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. 2002 May;76(9):4580-90.
doi: 10.1128/jvi.76.9.4580-4590.2002.

Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors

Anne-Kathrin Zaiss  1 , Qiang Liu, Gloria P Bowen, Norman C W Wong, Jeffrey S Bartlett, Daniel A Muruve
Affiliations
Free PMC article

Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors

Anne-Kathrin Zaiss et al. J Virol. 2002 May.
Free PMC article

Abstract

Adenovirus vectors induce acute inflammation of infected tissues due to activation of the innate immune system and expression of numerous chemokines and cytokines in transduced target cells. In contrast, adeno-associated virus (AAV) vectors are not associated with significant inflammation experimentally or clinically. We tested the ability of AAV vectors to induce the expression of chemokines in vitro and to activate the innate immune system in vivo. In human HeLa cells and murine renal epithelium-derived cells (REC cells) the adenovirus vector AdlacZ induced the expression of multiple inflammatory chemokines including RANTES, interferon-inducible protein 10 (IP-10), interleukin-8 (IL-8), MIP-1beta, and MIP-2 in a dose-dependent manner. The use of AAVlacZ did not induce the expression of these chemokines above baseline levels despite 40-fold-greater titers than AdlacZ and greater amounts of intracellular AAVlacZ genomes according to Southern and slot blot analysis. This finding confirmed that the lack of AAVlacZ induction of chemokine was not due to reduced transduction. In DBA/2 mice, the intravenous administration of 2.5 x 10(11) particles of AAVlacZ resulted in the rapid induction of liver tumor necrosis factor alpha (TNF-alpha), RANTES, IP-10, MIP-1beta, MCP-1, and MIP-2 mRNAs. However, 6 h following injection, chemokine mRNA levels returned to baseline. As expected, administration of 10-fold less AdlacZ caused an induction of liver TNF-alpha and chemokine mRNAs that persisted for more than 24 h posttransduction. Whereas intravenous administration of 2.5 x 10(11) particles of AAVlacZ triggered a transient infiltration of neutrophils and CD11b(+) cells into liver, this response stood in contrast to widespread inflammation and toxicity induced by AdlacZ. Kupffer cell depletion abolished AAVlacZ but not AdlacZ-induced chemokine expression and neutrophil infiltration. In summary, these results show that AAV vectors activate the innate immune system to a lesser extent than do adenovirus vectors and offer a possible explanation for the reduced inflammatory properties of AAV compared to adenovirus vectors.

