Defining a Novel Role for the Coxsackievirus and Adenovirus Receptor in Human Adenovirus Serotype 5 Transduction In Vitro in the Presence of Mouse Serum

Abstract

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.

Human Adenovirus Serotype 5 Is Sensitive to IgM-Independent Neutralization In Vitro and In Vivo

“…Interestingly, murine sera from different immunoglobulin-deficient mice strains ( Rag2 -/- , Ighm tm1Che ), behaved in a similar manner, suggesting that at least some of the complement molecules directly attach to HAdV-5 capsids without requiring the presence of immunoglobulins (Figure 3). Furthermore, HAdV-5 was neutralized by immunocompetent C57BL/6 serum in the absence of FX, as previously demonstrated [11,21]. However, regardless of the presence of FX, some of the molecules involved in the immune response were able to bind to the virions.…”

“…It has been previously reported that HAdV-5 specifically binds to murine and human FX, and that the capsid:FX interaction inhibits the neutralization mediated by IgM and complement components [11,21]. A previous study based on particle size tracking demonstrated that purified human IgM and IgG were able to bind to HAdV-5 virions, with IgM only binding in a FX-dependent manner [14].…”

Adenoviral vector with shield and adapter increases tumor specificity and escapes liver and immune control

“…The shield might also block a soluble blood factor that binds to the virion and retargets the particle to CAR-expressing tissue. Recently, the presence of such a factor has been described, but not identified, in murine Rag2 -/- serum 56 . In conclusion, the combination of retargeting and shielding improved the tumor-to-liver ratio upon intratumoral injection from 300 to 7200 for the EGFR + and from 1300 to 1,100,000 for the HER2 + xenograft.…”

Identification of chicken CAR homology as a cellular receptor for the emerging highly pathogenic fowl adenovirus 4 via unique binding mechanism

“…Most AdVs have one unique fibre that attaches to variable cellular receptors. HAdV-2 and HAdV-5 in subgroup C use the high-affinity human coxsackie and adenovirus receptor (CAR) as the cellular receptor to infect host cells [13,14], HAdV-37 uses integrins to infect human corneal cells [15], mouse adenovirus type 1 utilizes integrin and heparin sulfate as cellular receptors [16], bovine adenovirus serotype 3 utilizes sialic acid as a receptor for viral entry [17], and porcine adenovirus serotype 3 internalization is independent of CAR and integrin, but uses some other receptors [18]. However, HAdV-52, HAdV-40 [19], and HAdV-41 [20] are equipped with two different fibre proteins, only the long fibre type is inserted into each penton and binds the CAR, whereas the short fibre binds to sialylated glycoproteins during infection [21,22].…”