Pre-existing immunity against Ad vectors: Humoral, cellular, and innate response, what's important?

Fausther-Bovendo, Hugues; Kobinger, Gary P

The importance of nAbs in reducing Ad vector immunogenicity has been extensively demonstrated. Passive transfer of serum from Ad immune mice or purified nAbs against Ad decreases Ad transgene expression, as well as transgene specific cellular and humoral response in rodents. 15, 16 Depletion of Ab against the three main Ad capsid proteins (fiber, penton, hexon) by affinity chromatography in Ad immune serum, greatly reduces the inhibitory effect of these sera on vaccine vector efficacy, as demonstrated by an almost complete restoration of transgene expression level. 15 Although nAbs targeting the main 3 capsid proteins have been detected in vitro, 15,17-19 they do not equally contribute to Ad vector neutralization in vivo. The relative contribution of each subset of nAbs has been teased apart by vaccinating Ad preimmuned animals with capsid chimeric Ad vectors. From these studies, nAbs targeting the hexon protein appears to play a dominant role in vivo. [18] [19] [20] [21] [22] Hexon specific nAbs are directed against exposed loops on the surface of the virus particle. These exposed loops are also known as hypervariable regions (HVR). Replacing the entire HAd5 hexon sequence 22 or simply the exposed epitopes of HAd5 hexon 21 with hexon or HVR from a different serotype, was sufficient to overcome pre-existing HAd5 immunity in rodents. In addition, a three amino acid substitution mutation in one of the HVR was able to greatly reduce in vitro neutralization by a NHP polyclonal serum raised against chimpanzee Ad serotype 68 (ChAd68) derived vector. 19 Conversely, addition of the hexon domain of simian Ad serotype 23 (SAd23/Pan6) into a SAd24/Pan7 vector rendered the chimeric vector susceptible to pre-existing SAd23/Pan6 immunity, as illustrated by reduce transgene expression in rodents. 18 In contrast, nAbs against the Ad fiber only play a limited role in in vivo neutralization. NAbs generated after a single HAd7 injection can only weakly neutralize chimeric HAd5 vector expressing HAd7 fiber, indicating that most nAbs were generated against other capsid proteins. Conversely, nAbs generated after a single HAd5 injection can readily neutralize chimeric HAd5 vector expressing HAd7 fiber. 23 In vivo, pre-existing ChAd6 immunity inhibited expression of the transgene from chimeric SAd24/Pan7 vectors that possess the ChAd6 hexon protein but not those that possess the ChAd6 fiber protein. 18 Passive transfer of mouse serum containing Abs against a chimeric HAd35 expressing HAd5 fiber, did not impact cellular and humoral immune responses against the transgene generated after HAd5 injection. 20 One caveat of the above studies is that in vivo, nAbs targeting the fiber are less common after a single injection 23 but are more readily detectable after two or more immunizations with the same adenovirus. 15, 17, 20 However, in most animal studies, pre-existing immunity is generated by a single Ad injection. The induced Ab response is probably of limited breath and neutralizing titer compared with humoral responses generated in individuals after repeated natural infections with replication competent Ad. As a result, these studies may underestimate the role played by nAbs targeting Ad fiber; especially as nAbs against Ad fiber and penton can act synergistically. 17 Although nAbs against Ad strongly reduce Ad vector immunogenicity, they are serotype specific with limited to no crossneutralization of different Ad serotypes (Fig. 1A) . The lack of cross-neutrali