Defining a Novel Role for the Coxsackievirus and Adenovirus Receptor in Human Adenovirus Serotype 5 Transduction In Vitro in the Presence of Mouse Serum.
Defining a Novel Role for the Coxsackievirus and Adenovirus Receptor in Human Adenovirus Serotype 5 Transduction In Vitro in the Presence of Mouse Serum.
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DOI：
10.1128/jvi.02487-16
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发表时间：
2017-06-15
影响因子：
5.4
通讯作者：
Baker AH
中科院分区：
文献类型：
--
作者：
Lopez-Gordo E;Doszpoly A;Duffy MR;Coughlan L;Bradshaw AC;White KM;Denby L;Nicklin SA;Baker AH
人腺病毒血清型5（HAdV-5）载体在血管内递送时主要具有肝嗜性，导致免疫激活和毒性。凝血因子X（FX）与HAdV-5结合介导肝脏转导，并在小鼠中提供病毒体中和保护。FX在缺乏IgM抗体或补体的小鼠中不适于肝脏转导，表明存在替代转导途径。为了鉴定介导HAdV-5 FX非依赖性进入的新因子，我们在来自免疫活性C57 BL/6或缺乏IgM抗体的免疫受损小鼠（Rag 2−/−和NOD-scid-gamma [NSG]）的血清存在下研究了HAdV-5的体外转导。来自所有三种小鼠品系的血清增强了A549细胞的HAdV-5转导。虽然HAdV-5-FX与FX结合蛋白（X-bp）相互作用的抑制在C57 BL/6血清存在下抑制了转导，但它对Rag 2−/−或NSG血清存在下观察到的增强转导的影响可以忽略不计。Rag 2−/−血清也增强了FX结合缺陷型HAdV-5 HVR 5 * HVR 7 * E451 Q（AdT*）的转导。有趣的是，Rag 2−/−血清在CHO-CAR和SKOV 3-CAR细胞（转染以稳定表达人柯萨奇病毒和腺病毒受体[CAR]的CHO或SKOV 3细胞）中以FX非依赖性方式增强HAdV-5转导。此外，用可溶性HAdV-5纤维球阻断CAR抑制了A549细胞中小鼠血清增强的转导，表明CAR的潜在作用。在Rag 2−/−血清存在下，HAdV-5 KO 1和HAdV-5/F35（CAR结合缺陷）的转导与HAdV-5的转导相当，表明HAdV-5和CAR之间不需要直接相互作用。这些数据表明，FX可以保护HAdV-5免于中和，但在免疫受损小鼠血清存在下对HAdV-5转导的贡献最小。或者，转导通过能够将HAdV-5桥接至CAR的未鉴定的小鼠血清蛋白发生。重要性血管内注射HAdV-5载体可导致急性肝毒性、转氨酶升高、血小板减少和血管内皮损伤，这说明HAdV-5介导的系统性基因治疗尚需克服的挑战。CAR和小鼠血清中可能存在的未鉴定因子可能是HAdV-5转导的重要介体，这一发现强调了仍然需要更好地理解定义腺病毒免疫识别和细胞摄取机制之间相互作用的复杂生物学。这些发现对于进一步优化和开发用于基因治疗的基于HAdV-5的腺病毒载体具有重要意义。
Human adenoviral serotype 5 (HAdV-5) vectors have predominantly hepatic tropism when delivered intravascularly, resulting in immune activation and toxicity. Coagulation factor X (FX) binding to HAdV-5 mediates liver transduction and provides protection from virion neutralization in mice. FX is dispensable for liver transduction in mice lacking IgM antibodies or complement, suggesting that alternative transduction pathways exist. To identify novel factor(s) mediating HAdV-5 FX-independent entry, we investigated HAdV-5 transduction in vitro in the presence of serum from immunocompetent C57BL/6 or immunocompromised mice lacking IgM antibodies (Rag 2−/− and NOD-scid-gamma [NSG]). Sera from all three mouse strains enhanced HAdV-5 transduction of A549 cells. While inhibition of HAdV-5–FX interaction with FX-binding protein (X-bp) inhibited transduction in the presence of C57BL/6 serum, it had negligible effect on the enhanced transduction observed in the presence of Rag 2−/− or NSG serum. Rag 2−/− serum also enhanced transduction of the FX binding-deficient HAdV-5HVR5*HVR7*E451Q (AdT*). Interestingly, Rag 2−/− serum enhanced HAdV-5 transduction in a FX-independent manner in CHO-CAR and SKOV3-CAR cells (CHO or SKOV3 cells transfected to stably express human coxsackievirus and adenovirus receptor [CAR]). Additionally, blockade of CAR with soluble HAdV-5 fiber knob inhibited mouse serum-enhanced transduction in A549 cells, suggesting a potential role for CAR. Transduction of HAdV-5 KO1 and HAdV-5/F35 (CAR binding deficient) in the presence of Rag 2−/− serum was equivalent to that of HAdV-5, indicating that direct interaction between HAdV-5 and CAR is not required. These data suggest that FX may protect HAdV-5 from neutralization but has minimal contribution to HAdV-5 transduction in the presence of immunocompromised mouse serum. Alternatively, transduction occurs via an unidentified mouse serum protein capable of bridging HAdV-5 to CAR. IMPORTANCE The intravascular administration of HAdV-5 vectors can result in acute liver toxicity, transaminitis, thrombocytopenia, and injury to the vascular endothelium, illustrating challenges yet to overcome for HAdV-5-mediated systemic gene therapy. The finding that CAR and potentially an unidentified factor present in mouse serum might be important mediators of HAdV-5 transduction highlights that a better understanding of the complex biology defining the interplay between adenovirus immune recognition and cellular uptake mechanisms is still required. These findings are important to inform future optimization and development of HAdV-5-based adenoviral vectors for gene therapy.