TRIM21 mediates antibody inhibition of adenovirus-based gene delivery and vaccination

Abstract

Adenovirus has enormous potential as a gene-therapy vector, but preexisting immunity limits its widespread application. What is responsible for this immune block is unclear because antibodies potently inhibit transgene expression without impeding gene transfer into target cells. Here we show that antibody prevention of adenoviral gene delivery in vivo is mediated by the cytosolic antibody receptor TRIM21. Genetic KO of TRIM21 or a single-antibody point mutation is sufficient to restore transgene expression to near-naïve immune levels. TRIM21 is also responsible for blocking cytotoxic T cell induction by vaccine vectors, preventing a protective response against subsequent influenza infection and an engrafted tumor. Furthermore, adenoviral preexisting immunity can lead to an augmented immune response upon i.v. administration of the vector. Transcriptomic analysis of vector-transduced tissue reveals that TRIM21 is responsible for the specific up-regulation of hundreds of immune genes, the majority of which are components of the intrinsic or innate response. Together, these data define a major mechanism underlying the preimmune block to adenovirus gene therapy and demonstrate that TRIM21 efficiently blocks gene delivery in vivo while simultaneously inducing a rapid program of immune transcription.

Keywords: TRIM21; adenovirus; gene therapy; host–pathogen; viral vector.

Fig. 1.

Diminished transgene expression in the presence of anti-Ad5 antibodies is mediated by TRIM21. (A) Viral copy number at the indicated time points postinfection. (B) Model of human IgG1. Key residues involved in binding to FcγRs are depicted in red (L234/L235), and the TRIM21- and FcRn-binding sites are depicted in blue (H433 in light blue, N434 and H435 in dark blue). (C) Relative infection of Ad5-Luc in mouse liver in WT animals using 2.5 µg WT rh9C12 and FcγR-binding mutant LALA. (D) Relative infection of Ad5-Luc in mouse liver (Left) and spleen (Right) in WT and T21-KO animals using 1 µg WT rh9C12 and TRIM21 nonbinding mutant H433A. (E) Viral copy number 4 h postinfection with anti-Ad5 immune serum (diluted 1:500). (F) Relative infection of Ad5-Luc in mouse liver using the indicated dilutions of anti-Ad5 immune serum in WT and T21-KO animals. (G) Absolute levels of infection measured in relative light units (RLUs) in clodronate-treated or control mice. (H) Relative infection in clodronate-treated or control mice using 1 µg WT rh9C12 and TRIM21 nonbinding mutant H433A. Error bars depict mean ± SEM. Groups consisted of n = 3–10 mice.

Fig. 2.

TRIM21 blocks antigen-specific CD8 T cell responses in an antibody-dependent manner. (A) SIINFEKL (SL8)-specific CD8 T cell frequency in the blood of mice i.v. immunized with Ad5-Ova [10⁸ particles (pts)] in the presence of WT rh9C12 or H433A. (B) SIINFEKL (SL8)-specific CD8 T cell frequency in the blood of WT and T21-KO mice immunized with Ad5-Ova in the presence of WT rh9C12. Error bars depict mean ± SEM. Groups consisted of n = 4–5 mice. (C) Mice were immunized with Ad5-Ova with or without WT rh9C12 or H433A and infected with PR8-SL8 on day 10 post immunization. Viral titer in the lung was determined 3 d postinfection by qPCR. Error bars depict mean ± SEM. Groups consisted of n = 5–6 mice. (D) WT (Left) or T21-KO (Right) animals were immunized with Ad5-Ova only or in the presence of WT rh9C12 and injected with MCA-ovalbumin tumor cells on day 7 post immunization. Error bars depict mean ± SEM. Groups consisted of n = 5–8 mice. Tumor growth curves were analyzed by comparing area under the curve by one-way ANOVA with a Tukey post hoc test.

Fig. 3.

Anti-Ad5 antibodies elicit a potent inflammatory response. (A) Fold induction of cytokines in mouse liver at the indicated time points in response to Ad5 alone (2 × 10¹⁰ pts) or Ad5 and WT rh9C12. (B) Fold induction of the indicated cytokines in response to Ad5 alone or and Ad5 and anti-Ad5 immune serum 4 h postinfection. (C) Fold induction of the indicated cytokines in the liver of WT (black bars) and T21-KO (gray bars) mice 4 h postinfection. Error bars depict mean ± SEM. Groups consisted of n = 3–5 mice.

Fig. 4.

TRIM21 positively regulates innate immunity in response to Ad5 infection in the presence of antibody. (A) Venn diagram comparing differential gene expression between WT and T21-KO animals in uninfected mice, mice infected with Ad5 (2 × 10¹⁰ pts), and mice infected with Ad5 in the presence of rh9C12-WT. The gene common to all groups (i.e., intersection of the Venn diagram) is Trim21. (B) Classification of 995 differentially expressed genes upon virus infection in WT mice. Pie chart colors correspond to the relative number of up- and down-regulated genes separately for each classification according to (genes^up − genes^down)/genes^total. (C) Heat map of differentially expressed immune genes (GO:0002376) between WT mice infected with Ad5 and WT mice infected with Ad5 + rh9C12 WT. Row Z-scores were calculated from rlog-transformed read counts for the conditions shown (n = 4 each). Row Z-scores for WT mice in the following conditions are shown: uninfected, Ad5 alone, Ad5 + rh9C12-WT, and Ad5 + rh9C12-H433A. (D) TRIM21-dependent differential gene expression. Venn diagram comparing differential gene expression between WT mice infected with Ad5 and rh9C12-WT or rh9C12-H433A and between WT and T21-KO mice infected with Ad5 and rh9C12-WT (Top), and classification of the 416 TRIM21-dependent differentially expressed genes (Bottom Left), as well as the classification of the 183 immunity genes (Bottom Right). Pie chart colors as in B. Pie chart classifications: category “immunity” was based on GO term “immune system process” (GO:0002376), whereas all other categories were assigned based on UniProt descriptions of the relevant gene.