Data Sheet 1_Human saliva exerts strong type-dependent effects on adenovirus infectivity.docx

BackgroundThe development of mucosal adenovirus (Ad) vaccine vectors is considered one of the next frontiers to protect vulnerable patients from respiratory and gastrointestinal pathogens. An efficient delivery to or through the oral cavity necessitates a thorough understanding of Ad interactions with saliva for oral, buccal or sublingual vaccine delivery, which could additionally prove instrumental in the containment of natural Ad infections but remains unexplored. Therefore, we investigated the influence of saliva on Ad infectivity, emphasizing its intrinsic antiviral role against particular Ad types in various epithelial cell cultures.
MethodsA saliva pool was created from healthy donors (n=16) and incubated with ChAdOx1 or human Ads from 20 different types prior to infection of human immortalized epithelial cells. All human Ads used were replication-competent and expressed a GLN cassette containing a green-fluorescent protein, nano-luciferase, and neomycin resistance. Loss-of-function experiments were conducted by immunoprecipitation or enzymatic digestion of specific saliva components to decipher related mechanisms.
ResultsTemporal and inter-individual variability in saliva samples were observed, validating the use of a saliva pool to represent the population. Saliva strongly influenced Ad infectivity, in general through inhibiting species B types and enhancing species D and E Ads, that include the vaccine vector platforms Ad26 and ChAdOx1. Interestingly, Ad20 presented the highest infectivity enhancement, as well as superior to average salivary mucus crossing rates. Furthermore, saliva immunoglobulins and human neutrophil peptides marginally influenced the Ad infectivity, while sialic acid inhibited all tested Ad types.
ConclusionSaliva may have a protective role against infection by certain types of Ads. This discovery highlights a potential limitation in the efficacy of next-generation oral Ad vaccine vectors. Consequently, our study underscores the importance of identifying and utilizing saliva-resistant Ad vectors to optimize Ad-based vaccination strategies.

研究背景
黏膜腺病毒（adenovirus, Ad）疫苗载体的开发被视为保护脆弱人群免受呼吸道和胃肠道病原体侵害的下一前沿方向之一。若要通过口腔实现高效的疫苗递送（涵盖口服、颊部或舌下给药途径），需充分阐明腺病毒与唾液的相互作用机制；该研究方向或可助力自然腺病毒感染的防控，但目前尚未得到探索。因此，本研究探究了唾液对腺病毒感染性的影响，重点关注其在多种上皮细胞培养体系中对特定腺病毒亚型的固有抗病毒作用。

研究方法
本研究从16名健康供者中收集唾液并制备混合唾液样本，将其与ChAdOx1或20种不同亚型的人腺病毒共孵育，随后感染人永生化上皮细胞。本研究使用的所有腺病毒均为复制能力完整的毒株，且表达带有绿色荧光蛋白、纳米荧光素酶与新霉素抗性基因的GLN表达盒。通过对特定唾液成分进行免疫沉淀或酶解消化开展功能丧失实验，以解析相关作用机制。

研究结果
本研究观察到唾液样本存在时间维度与个体间的差异，验证了使用混合唾液样本可代表群体特征。唾液可显著影响腺病毒的感染性：总体而言，唾液会抑制B亚群腺病毒的感染，同时增强D亚群与E亚群腺病毒的感染，其中包括Ad26与ChAdOx1这两类疫苗载体平台。值得注意的是，Ad20的感染增强效果最为显著，且其唾液黏液穿透率高于平均水平。此外，唾液免疫球蛋白与人中性粒细胞肽对腺病毒感染性仅存在轻微影响，而唾液酸可抑制所有受试腺病毒亚型的感染。

研究结论
唾液对某些亚型腺病毒的感染可能具有防护作用。这一发现揭示了下一代口服腺病毒疫苗载体效力可能存在的局限性。综上，本研究凸显了筛选并利用抗唾液腺病毒载体以优化基于腺病毒的疫苗接种策略的重要性。