發展以醣質為抗原之HIV-1疫苗: 設計、合成、以及免疫評估

Abstract

發展以醣質為抗原之HIV-1疫苗: 設計、合成、以及免疫評估 愛滋病是由於受到人類免疫缺陷病毒（HIV）的感染，導致後天性細胞免疫功能出現缺陷所造成的一種致命性疾病。發展出有效抗HIV疫苗，一直被認為是最有希望控制全球愛滋病毒/愛滋病流行的一種方式。然而疫苗發展主要的挑戰在於如何設計出抗原結構，進而產生出廣泛性中和抗體，有效的殺滅病毒。愛滋病毒用於躲避抗體毒殺的主要機制，乃是以多種不同的N型鍵結寡醣來遮蔽其位於病毒表面的醣蛋白gp120。最近的研究顯示 在愛滋病患體內分離出一種新類型的廣泛性中和抗體，能夠經由辨識醣蛋白gp120 上的N型鍵結寡醣，進而對於HIV-1 病毒株以及它的各種突變類型皆能有效的加以毒殺。此結果揭示利用特殊結構的寡醣分子作為疫苗抗原，來產生廣泛性中和抗體治療愛滋病的可行性。若能深入了解抗體與N型鍵結寡醣間的辨識機制，將有助於設計出最有效的疫苗抗原。 本論文描述利用化學以及酵素的合成方式，來合成各種與醣蛋白gp120 相關的N型鍵結寡醣結構，包括各種高甘露醣型、混合型、以及複雜型的N型鍵結寡醣。這些N型鍵結寡醣分子將用於建立醣分子庫，並結合醣晶片的技術，來剖析這些醣分子與各種已知抗HIV-1廣泛性中和抗體 (PG9, PG16, PGTs 121, 128, 141-145)間的結合專一性。此研究發現利用氧化鋁包覆玻片的醣晶片的技術，可以偵測到抗體與醣分子間微弱但非常重要的結合作用力。分析結果顯示，有些高效價的抗HIV-1廣泛性中和抗體，對於非均質的醣分子混合體 (包含兩種醣分子以不同比例混合)的結合力較對於均質的醣分子更強。這種現象可能是為了因應病毒表面醣蛋白非均質化的醣化情形，宿主人類的免疫系統所採取的對應手段。 經由以上醣晶片研究結果，提供了設計疫苗所需醣抗原分子的基本結構，依照這些基本結構可用來設計與合成疫苗所需的醣共軛體，繼而用以進行免疫學上的研究，觀察此疫苗是否能誘導產生廣泛性中和抗體，以及抗體是否能與各種HIV 病毒上的醣蛋白gp120作結合，進而消滅病毒。 關鍵詞: HIV-1疫苗，N型鍵結寡醣，以醣質為基礎的疫苗抗原，廣泛性中和抗體，醣共軛體，醣合成。

Abstract An HIV vaccine continues to be the best hope to end the HIV pandemics. The challenge of HIV vaccine development is to design the immunogens to induce the antibodies that can have broadly neutralizing activities. An effective mechanism that HIV uses to avoid attacks of neutralizing antibodies is to coat envelope gp120 with heterogeneous N-linked carbohydrate structures. Recently, a class of broadly neutralizing antibodies (bNAbs), isolated from HIV positive donors, was shown to be potent in neutralizing primary HIV-1 strains across clades by targeting the glycan dependent epitopes on gp120 surface, demonstrating the feasibility of inducing such antibodies. Understanding the molecular mechanism of the HIV-gp120 glycan coat recognition by bNAbs may guide effective immunogen design. This thesis describes the efficient chemical and enzymatic syntheses of diverse HIV-1 gp120 related high-mannose, hybrid, and complex type N-glycans. A well defined library of natural and unnatural N-glycan structures was used to develop glycan microarray platform for profiling glycan binding specificities of HIV-1 bNAbs, PG9, PG16, PGTs 121, 128, and 141-145. With the next generation array on aluminium oxide-coated glass (ACG) slide, the critical low affinity antibody-carbohydrate interactions were detected. In addition, we showed that a few highly potent HIV-1 broadly neutralizing antibodies exhibit strong binding to closely spaced hetero-glycans on array surface, suggesting that this phenomenon might be an important adaptation of host immune system to deal with heterogeneously glycosylated HIV-1 spike. The microarray studies of glycan binding specificities of HIV-1 bNAbs established a structural basis for designs of immunogens based on epitopes recognized by these antibodies. Finally, the immunological evaluation of carbohydrate-based immunogens was performed to investigate whether the synthetic glycoconjugates can induce broadly neutralizing antibodies that can cross react with HIV-gp120. Keywords HIV-1 vaccine, N-linked oligosaccharides, carbohydrate-based immunogens, broadly neutralizing antibodies, glycoconjugates, carbohydrate synthesis.