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

Andor Doszpoly,Andrew H Baker,Fernando De La Cuesta,Stuart A Nicklin,Cécile Bénézech,Estrella Lopez-Gordo

doi:10.3390/v11070616

Abstract

Human adenovirus 5 (HAdV-5) is used as a vector in gene therapy clinical trials, hence its interactions with the host immune system have been widely studied. Previous studies have demonstrated that HAdV-5 binds specifically to murine coagulation factor X (mFX), inhibiting IgM and complement-mediated neutralization. Here, we examined the physical binding of immune components to HAdV-5 by nanoparticle tracking analysis, neutralization assays, mass spectrometry analysis and in vivo experiments. We observed that purified mouse Immunoglobulin M (IgM) antibodies bound to HAdV-5 only in the presence of complement components. Active serum components were demonstrated to bind to HAdV-5 in the presence or absence of mFX, indicating that immune molecules and mFX might bind to different sites. Since binding of mFX to HAdV-5 blocks the neutralization cascade, these findings suggested that not all complement-binding sites may be involved in virion neutralization. Furthermore, the data obtained from serum neutralization experiments suggested that immune molecules other than IgM and IgG may trigger activation of the complement cascade in vitro. In vivo experiments were conducted in immunocompetent C57BL/6 or immuno-deficient Rag2-/- mice. HAdV-5T* (a mutant HAdV-5 unable to bind to human or mFX) was neutralized to some extent in both mouse models, suggesting that murine immunoglobulins were not required for neutralization of HAdV-5 in vivo. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis of HAdV-5 and HAdV-5T* after exposure to murine sera showed stable binding of C3 and C4b in the absence of mFX. In summary, these results suggest that HAdV-5 neutralization can be mediated by both the classical and alternative pathways and that, in the absence of immunoglobulins, the complement cascade can be activated by direct binding of C3 to the virion.

Highlights

Human adenoviruses have been used as gene therapy vectors for the past four decades
The high hepatic tropism of Human adenovirus 5 (HAdV-5) is putatively mediated by the binding of the capsid hexon hypervariable regions (HVRs) of HAdV-5 hexon with blood coagulation factor X (FX) [8], which in turn interacts with heparan sulphate proteoglycans (HSPGs) present on the surface of hepatocytes [9,10], and results in virion accumulation in the liver
We recently reported that binding of human FX to the HAdV-5 capsid prevents binding of human Immunoglobulin M (IgM) but not binding of human IgGs [14]

Summary

Introduction

Human adenoviruses have been used as gene therapy vectors for the past four decades. Adenoviral vectors have large DNA packaging capacity (7.5–36 kbp), can transduce both quiescent and dividing cells, and present a minimal risk of integration of vector DNA into the host [1]. Use is hampered by several factors such as the high level of pre-existing neutralizing antibodies against HAdV-5 virions in the clinical population [2,3] and hepatic tropism following intravenous administration, which can lead to acute liver toxicity in humans, non-human primates and rodent models [4,5,6,7]. The high hepatic tropism of HAdV-5 is putatively mediated by the binding of the capsid hexon hypervariable regions (HVRs) of HAdV-5 hexon with blood coagulation factor X (FX) [8], which in turn interacts with heparan sulphate proteoglycans (HSPGs) present on the surface of hepatocytes [9,10], and results in virion accumulation in the liver. When FX is bound to the virions it serves as a shield to protect the virus against immune neutralization both in vitro and in vivo

Methods

Results

Conclusion