Use of DAF-Displaying Adenovirus Vectors Reduces Induction of Transgene- and Vector-Specific Adaptive Immune Responses in Mice

Abstract

Adenovirus (Ad)-based vectors are attractive candidates for a variety of gene-transfer applications. In this study, we found that decay-accelerating factor (DAF)-displaying Ads induce significantly decreased cellular immune responses to transgenes expressed from the vectors in both Ad5-naive and Ad5-immune mice. Specifically, we found a diminished ability of splenocytes to secrete interferon-g after recall exposure to multiple peptides derived from antigens expressed by DAF-displaying Ads. We also confirmed that DAF-displaying Ads induce decreased numbers of antigen-specific, CD8þ effector memory and central memory CD8 þ T cells, thereby uncovering a unique role of complement in modulating the induction of robust memory T-cell responses. We also confirmed that DAF-displaying Ads generate significantly reduced titers of Ad capsid-specific neutralizing antibodies after gene transfer in vivo. In conclusion, DAF-displaying Ad5-based vectors exhibit decreased induction of complement-dependent, innate immune responses, resulting in both an improved safety profile and a decreased propensity to induce humoral and cellular adaptive immune responses to Ad capsid proteins and Ad vector-expressed transgene products. This attractive combination of features will be beneficial in a variety of clinically relevant gene-transfer applications.

Stimulation of Innate Immunity byIn VivoCyclic di-GMP Synthesis Using Adenovirus

“…4). These results are consistent with previous studies demonstrating that the adenovirus vector alone is capable of altering gene expression in the liver (26,27). The expression of four genes was significantly (P Ͻ 0.05) higher in the Ad5-VCA0956 treatment than in the Ad5-VCA0956* treatment (Fig.…”

“…The VCA0956 gene was amplified from the Vibrio cholerae El tor strain C6706 using the DNA polymerase Phusion (New England BioLabs) and the oligonucleotides 5=-ATAGGTACCCCA CCGTGATGACAACTGAAGATTTCA-3= and 5=-ATACTCGAGTTAGA GCGGCATGACTCGAT-3= (IDT). This product was then inserted into the plasmid pShuttle-CMV (cytomegalovirus) (26) by digestion with KpnI and XhoI (Fermentas) and ligated with a T4 DNA ligase (Invitrogen). Escherichia coli strain DH10B (Invitrogen) was used for harboring plasmid DNA, and sequence fidelity was confirmed by sequencing (Genewiz).…”

Vaccines Expressing the Innate Immune Modulator EAT-2 Elicit Potent Effector Memory T Lymphocyte Responses despite Pre-Existing Vaccine Immunity

“…2). Two immunizations with Ad5-null induced Ad5-neutralizing Ab titers that were .1/200, a level that closely parallels levels of pre-existing Ad5 immunity noted in human populations as previously described (26,28,29). To confirm that experimental and control groups developed equivalent amounts of anti-Ad5-specific immunity, blood samples were collected 1 d before vaccination (27 d after rAd5-null injections), and ELISA analysis for total Ad5-specific IgG was completed.…”

“…All viruses were found to be replication-competent adenovirus-free by both replication-competent adenovirus PCR (E1 region amplification) and direct sequencing methods as previously described (26).…”

“…ELISA-based titering experiments were essentially completed as previously described (26,28). In brief, 5 3 10 8 vps Ad5 vector per well or 0.2 mg recombinant Gag protein per well (each diluted in PBS) was used to coat wells of a 96-well plate overnight at 4˚C.…”