E1(+)/mCherry Human Adenovirus (Serotype 5)

The complement system is essential for antimicrobial defense. In the classical pathway, pathogen-bound antibodies recruit the C1 complex (C1qC1r2C1s2), which initiates a lytic cascade involving C2, C3, C4, and C5 and triggers microbial clearance. Here, researchers demonstrate a C4-dependent antiviral mechanism that is independent of downstream complement components. C4 inhibits human adenovirus infection by directly inactivating viral capsids. Through the classical pathway, antibodies catalyze rapid C4 activation and capsid deposition by lytic C4b. Capsid-deposited C4b neutralizes infection independently of C2 and C3 but requires C1q antibody engagement. C4b inhibits capsid disassembly, preventing endosomal escape and cytoplasmic entry. Mice lacking C4 exhibit higher viral burdens. Furthermore, complement synergizes with the Fc receptor TRIM21 to block transduction of adenoviral gene therapy vectors, but Fab viral shielding can be partially restored. These results suggest that the complement system can be altered to prevent viral infection and enhance viral gene therapy efficacy.

To test complement activation directly, the researchers incubated virus and antibodies with NHS and monitored the generation of C4b over time (Figure 1A). They observed that C4 was cleaved after only 1 minute of incubation, and after 15 minutes, most of the C4 in the serum was converted to C4b. This conversion was largely dependent on an intact complement cascade, as cleavage of C4 in C1q-depleted serum was not observed (Figure 1A). Next, the researchers took advantage of the fact that Ad5 can be engineered to express different transgenes (Figure 1B). mCherry Human Adenovirus (Serotype 5) (Ad5-mCherry) was incubated with antibodies and NHS for 30 minutes to allow complement activation and C4a generation as well as C4b deposition, while Ad5-GFP was maintained under control conditions. Ad5-mCherry was added to the cells, followed by Ad5-GFP. If neutralization was caused by effects on target cells, equal neutralization would be expected from Ad5-mCherry and Ad5-GFP. However, if neutralization was caused by direct modification of the viral capsid via C4b deposition, only neutralization of Ad5-mCherry would be expected. The researchers observed the latter result; Ad5-mCherry was strongly neutralized in a C1q-dependent manner, whereas Ad5-GFP infection was unaffected (Figure 1B), indicating that neutralization requires direct modification of the viral capsid.

Figure 1. Activation of the Complement Cascade in Presence of Ad5 and 9C12 Results in Deposition of C4b on the Ad5 Capsid. (Bottermann M, et al., 2019)