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

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.

Innate immunity to adenovirus: lessons from mice

“…Recently, Tian et al demonstrated that the most abundant Gla domain-containing plasma protein, prothrombin (FII), is able to bind to species C HAdv-C1, HAdv-C2, HAdv-C6, and HAdv-C57 with higher affinity than FX [19], thus potentially pointing to a high significance of HAdv interactions with blood coagulation factors for certain step(s) in the virus life cycle. Because natural IgM are one of the most abundant plasma proteins, the binding of IgM and coagulation FX to HAdv-C5 appears to be a competing process, since low amounts of IgM were still found to associate with HAdv-C5 virions (as demonstrated by mass spectrometry of HAdv-C5 virions incubated with mouse serum) even in the presence of coagulation FX [21]. Because natural IgM are one of the most abundant plasma proteins, the binding of IgM and coagulation FX to HAdv-C5 appears to be a competing process, since low amounts of IgM were still found to associate with HAdv-C5 virions (as demonstrated by mass spectrometry of HAdv-C5 virions incubated with mouse serum) even in the presence of coagulation FX [21].…”

“…Indeed, HAdv-C5 mutants unable to bind coagulation FX were shown to be inactivated in fresh mouse and human serum via a natural IgM-and complement-dependent mechanism (reviewed in detail in this issue by Allen & Byrnes [20]). Because natural IgM are one of the most abundant plasma proteins, the binding of IgM and coagulation FX to HAdv-C5 appears to be a competing process, since low amounts of IgM were still found to associate with HAdv-C5 virions (as demonstrated by mass spectrometry of HAdv-C5 virions incubated with mouse serum) even in the presence of coagulation FX [21]. Although coagulation FX and natural IgM are the principal blood factors that bind to HAdv in the blood after intravenous virus administration to nonimmune hosts, these factors do not protect the virus from neutralization by HAdv-specific neutralizing antibodies in HAdv-C5 immune hosts.…”

“…Although coagulation FX does certainly protect HAdv-C5 from inactivation by the complement [26], a role of coagulation FX in mediating Ad entry in hepatocytes cannot be completely ruled out. Specifically, the efficacy of liver-directed gene transfer after intravenous injection is consistently higher for FX-binding WT HAdv-C5based vectors, compared to virus mutants unable to bind FX, when it was tested in Rag2 À/À [21], Rag1 À/À , J H D À/À , C1qa À/À , and C4b À/À mice [26]. These data indicate that when complement-mediated virus inactivation mechanisms are dysfunctional, the HAdv-C5 that is able to bind FX transduces hepatocytes in vivo with higher efficacy than viruses ablated for FX binding.…”

The imitation game: a viral strategy to subvert the complement system

“…In addition to the above viruses, the sensitivity of human adenovirus 5 to AP-mediated neutralization was also examined recently. The results showed that the pathway is indeed important for the neutralization of the virus [45]. It is known that AP synergizes with CP and LP in targeting pathogens.…”

Interaction of adenovirus with antibodies, complement, and coagulation factors

“…AdV‐5 is normally stable when incubated in mouse serum but, when FX is blocked, AdV‐5 strongly activates complement and becomes neutralized via a mechanism that requires both natural IgM and the classical complement pathway . In some circumstances, AdV‐5 may also activate complement independently of antibodies, via the alternative pathway . Following complement activation, C3b and C4b become covalently attached to AdV‐5, and this opsonization by complement can both interfere with virus binding to cells and induce intracellular neutralization .…”