探討針對A型流感病毒之廣效中和人類單株抗體

Abstract

自西元2013年中國爆發新型重組禽流感H7N9病毒感染，截至去年已有五波的H7N9流感病毒爆發。雖然現今已有許多可中和不同病毒株之廣效性中和抗體，然而因為不同病毒亞型的HA仍有些結構上的差異，導致許多廣效性中和抗體對於H7N9病毒之中和能力較差。因此，本篇研究之目的為探討可中和H7N9流感病毒的抗體之特性。首先，我們將一位感染H7N9病毒的病人周邊血單核細胞運送至財團法人生物技術開發中心(DCB)，以生產多個人類單株抗體後，先以H7N9類病毒顆粒之結合能力來篩選抗體，再藉由微量抗體中和反應試驗(Microneutralization assay，mNT)找到兩支對於H7N9病毒具有中和能力之抗體，分別為HNP6 C3HK和HNP2 C11HL2。我們經由細胞免疫螢光試驗比較此二抗體對於流感病毒表面蛋白質HA、NA及M蛋白質的結合能力，發現HNP6 C3HK及HNP2 C11HL2是透過結合HA蛋白質而發揮其中和能力；此外，此二抗體亦對2017年所分離出具有多鹼性胺基酸插入之高致病性H7具有結合能力。關於廣效結合性的部分，我們發現此二抗體可結合Group 2的HA，包含H3、H7及H10，然而對於Group 1的HA，兩支抗體皆無法結合H1，僅有HNP2 C11HL2可結合H9。進一步中和實驗的結果顯示HNP6 C3HK 可抑制分別來自2007，2014和2017年之H3N2 病毒，而HNP2 C11HL2只具有抑制2007年H3N2病毒之能力。為了確認抗體之結合位點位在HA stem上，我們利用將全長HA經胰蛋白酶和二硫蘇糖醇(DTT)作用後得到HA stem之結構，並測試抗體的結合能力，可以發現此二抗體的結合皆下降。我們亦參考文獻將HA做特定修飾後僅保留stem之結構，然而發現此合成stem並非正確結構，因而無法被已知結合於stem結構之抗體所辨認。進一步探討HNP6 C3HK和HNP2 C11HL2於HA蛋白質之結合位點，我們將許多HA stem區域之特定位點做單點突變後，比較抗體結合能力的變化。由細胞免疫螢光試驗發現S29、N46、I357和D358突變後會影響抗體結合能力，I357及D358之突變甚至會導致HNP6 C3HK結合完全消失；我們亦使用流式細胞術量化相對於未突變之HA，此二抗體對於帶有S29A，N46H，I357A及D358A之HA的結合百分比，並得到與細胞免疫螢光試驗一致的結果。為了確認I357及D358為此二抗體之結合位點，我們透過比較此二抗體對於未含突變及帶有此二突變的重組H7N9病毒之中和能力，發現HNP6 C3HK和HNP2 C11HL2對於分別帶有I357A和D358A之重組病毒失去其抑制之能力。在探討抗病毒機制的部分，我們發現若先將HNP2 C11HL2與成熟之HA作用才進行酸性環境刺激與DTT處理，可以抑制HA1與HA2的解離。最後我們透過測試抗體依賴的細胞介導之細胞毒性作用發現，HNP6 C3HK可藉由結合Fc γRIIIa接受器活化下游反應。綜言之，由感染H7N9病毒的病人檢體所合成之中和性抗體HNP6 C3HK和HNP2 C11HL2對於H3、H7和H10有結合能力且可中和H3N2與H7N9病毒。另外，位於融合肽(Fusion peptide)之357和358對於此二抗體之結合與中和相當重要。

Reassortant avian influenza A H7N9 virus has caused 5 epidemic waves in China since 2013. Although there have been several broadly neutralizing antibodies (bnAb) showing cross-reactivity against various influenza viruses, most bnAbs exhibit less effective antiviral activity against H7N9 virus due to the differences in structure and glycosylated modification patterns between hemagglutinin proteins of group 1 and group 2 influenza virus. This study aimed to investigate the characteristics of two neutralizing antibodies that effectively neutralize H7N9 virus. First, a number of human monoclonal antibodies (HuMAbs) were generated from the peripheral blood mononuclear cells (PBMCs) of one H7N9-infected patient by the Development Center of Biotechnology (DCB). After initial binding screening using the virus-like particles, micro-neutralization assay was performed to determine the neutralizing abilities of selected HuMAbs against H7N9 viruses. Two HuMAbs, HNP6 C3HK and HNP2 C11HL2, exhibited efficient neutralizing abilities. In immunofluorescence assay, HNP6 C3HK and HNP2 C11HL2 showed binding activity to HA protein. Besides, highly pathogenic H7 bearing poly-basic amino acids insertion in the cleavage site could also be recognized by these two HuMAbs. In addition, both antibodies had cross-binding ability to H3, H7, H10 of group 2 influenza viruses and HNP2 C11HL2 showed reactivity to H9 of group1 influenza viruses. Furthermore, HNP6 C3HK exhibited broadly neutralizing ability to H3N2 viruses isolated in 2007, 2014 and 2017, and HNP2 C11HL2 can inhibit H3N2 virus in 2007. To confirm that the binding sites of these two antibodies are located on the stem region, the binding abilities of these two HuMAbs to trimeric stem structure generated by sequential treatment of trypsin and DTT on HA0 were determined. In this experiment, the binding activity of both antibodies decreased upon trypsin and DTT treatment. A H7 stem structure was also synthesized based on the study results from other study, but the stem structure could not be recognized by a reference antibody targeting to conformational epitopes in the stem region. Next, the residues critical for HNP6 C3HK and HNP2 C11HL2 binding were determined by site-directed mutagenesis and immunofluorescence assay (IFA). The substitution at resides S29, N46, I357 and D358 reduced the binding of these two antibodies. Substitutions of I357A and D358A even abolished the binding of HNP6 C3HK. The binding abilities of both antibodies to four mutant variants were also quantified by flow cytometry and the results were consistent with the IFA. To further confirm these findings, the neutralizing abilities of HNP6 C3HK and HNP2 C11HL2 against recombinant H7N9 mutant viruses carrying I357A or D358A substitutions were examined. Both antibodies lost neutralizing abilities to mutant viruses as compared to wild type recombinant viruses. The study results were further confirmed by the finding that the binding of HNP2 C11HL2 to the HA prior to the treatment of low pH-trigger and DTT could block the dissociation of HA1 and HA2. Finally, HNP6 C3HK was shown to exhibit the capability of inducing antibody-dependent cellular cytotoxicity (ADCC) through binding to FcγRIIIa receptor. In conclusion, HNP6 C3HK and HNP2 C11HL2 generated from B cells of a H7N9-infected patient showed binding abilities to H3, H7 and H10 and broadly neutralizing activities against H7N9 and H3N2 viruses. I357 and D358 were critical for the binding of both antibodies.