家禽傳染性支氣管炎冠狀病毒變異株之研究與抗病毒奈米疫苗之開發驗證

Abstract

家禽傳染性支氣管炎病毒（IBV）對全世界的家禽業構成威脅。本研究從台灣型IBV變異株的分析出發（Unit 3），隨後著重於發展和評估一種能有效誘發雞免疫反應的抗病毒奈米疫苗平台（Unit 4, 5, 6）。 從接種台灣型弱毒IBV疫苗的肉雞中分離到一株致病的台灣型IBV 3575/08毒株。儘管3575/08毒株與台灣型本土株的結構基因高度同源，但3575/08株仍表現出明顯的抗原性差異，並具有更強的呼吸道和腎臟致病性。3575/08株感染會延遲雞的先天與適應性免疫反應基因的表現，讓感染雞隻更早開始排出病毒，且排毒時間持續更長。這個新的IBV毒株具有已知的本土基因型，但在抗原性、致病性和宿主免疫原性等方面卻表現出明顯的改變。變異株的出現表示IBV在田間正在持續發生變異。 在病毒的不斷變異之下， IBV的疫苗製劑需要發展更創新、有效的方式。然而，由於家禽疫苗佐劑的選擇有限，在接種路徑上有很大的限制，且往往效果不佳。為了構建了一種安全有效的新型佐劑，本研究以禽TLR21促進劑CpG ODN 2007作為佐劑，與PLGA的中空奈米顆粒平台結合，形成包覆CpG的奈米顆粒（CpG-NP）。該奈米顆粒能持續釋放CpG達96小時以上。在雞骨髓來源樹突狀細胞（BMDCs）細胞試驗中，CpG-NP能有效地被DC吞噬，誘導DC成熟，促進樹突的形成。在針對84個免疫相關基因的檢測中發現，CpG-NP比起游離CpG可增強更多的基因表達。這些上調的基因表明免疫傾向於Th1反應，並具有促進黏膜免疫反應的作用。CpG-NP也在DF-1細胞上表現出具有抗H6N1禽流感病毒感染的保護作用。因此，CpG-NP對雞具有高效、持久的免疫刺激作用。CpG-NP隨後與IBV次單位抗原（受體結合位，RBD）混合進行皮下注射免疫，而另外IBV抗原或以游離的方式存在，或與奈米顆粒表面進行連結。與游離的CpG佐劑相比，以CpG-NP為佐劑的這兩種疫苗劑型均能顯著提高雞的體液免疫反應，而在活毒攻毒模式中，以CpG-NP為佐劑的次單位抗原疫苗產生了良好的保護效力。 為了避免還原劑對抗原構型的損害，同時又實現抗原和佐劑在奈米疫苗平台上的共遞送，奈米顆粒同時包裹CpG和IBV重組次單位抗原（rRBD-CpG-NP）的策略替代了表面連結抗原的方式，以此對雞進行眼鼻內免疫的效果評估。在試驗中，RBD-CpG-NP組淚液中抗原特異性IgA和IgG是CpG和RBD均為游離態組別的3倍和6倍。通過對免疫相關基因的檢測和抗體分泌細胞的染色，證實哈氏腺的B細胞分化和增殖是抗體提升的原因。IBV攻毒後，RBD-CpG-NP免疫組的雞臨床症狀較輕。在呼吸系統，RBD-CpG-NP疫苗能保護氣管纖毛活性，減少病毒在肺組織中的複製。奈米顆粒疫苗免疫還可減少全身性感染，從而減少腎損傷和泄殖腔的排毒。因此，這個以雞黏膜為標靶的抗原與佐劑共遞送奈米疫苗對雞具有良好的免疫刺激作用，對IBV感染產生有效保護。 以趨化因子修飾奈米顆粒表面可進一步增強奈米疫苗對黏膜和淋巴組織的靶向性。我們利用聚多巴胺進行蛋白質在奈米顆粒的表面修飾，將C-C趨化因子CCL19與CpG-NP連結（CCL19-NP），且能保留CCL19的正確構型，以BMDC細胞試驗測試CCL19-NP的趨化性。結果發現相較於游離的CCL19，CCL19-NP可顯著提升DC的遷移能力，增強DC的攝取能力，進而刺激更多的促發炎和抗病毒細胞因子的表現。 綜合以上，為了因應不斷出現的IBV變異株，本研究分析了IBV變異株的基因組及其與宿主免疫系統的相互作用，發展和評估了一種具有較強抗病毒和免疫刺激能力的奈米顆粒疫苗，能夠標靶性的針對IBV的感染部位刺激黏膜免疫，此疫苗的內容物和表面修飾可根據不同需要而彈性改變，在抗病毒研究和疫苗研製和抗病毒中具有廣泛的應用前景。

