探討豬隻凝集素mKCR, Nkp44, Siglec-9, TLT2於豬流行性下痢病毒感染中之角色暨研發脂質奈米微粒包覆之豬流行性下痢病毒棘蛋白S1結構域信使核糖核酸疫苗

Abstract

豬流行性下痢病毒（Porcine epidemic diarrhea virus, PEDV）可引發新生仔豬嚴重的急性水樣腹瀉與嘔吐，並導致極高的致死率，對全球養豬產業造成巨大的經濟損失。然而，關於該病毒的主要功能性受體，至今在學術界仍存在諸多爭議。由於PEDV棘蛋白（Spike protein）表面覆蓋有極為複雜且高度醣基化的結構，宿主細胞表面的「醣基-凝集素交互作用」被普遍認為在病毒初期的附著與入侵過程中扮演關鍵角色。為了系統性地探討這些宿主因子，本研究透過親和力酵素免疫分析法篩選可能與PEDV重組棘蛋白進行交互作用的醣結合凝集素（lectins）。本研究優先篩選出四種關鍵的候選凝集素（mKCR、Nkp44、Siglec-9 與 TLT2）。經由生物資訊學分析進一步確認了這些候選分子在醣類辨識結構域（CRD）內具有高度的序列保守性，並成功鑑定出其在豬隻基因組中的特異性同源基因。為了探討這些豬源凝集素是否具備與病毒棘蛋白交互作用，本研究建構豬源凝集素mKCR、Nkp44、Siglec-9 與 TLT2表現載體，並將其導入具病毒感受性細胞與非感受性的細胞模型中。細胞的蛋白質表現分析證實，各候選凝集素均能成功於細胞中表達。然而，在隨後的病毒攻毒試驗中，分子檢測與病毒抗原染色結果均顯示，無論細胞是否表達這些豬源凝集素，非感受性細胞模型中皆完全無法偵測到任何病毒複製或病毒蛋白質合成的訊號。同樣地，在外源性表達這些凝集素的天然感受性細胞模型中，其內部病毒載量亦未較對照組提升。上述發現皆表明，雖然mKCR、Nkp44、Siglec-9與TLT2能與病毒棘蛋白上的醣基產生相互作用，在細胞表面提供物理性的螯合位點，但它們在感染鏈中僅扮演非感受性附著因子（Non-permissive attachment factors）的角色。它們可能透過多價結合的親和力將病毒顆粒錨定於細胞膜表面，充當病毒初期綁定階段角色，但本身並不具備獨立啟動膜融合或媒介具複製能力之病毒進入細胞的功能。此外，在探討病毒附著機制的同時，本研究第二部分亦針對 PEDV 高突變率與變異株流行之特性，研發出一款新型的信使核糖核酸（mRNA）疫苗預防策略。為克服過往實驗室使用全長棘蛋白作為抗原時所面臨的結構複雜性、細胞代謝負擔以及體內免疫保護力不彰等限制，本研究將抗原標的聚焦於包含主要中和性抗原決定之受體結合結構域的S1次單位。本研究成功製備出經核苷酸修飾的S1 mRNA，並利用微流體混合系統將其高效封裝於脂質奈米顆粒（LNPs）中。經由細胞試驗證實，脂質奈米顆粒的包覆能顯著提升細胞內運送效率，並於驅使細胞表達目標。更為關鍵的是，長期穩定性監測證明，此款 LNP-mRNA 疫苗配方在常規冷藏條件下能長期維持其結構完整性與轉譯能力，確保了抗原表達效能的持續與穩定。總結來說，本研究不僅首次闡明四種新型豬隻凝集素於PEDV感染細胞功能性角色，亦成功產製脂質奈米微粒包覆之流行性下痢病毒棘蛋白S1結構域信使核糖核酸疫苗平台，具高效、穩定且具備物流可行性的候選疫苗之潛力，為全球養豬業控制冠狀病毒疫情提供了創新的應對方案 。

Porcine epidemic diarrhea virus (PEDV) causes severe acute watery diarrhea, acute vomiting, and high mortality in neonatal piglets, resulting in catastrophic economic losses in the global swine industry. However, controversy persists regarding its primary functional entry receptors. Given that the PEDV spike (S) protein possesses a highly glycosylated surface landscape, host glycan-lectin interactions are widely hypothesized to play a crucial role in initial viral attachment and entry into host cells. To systematically identify these host factors, this study utilized a high-throughput affinity ELISA screening to evaluate potential interactions between carbohydrate-binding lectins and the recombinant PEDV spike protein. Based on this screening, four key candidate porcine lectins, mKCR, Nkp44, Siglec-9, and TLT2, were prioritized for further evaluation. Bioinformatic analysis confirmed high sequence conservation within their carbohydrate-recognition domains (CRDs) and successfully identified their specific homologous genes within the porcine genome. To investigate the potential function of these porcine lectins in facilitating viral entry, corresponding porcine expression vectors were constructed and introduced into both naturally permissive and strictly non-permissive cellular models. Cellular and protein expression analyses verified that each candidate lectin was successfully expressed within the cytoplasm. However, in subsequent viral challenge assays, molecular detection and viral antigen staining consistently revealed a complete absence of viral replication or viral protein synthesis in the non-permissive cellular model, regardless of candidate lectin expression. Similarly, the expression of these lectins in naturally permissive models failed to enhance or alter baseline intracellular viral loads compared to the control group. These findings collectively demonstrate that while mKCR, Nkp44, Siglec-9, and TLT2 interact with the viral glycan shield to provide physical sequestration sites on the host plasma membrane, they function strictly as non-permissive attachment factors. By anchoring virions to the cell surface through multivalent avidity, they might act as initial tethering phase of the infection cycle but are insufficient to independently trigger membrane fusion or mediate productive viral entry. Complementing these mechanistic insights into viral attachment, this study developed a messenger RNA (mRNA) vaccine strategy hoping to conquer rapid viral mutations and evolving field strains. To overcome the inherent structural complexity, cellular metabolic burden, and suboptimal protective efficacy associated with full-length spike protein formulations previously evaluated in our laboratory, this platform strategically pivoted to the S1 subunit, which concentrates the primary neutralizing epitopes and the receptor-binding domain. Nucleoside-modified S1 mRNA was generated via in vitro transcription and encapsulated into lipid nanoparticles (LNPs) using a specialized microfluidic mixing system. Cellular delivery assays demonstrated that LNP encapsulation significantly enhanced intracellular delivery, driving robust target protein expression within the cytoplasm. Crucially, long-term stability monitoring proved that the formulated LNP-mRNA vaccine successfully retained its structural integrity and translational capability under standard refrigeration conditions, ensuring consistent and sustained antigen expression efficiency over time. In conclusion, this study not only provides the first functional characterization of these four novel porcine lectins during PEDV infection, but also successfully develops a lipid nanoparticle (LNP)-encapsulated PEDV S1-based mRNA vaccine platform. This platform demonstrates significant potential as a highly efficient, stable, and logistically viable vaccine candidate, offering an innovative strategy for the global swine industry to control coronaviral outbreaks.