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Figures

FIG. 1.
FIG. 1.
Adenovirus and AAV vector-induced chemokine expression in vitro determined by an RNase protection assay of AdlacZ- or AAVlacZ-transduced cells. (A) REC cells were transduced with increasing titers of AdlacZ (1 × 103 to 70 × 103 part/cell) or AAVlacZ (15 × 103 to 200 × 103 part/cell). AdlacZ induced the expression of RANTES, MIP-1β, MIP-2, and IP-10 in a dose-dependent manner. In contrast, AAVlacZ failed to induce chemokine expression above baseline at titers 40-fold greater. (B) HeLa cells transduced with increasing titers of AdlacZ (1 × 103 to 70 × 103 part/cell) are induced to express RANTES, IP-10, MIP-1α, and IL-8 in a dose-dependent manner. AAVlacZ (15 × 103 to 200 × 103 part/cell) does not induce chemokine expression in HeLa cells. VH, sucrose vehicle.
FIG. 2.
FIG. 2.
Transduction efficiency of adenovirus and AAV vectors in vitro. (A) Slot blot analysis of total DNA from AdlacZ- and AAVlacZ-transduced REC and HeLa cells. Probing for the lacZ reporter gene confirms that cellular viral genome content correlates directly with vector titer. (B) Southern blot of transduced cells probing for the lacZ reporter gene. Total DNA from AdlacZ-transduced REC and HeLa cells reveals a 7,500-bp fragment of adenovirus DNA containing the lacZ gene. AAVlacZ-transduced cells demonstrate multiple genome conformations characteristic of early AAV infection. SS, single-stranded vector genome; M, monomer.
FIG. 2.
FIG. 2.
Transduction efficiency of adenovirus and AAV vectors in vitro. (A) Slot blot analysis of total DNA from AdlacZ- and AAVlacZ-transduced REC and HeLa cells. Probing for the lacZ reporter gene confirms that cellular viral genome content correlates directly with vector titer. (B) Southern blot of transduced cells probing for the lacZ reporter gene. Total DNA from AdlacZ-transduced REC and HeLa cells reveals a 7,500-bp fragment of adenovirus DNA containing the lacZ gene. AAVlacZ-transduced cells demonstrate multiple genome conformations characteristic of early AAV infection. SS, single-stranded vector genome; M, monomer.
FIG. 3.
FIG. 3.
Adenovirus and AAV vector-induced chemokine and cytokine expression in vivo. RNase protection assay of DBA/2 mouse liver RNA following the intravenous administration of 2.5 × 109, 2.5 × 1010, and 2.5 × 1011 particles of AdlacZ or AAVlacZ. (A) Chemokine mRNA expression. One, six, and twenty-four hours following injection, AdlacZ induced the expression of RANTES, MIP-1β, MIP-2, MCP-1, and IP-10 in a dose-dependent manner. AAVlacZ induced the expression of proinflammatory chemokines only 1 h following the administration of 2.5 × 1011 particles. Chemokine mRNA was not increased in mouse liver 6 and 24 h following the administration of 2.5 × 1011 particles of AAVlacZ. (B) Cytokine mRNA expression. LTB-β and TNF-α mRNAs are induced in a pattern similar to that of chemokines following AdlacZ and AAVlacZ administration. Data are representative samples of experiments performed with three animals per time point.
FIG. 4.
FIG. 4.
Analysis of adenovirus and AAV vector transduction in vivo. (A) Slot blot analysis of mouse liver total DNA 1, 6, and 24 h following transduction with 2.5 × 1011 particles of AdlacZ or AAVlacZ. Probing for the lacZ reporter gene reveals equivalent amounts of AAV and adenoviral genome DNA in transduced mouse livers. (B) Southern blot analysis of mouse liver total DNA 1, 6, or 24 h after injection of 2.5 × 1011 particles of AdlacZ or AAVlacZ. Probing for the lacZ gene reveals a 7,500-bp fragment of adenovirus DNA in AdlacZ-transduced mice. AAVlacZ-transduced livers demonstrate multiple genome conformations characteristic of early AAV infection. SS, single-stranded vector genome; M, monomer. Data are representative samples of experiments performed with three animals per time point.
FIG. 5.
FIG. 5.
Leukocyte infiltration following adenovirus or AAV vector transduction in vivo. (A) Leder (esterase) stain of liver sections showing infiltrating neutrophils in normal (+KC) or Kupffer cell-depleted (−KC) DBA/2 mice 1 h following the intravenous administration of 2.5 × 1011 particles of AdlacZ or AAVlacZ. (B) CD11b+ immunohistochemistry of liver sections showing infiltrating leukocytes in normal (+KC) or Kupffer cell-depleted (−KC) DBA/2 mice 1 h following the intravenous administration of 2.5 × 1011 particles of AdlacZ or AAVlacZ (magnification, ×40). Arrowheads indicate one representative stained cell. Quantitative analysis of neutrophil (C) and CD11b+ (D) cell infiltration in mouse liver 1, 6, or 24 h after intravenous administration of 2.5 × 1011 particles of AdlacZ or AAVlacZ. ▪, with Kupffer cells; ░⃞, depleted of Kupffer cells. Values represent mean cells per high power field ± SD (n = 3).
FIG. 6.
FIG. 6.
Liver morphology following adenovirus or AAV vector transduction in vivo. (A) Liver necrosis (seen as pale patchy areas) is detected at 24 h in mice administered 2.5 × 10 11 particles of AdlacZ, but not in AAV- or vehicle-treated animals (hematoxylin and eosin stain; magnification, ×20). Data are representative samples of experiments performed with three animals. (B) Serum aspartate aminotransferase (AST/GOT) (▪) and alanine aminotransferase (ALT/GPT) (░⃞) levels in mice 24 h following the administration of 2.5 × 1011 particles of AdlacZ or AAVlacZ. VH, vehicle-treated animals. Values represent mean SF units ± SD (n = 3).
FIG. 7.
FIG. 7.
Adenovirus and AAV vector-induced chemokine and cytokine expression in Kupffer cell-depleted mice. (A) Kupffer cell immunohistochemistry in mouse liver following gadolidium chloride treatment. Liver Kupffer cells are reduced over 90% in mice receiving GdCl3. (B) RNase protection assay of mouse liver RNA, 1 h following intravenous administration of 2.5 × 1011 particles of AdlacZ or AAVlacZ in normal mice (+KC) or GdCl3-treated mice (−KC). Kupffer cell depletion abolishes AAVlacZ-induced chemokine mRNA expression at 1 h. VH, vehicle. Data are representative samples of experiments performed with four animals per group (results for two representative animals are shown). (C) LTB-β and TNF-α mRNA expression in GdCl3-treated mice (−KC) 1 h following the administration of 2.5 × 1011 particles of AdlacZ or AAVlacZ.
FIG. 8.
FIG. 8.
Analysis of adenovirus and AAV vector transduction in Kupffer cell-depleted mice. (A) Slot blot analysis of mouse liver total DNA 1 h following the administration of 2.5 × 1011 particles of AdlacZ or AAVlacZ to normal (+KC) and GdCl3-treated (−KC) animals. Probing for the lacZ reporter gene reveals equivalent amounts of AAV and adenoviral genome DNA in transduced mouse livers. Data are representative samples of experiments performed with four animals per group (results for two representative animals are shown). (B) Southern blot analysis of mouse liver total DNA, 1 h following the administration of 2.5 × 1011 particles of AdlacZ or AAVlacZ to Kupffer cell-depleted mice. Probing for the lacZ gene reveals a 7,501-bp fragment of adenovirus DNA in AdlacZ-transduced mice. AAVlacZ-transduced livers demonstrate multiple genome conformations, characteristic of early AAV infection. SS, single-stranded vector genome; M, monomer; VH, vehicle.
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