Avian coronavirus infectious bronchitis virus (IBV) poses economic threat to the poultry industry worldwide. This study starts with the investigation on a Taiwan IBV variant (Unit 3), and followed by the development and assessment of antiviral nanoparticle vaccines that aim to elicit effective immunity in chickens (Unit 4, 5 and 6). A pathogenic Taiwan type IBV 3575/08 was isolated from broilers vaccinated with the Taiwan type live attenuated vaccine. Despite the high degree of homology between 3575/08 strain and the Taiwan type vaccine strain in their structural genes, 3575/08 was observed to exhibited distinct antigenicity, and showed strong respiratory and renal pathogenicity. The 3575/08 strain was also found to delay the expression of a subset of innate and adaptive immune genes that result in early and prolonged viral shedding. This new IBV strain harbors a known local genotype but displays remarkably altered antigenicity, pathogenicity and host defenses revealed continuous mutation of IBV in the field. To control the IBV variants infection in chicken, novel vaccine is urgent in need. However, due to limited choices of adjuvants of poultry vaccine, novel vaccine formulations are unable to adapt to various inoculation routes and usually ineffective. Thus, a safe and effective novel adjuvant by incorporating avian toll-like receptor 21 (TLR21) agonist CpG ODN 2007 with a PLGA-based hollow nanoparticle platform (CpG-NP) was engineered. The CpG-NPs capable of sustained release of CpG for up to 96 hours. With the ex vivo model of chicken bone marrow derived dendritic cells (BMDCs), CpG-NP was found to effectively engulfed by dendritic cell (DC) and therefore induce DC maturation, promote dendrite formation. When testing the mRNA expression of 84 immune related genes, CpG-NP was found to enhance more genes compare to free CpG. These upregulated genes suggest immune skewing toward T helper cell 1 bias and evidence of improved mucosal immunity upon vaccination with the CpG-NP. The CpG-NP also showed protective effects to DF-1 cells against avian influenza virus H6N1 infection. Thus, the novel hollow CpG-NP formulation was demonstrated to exhibit effective and long-lasting immunostimulatory ability for chickens. The CpG-NP was subsequently coupled with soluble or surface conjugated IBV subunit antigen (receptor-binding domain, RBD) for subcutaneous administration. Chickens were immunostimulated to acquire significantly higher humoral immune response in both of these two vaccine formulations as compared to antigen alone or free form CpG adjuvanted group. Furthermore, the CpG-NP adjuvanted antigen group elicited protective immunity against viral challenge. To achieve the co-delivery of antigen and adjuvant using nanoparticles, recombinant IBV subunit antigen and CpG were con-encapsulated (rRBD-CpG-NP) and evaluated in chickens using the mucosal administration route. With this proprietary formulation, the antigen-specific IgA and IgG in tears showed 3-fold and 6-fold increase in rRBD-CpG-NP group, respectively, as compared to rRBD + free CpG group. Through the PCR array on immune related genes and the antibody-secreting cell staining, greater Harderian gland B cell differentiation and proliferation were confirmed to be responsible for the superior antibody production. After IBV challenge, the rRBD-CpG-NP immunized chickens showed milder clinical signs. In the respiratory system, rRBD-CpG-NP vaccine protected the tracheal ciliary activity and reduced virus replication in the lung. Immunization of nanoparticle vaccine in chickens also led to alleviated systemic symptoms as observed improved renal lesions and virus shedding in the cloaca. This chicken mucosal-targeting nanoparticle vaccine, co-delivering the adjuvant and antigen, shows promising immunostimulatory effect to against IBV infection. Mucosal and lymphoid tissue targeting can be further enhanced by nanoparticle surface modification with chemokines. Novel technology involved in this surface functionalization using polydopamine retains the native protein structure on the nanoparticle surface. Here, the C-C chemokine ligand, CCL19, was conjugated to the CpG-NP (CCL19-NP) to test the chemotaxis to BMDC ex vivo. As compared to the free CCL19, the CCL19-NP was found to promote DC migration and enhance DC uptake, thus stimulated more proinflammation and antiviral cytokines expression. Overall, to deal with the emerging variants of IBV, this study analyzed the variant strain’s genome and the interaction with host immune system, then an infection site, mucosa, targeting nanoparticle vaccine eliciting potent antiviral and immunostimulatory ability was developed and assessed. This vaccine’s contents and surface can be tailored as demands, demonstrating broad application in antiviral and vaccine development.