及治療該疾病，但是病毒不斷變異可能導致抗體或疫苗失效。當病毒表面棘蛋白 (spike protein；S蛋白) 的S1 subunit結合宿主細胞ACE2受體，S2 subunit會引發病毒與宿主細胞產生膜融合。抗體藥物主要透過結合S 蛋白S1subunit的受體結合域 (receptor-binding domain；RBD) 防止病毒與宿主細胞結合，然而該區域的高突變率會造成中和性抗體失效及疫苗保護力下降。相較之下負責引發膜融合的S2 subunit 具有較高保守性，然而針對此區域的抗體卻較少被發表。本研究針對S2subunit開發單株抗體，成功製備出三株中和性抗體S2-4D、S2-5D 及S2-8D，其抗原決定位 (epitope) 在SARS-CoV-2 變異株中具有100%保守性。S2-4D、S2-5D 及S2-8D 可以透過阻止S 蛋白引起的膜融合現象抑制病毒感染，並且對於突變株仍具有廣效性。血清學實驗顯示部分COVID-19 病人血清中亦含有此類抗體。進一步將S2-4D、S2-5D 及S2-8D 的抗原決定位S(1127-1167) 製備為疫苗，可以在小鼠體內成功誘導產生中和性血清，並且可以抑制膜融合現象，因此該片段具有發展成廣效性疫苗的潛力。

Zika virus (ZIKV) infection may cause severe neurological complications. However, there are no FDA-approved vaccines and antivirals for Zika viral disease. Moreover, it can be misdiagnosed as other infectious diseases since its clinical presentations closely resemble other Flavivirus. The ZIKV envelope protein (E) mediates host receptor binding and membrane fusion, and is therefore the major target for neutralizing antibodies. In this study, four monoclonal antibodies (mAbs) (A1, B, C1, and 9E-1) targeting the ZIKV envelope protein have been developed by using hybridoma technology. Although these four mAbs cannot neutralize ZIKV, mAbs B and 9E-1 can bind ZIKV. Therefore, mAbs B and 9E-1 have been developed as the lateral flow immunochromatographic assay for specific detection of ZIKV. Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 760 million confirmed cases. Though vaccines and antivirals for this disease are currently available, the newly emerged variants may render them ineffective. After the S1 subunit of SARS-CoV-2 spike (S) protein binds to host ACE2 receptor, the spike S2 subunit trigger virus-host cell membrane fusion. Most neutralizing antibodies (nAbs) target the receptor binding domain (RBD) of S1 subunit to inhibit virus-host cell binding, however, they lost their neutralizing activity against the newly emerged SARS-CoV-2 variants with sequence mutations at RBD. In contrast, the nAbs targeting the relatively conserved S2 subunit were poorly defined and investigated. Here we report three S2-specific nAbs, S2-4D, S2-5D, and S2-8D, which targeted a highly conserved epitope at S2 subunit of S protein, performed potent neutralizing activity against SARS-CoV-2 infection through blocking the S protein-mediated virus-host cell membrane fusion. Notably, these nAbs exhibited broadly neutralizing activity against SARS-CoV-2 variants. Furthermore, the human sera obtained from COVID-19 patients contain similar antibodies. Antisera collected from mice immunized with the identified epitope peptides S(1127-1167) of these three nAbs also showed potent neutralizing activity and can inhibit membrane fusion, indicating that the highly conserved binding epitope of S2-4D, S2-5D, and S2-8D is a potential vaccine candidate and may provide protection against newly emerged SARS-CoV-2 variants.