嚴重特殊傳染性肺炎(COVID-19)是具有高度傳染性且致命的疾病，其致病原是一種新型冠狀病毒(SARS-CoV-2)，可透過飛沫的途徑從呼吸道感染人體。自從2019年12月在中國武漢被發現後，短時間內就在世界各地造成大流行。根據世界衛生組織的統計數據，直到2020年底全球已有超過8千萬確診病例包括190萬名患者死亡。雖然當時已有安全有效的疫苗可供大規模施打，但多種新型疫苗仍存在製造技術門檻太高, 或是需要超低溫保存及運送的問題。對此，開發以蛋白質為抗原的疫苗，具有克服以上問題的優勢。然而，若是蛋白質疫苗中沒有使用能協助刺激免疫反應的佐劑，則通常效果不彰，尤其不容易產生細胞毒殺型的T細胞(CTLs)。本篇論文研究透過特定製劑的方式，使得含有棘蛋白(spike protein)的疫苗，不僅能提供有效的保護力，並能符合由世界衛生組織(World Health Organization, WHO) 提出的建議, 要求COVID-19疫苗需能產生Th1免疫反應。首先，我們設計並生產一個新型冠狀病毒的重組棘蛋白，其俱備穩定的三聚體結構，且與hACE2受體有高度的親和力。在動物試驗中，發現使用此三聚體的重組棘蛋白(S-Trimer 或 rTS) 作為抗原於三個不同劑量的條件下(0.5, 5, 20 μg)， 與一種乳液形式的佐劑(SWE)混合而成的疫苗製劑，其所能誘發的免疫反應顯著優於以鋁鹽為佐劑的對照組。值得注意的是，低劑量的S-Trimer (0.5 μg)搭配SWE佐劑，所引起的免疫反應相當於使用S-Trimer較高劑量的組別，卻更傾向Th1的反應；而且能保護倉鼠免於原始病毒株的感染；其免疫倉鼠後的血清仍可以有效中和變異的病毒株(Beta與Delta)。在另一項研究中，我們發現使用重組TS蛋白質和表達TS蛋白質的質體DNA在注射前與鋁鹽混合，可增強針對棘蛋白的體液和細胞免疫反應。通過評估免疫後小鼠血清中IgG2a/IgG1抗體的比例，以及由T細胞所分泌的細胞激素，也驗證結合TS DNA疫苗與TS蛋白質及鋁鹽的組合製劑，會誘發偏向Th1的免疫反應。攻毒試驗的結果顯示，被免疫這種組合式疫苗的倉鼠可以產生有效的抗體反應，感染之後體重減輕較少，並顯著減少肺部的病毒量和肺部組織的病變。總結，使用低劑量 rTS搭配SWE佐劑，或是利用鋁鹽同步遞送蛋白質與DNA的組合式疫苗，兩者皆能成功引起俱保護效果的免疫反應，而且能產生偏向Th1的免疫反應；特別是，接種組合式疫苗還能顯著促進俱抗原特異性的CTL反應。

The Coronavirus disease 2019 (COVID-19) is a highly contagious and lethal disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through air travel routes, it has globally and rapidly spread from Wuhan China. Till the end of 2020, this pandemic has resulted in over 83 million confirmed cases worldwide, with nearly 1.9 million deaths. Although, several vaccines against SARS-CoV-2 have been authorized and used for large-scale vaccination. The manufacturing difficulties and the storage of mRNA or viral vector vaccines at ultra-low temperatures limit global distribution. The utilization of protein subunit vaccines has proven advantageous in overcoming these obstacles, as it has been extensively studied and widely employed in numerous existing prophylactic vaccines for an extended period. Nevertheless, protein-based vaccines typically induce poor immune responses in the absence of a potent adjuvant and exhibit a limited ability to generate cytotoxic T lymphocytes (CTLs). The primary objective of this thesis is to examine the compositions of vaccines that utilize spike proteins in order to elicit immune responses that are protective and predominantly Th1-biased. Initially, a recombinant spike protein of the SARS-CoV-2 virus was engineered and synthesized using Chinese hamster ovary (CHO) cells. The resulting protein exhibited a stable trimeric structure and demonstrated a strong binding affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. The rodent studies showed that formulating this trimeric spike protein (S-Trimer or rTS) at different doses (0.5, 5, 20 ug) with a squalene-in-water emulsion (SWE) adjuvant elicited potent immunity that was superior to those induced by aluminum hydroxide (alum) adjuvant. Notably, low-dose (0.5 μg) of S-Trimer with SWE stimulates comparable immune responses that are biased toward Th1 in mice, protects hamsters from SARS-CoV-2 infection, and provides cross-neutralizing antibody activity against variants of concern (Beta and Delta strain). This study also demonstrated rTS protein and TS encoding DNA vaccine mixed with alum before injection enhances humoral and cellular immune responses. Through evaluating the IgG2a/IgG1 isotype profile and cytokine profile of the specific boosted T-cell response, it has been confirmed the combined formulation of TS DNA and protein with alum induced a Th1-biased immune response. Moreover, the viral challenge study showed that the hamsters immunized with this combined vaccine generate durable antibody responses, experience less body weight loss, and significantly reduce viral load in lung and lung lesions. In summary, either SWE adjuvating low-dose of rTS or the co-delivery of protein and DNA combined vaccine achieved protective immunity and induce a Th1-biased response; especially, the strategy of the combined vaccine significantly improves antigen-specific CTL response.

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