Manipulating Adenovirus Hexon Hypervariable Loops Dictates Immune Neutralisation and Coagulation Factor X-dependent Cell Interaction In Vitro and In Vivo

Abstract

Adenoviruses are common pathogens, mostly targeting ocular, gastrointestinal and respiratory cells, but in some cases infection disseminates, presenting in severe clinical outcomes. Upon dissemination and contact with blood, coagulation factor X (FX) interacts directly with the adenovirus type 5 (Ad5) hexon. FX can act as a bridge to bind heparan sulphate proteoglycans, leading to substantial Ad5 hepatocyte uptake. FX “coating” also protects the virus from host IgM and complement-mediated neutralisation. However, the contribution of FX in determining Ad liver transduction whilst simultaneously shielding the virus from immune attack remains unclear. In this study, we demonstrate that the FX protection mechanism is not conserved amongst Ad types, and identify the hexon hypervariable regions (HVR) of Ad5 as the capsid proteins targeted by this host defense pathway. Using genetic and pharmacological approaches, we manipulate Ad5 HVR interactions to interrogate the interplay between viral cell transduction and immune neutralisation. We show that FX and inhibitory serum components can co-compete and virus neutralisation is influenced by both the location and extent of modifications to the Ad5 HVRs. We engineered Ad5-derived HVRs into the rare, native non FX-binding Ad26 to create Ad26.HVR5C. This enabled the virus to interact with FX at high affinity, as quantified by surface plasmon resonance, FX-mediated cell binding and transduction assays. Concomitantly, Ad26.HVR5C was also sensitised to immune attack in the absence of FX, a direct consequence of the engineered HVRs from Ad5. In both immune competent and deficient animals, Ad26.HVR5C hepatic gene transfer was mediated by FX following intravenous delivery. This study gives mechanistic insight into the pivotal role of the Ad5 HVRs in conferring sensitivity to virus neutralisation by IgM and classical complement-mediated attack. Furthermore, through this gain-of-function approach we demonstrate the dual functionality of FX in protecting Ad26.HVR5C against innate immune factors whilst determining liver targeting.

Evaluation of D-dimer and lactate dehydrogenase plasma levels in patients with relapsed acute leukemia

“…cells (35). Coagulation disorders may also occur due to leukemia-associated complications, including infection or organic impairment (45).…”

Retargeting FX-binding-ablated HAdV-5 to vascular cells by inclusion of the RGD-4C peptide in hexon hypervariable region 7 and the HI loop

“…Through selective modification of the HVRs, we previously generated a vector (HAdV-5T*) devoid of FX interactions and consequently hepatocyte transduction by introducing point mutations in key FX-interacting amino acids of the hexon protein HVR, namely T270P and E271G (HVR5) and I421G, T423N, E454S, L426Y and E451Q (all in HVR7) (Alba et al , 2009). Conversely, it has been reported that FX may actually offer a protective role in gene delivery, by shielding HAdV-5 from immune-mediated attack by natural IgM and the classical complement system (Xu et al , 2013), which interact with HVRs, neutralizing the virus (Ma et al , 2015). The specific amino acids responsible for immune recognition remain unknown, and the impact in humans remains unconfirmed.…”

“…The specific amino acids responsible for immune recognition remain unknown, and the impact in humans remains unconfirmed. Incorporation of FX-binding HVRs from HAdV-5 into HAdV-26 (non-FX-binding Ad serotype) instilled liver transduction to this vector (Ma et al , 2015), reiterating the importance of FX in determining viral hepatic transduction. Therefore, retargeting of FX-ablated virus remains an area of interest for improving safety and efficacy of gene therapy vectors.…”

“…FX binding HAdV-5 has been shown to prevent IgM- and complement-mediated neutralization of the virus in vivo (Xu et al , 2013), with inhibitory serum components' binding sites within the HAdV-5 HVRs (1–3 and 5–7) (Ma et al , 20 15). We tested if this response was altered by the insertion of the RGD-4C peptide within these regions by investigating the sensitivity of the vectors to murine serum in vitro using a snake venom-derived FX-binding protein (X-bp), which binds to the Gla domain of FX, preventing its interaction with the Ad hexon (Waddington et al , 2008; Atoda et al , 1998).…”

Recent Advancements in Cardiovascular Gene Therapy and Vascular Biology

“…Recently, Ad serotype 49 was shown to induce higher vascular gene transfer efficiency than Ad5 after ex vivo delivery to coronary artery bypass graft tissues 25 Rabbits have also been used for restenosis, in-stent restenosis and vein graft stenosis studies 36 . Also, they have been useful for myocardial and peripheral skeletal muscle ischemia studies 37,38 .…”