研究以酵母聚醣作為佐劑促進腸病毒A71型與D68型之鼻黏膜疫苗免疫反應

Abstract

非小兒麻痺腸病毒中，特別是腸病毒A71型及腸病毒D68型，近年來因與全球急性無力脊髓炎的高度發生率及兒童高度死亡率相關，已成為臨床上極具重要性的病原體。鑑於黏膜表面在抵禦病毒初期入侵中扮演至關重要的角色，鼻腔疫苗因其可同時誘導局部及全身性黏膜免疫被認為是一個有前景的病原體預防策略。然而，鼻腔疫苗因其抗原往往無法突破黏膜免疫耐受性，以及產生持久且具保護力的記憶性免疫反應，使得其有效性受到很大限制。而酵母聚醣是一種酵母菌細胞壁的多醣成分，已知可與多種模式識別受體相互作用，協調先天性及適應性免疫反應。此外，其佐劑的潛力已在多個病毒疫苗模型中得到證實。在本研究中，我們旨在評估酵母聚醣作為腸病毒雙價鼻腔疫苗佐劑的應用性。 首先，我們分離並培養小鼠骨髓來源的樹突細胞，並於體外觀察有酵母聚醣存在的條件下其先天性免疫之刺激效應。透過誘導多種細胞激素的產生，顯示酵母聚醣在體外對骨髓來源的樹突細胞具有強效的活化能力。接著，我們製備兩種分別針對腸病毒A71型及腸病毒D68型的單價去活化疫苗，並加入酵母聚醣作為佐劑，經由鼻腔途徑多次給予C57BL/6小鼠，以研究其免疫調節作用。在三劑疫苗接種過後，酵母聚醣能顯著增強兩種疫苗在C57BL/6小鼠體內的免疫原性，包括誘導多個黏膜部位產生大量病毒特異性免疫球蛋白A、有效提高血清中和抗體的效價，以及促進抗原再刺激後脾臟中免疫球蛋白A產生性B細胞與白血球介素-17產生性T細胞的增生。此外，我們亦開發了含有酵母聚醣作為佐劑的腸病毒雙價疫苗，以驗證其在雙價疫苗中的效果。與單價疫苗結果一致，三次鼻腔的疫苗接種過後，以酵母聚醣作為佐劑能有效促進鼻黏膜病毒特異性免疫球蛋白A反應，同時誘發強勁的脾臟次級免疫反應。值得注意的是，酵母聚醣在雙價疫苗中的免疫增強效果在針對腸病毒D68型的特異性免疫中表現得更為顯著。 最後，我們使用兩種新生小鼠的腸病毒感染模型，評估含有酵母聚醣作為佐劑的單價及雙價腸病毒鼻黏膜疫苗的保護效果。在hSCARB2基因轉殖的新生小鼠中，在兩劑單價腸病毒A71型鼻黏膜疫苗接種過後，酵母聚醣作為佐劑於感染腸病毒A71型致死劑量後顯著提高了疫苗的保護效果，表現為提高的感染存活率、減輕的臨床症狀並減少肌肉組織的病理損傷。在ICR新生小鼠中，接受來自三次含有酵母聚醣作為佐劑之雙價腸病毒鼻黏膜疫苗接種供體的被動轉移血清後，不論是感染腸病毒A71型或是D68型致死劑量後均表現出延長的存活時間、較輕的臨床症狀及目標組織中較低的病毒載量。 總結而言，我們的研究結果支持以酵母聚醣作為腸病毒鼻腔疫苗佐劑的潛在應用，該策略為增強新生兒對腸病毒A71型及腸病毒D68型嚴重感染的防護效果提供了可行的方案，並推動鼻黏膜疫苗的蓬勃發展。

Non-polio enteroviruses, particularly EV-A71 and EV-D68, have become clinically significant in recent years due to their association with high frequencies of acute flaccid myelitis (AFM) and significant pediatric mortality worldwide. Given the critical role of mucosal surfaces in defending initial viral entry, intranasal vaccines have been proposed as a promising strategy to elicit both local and systemic mucosal immunity. However, the effectiveness of intranasal vaccines has been limited, as the antigens often fail to break mucosal tolerance and generate durable and protective memory responses. Zymosan, a yeast-derived cell wall component, is known to interact with multiple pattern recognition receptors (PRRs), coordinating both innate and adaptive immune responses. In addition, its adjuvant potential has been demonstrated in several viral vaccine models. In this study, we aimed to assess the applicability of zymosan as an adjuvant in an intranasal bivalent enterovirus vaccine. Initially, bone marrow-derived dendritic cells (BMDCs) were isolated and cultured in the presence of zymosan to evaluate its innate immunostimulatory effects. By inducing various cytokine productions, zymosan demonstrated potent activation of BMDCs in vitro. Subsequently, two inactivated monovalent vaccines targeting EV-A71 and EV-D68 were formulated with zymosan and administered intranasally to C57BL/6 mice to investigate the immunomodulatory effects of zymosan. Following a three-dose immunization regimen, zymosan significantly enhanced vaccine immunogenicity in vivo. This included robust induction of virus-specific IgA at multiple mucosal sites, elevated serum neutralizing antibody titers, and expansions of splenic IgA-producing B cells and IL-17-producing T cells upon antigen restimulation. Moreover, an EV-A71/EV-D68 bivalent vaccine with zymosan adjuvantation was developed to validate these findings in a bivalent vaccine setting. Consistent with the monovalent results, triple intranasal vaccination induced strong mucosal IgA responses, particularly at the nasal mucosa, as well as potent splenic secondary responses. Notably, the immunoenhancing effects of zymosan were more prominent in the context of EV-D68-specific immunity. Finally, two neonatal mouse models were employed to evaluate the protective efficacy of the zymosan-adjuvanted vaccines. In hSCARB2 transgenic neonatal mice, double vaccination with EV-A71 and zymosan significantly improved survival rates, mitigated clinical manifestations, and reduced histopathological damage following lethal EV-A71 challenge. In neonatal ICR mice, passive transfer of sera from zymosan-adjuvanted triple-vaccinated donors conferred significant protection to recipient mice, as evidenced by prolonged survival, reduced clinical severity, and decreased viral loads in target tissues. In summary, our findings support the application of zymosan as a potent nasal adjuvant in enterovirus vaccine formulations. This approach offers a viable strategy for enhancing early-life protection against severe EV-A71 and EV-D68 infections and contributes to the advancement of nasal spray-based vaccination platforms.