A limited innate immune response is induced by a replication-defective herpes simplex virus vector following delivery to the murine central nervous system

Abstract

Herpes simplex virus type 1 (HSV-1)-based vectors readily transduce neurons and have a large payload capacity, making them particularly amenable to gene therapy applications within the central nervous system (CNS). Because aspects of the host responses to HSV-1 vectors in the CNS are largely unknown, we compared the host response of a nonreplicating HSV-1 vector to that of a replication-competent HSV-1 virus using microarray analysis. In parallel, HSV-1 gene expression was tracked using HSV-specific oligonucleotide-based arrays in order to correlate viral gene expression with observed changes in host response. Microarray analysis was performed following stereotactic injection into the right hippocampal formation of mice with either a replication-competent HSV-1 or a nonreplicating recombinant of HSV-1, lacking the ICP4 gene (ICP4-). Genes that demonstrated a significant change (P < .001) in expression in response to the replicating HSV-1 outnumbered those that changed in response to mock or nonreplicating vector by approximately 3-fold. Pathway analysis revealed that both the replicating and nonreplicating vectors induced robust antigen presentation but only mild interferon, chemokine, and cytokine signaling responses. The ICP4- vector was restricted in several of the Toll-like receptor-signaling pathways, indicating reduced stimulation of the innate immune response. These array analyses suggest that although the nonreplicating vector induces detectable activation of immune response pathways, the number and magnitude of the induced response is dramatically restricted compared to the replicating vector, and with the exception of antigen presentation, host gene expression induced by the nonreplicating vector largely resembles mock infection.

Figure 1

Experimental design of vector injections into the mouse CNS for microarray analysis. Vehicle (mock), 8117/43 (ICP4−), or HSVlacZgC (gC−) was injected into the right hippocampus of mice (N = 9). Tissue was collected from the injection site and from the contralateral (uninjected) side of mice at 2 and 3 days. For each experimental group, triplicate RNA samples, each pooled from three animals were analyzed by Affymetrix and HSV-specific microarrays.

Figure 2

Coronal sections of mouse brains fixed and x-gal–stained 2 or 3 days following injection of either HSVlacZgC (gC−) or 8117/43 (ICP4−) HSV-1 viruses into the right CA1 region (boxes) of the hippocampus. Arrows indicate approximate injection track.

Figure 3

Immediate-early (IE), early (E), late (L), and latency-associated transcripts (LAT) HSV-1 viral gene expression as assayed by HSV-1–specific spotted arrays. Median resonance light scatter signal from triplicates representing viral gene expression 3 days p.i. of CNS tissue injected with (A) replication-competent HSVlacZgC (gC−) or (B) nonreplicating virus 8117/43 (ICP4−) 3 days p.i.

Figure 4

Supervised cluster analysis. HSVlacZgC (gC−)-, 8117/43 (ICP4−)-, and mock-injected arrays at 2 days (2d) or 3 days (3d) post injection. Red indicates up-regulation and blue indicates down-regulation of gene expression represented by fold-change.

Figure 5

IPA of genes altered by mock, 8117/43, or genes altered by both mock and 8117/43 versus uninjected samples. (A) Selected biological functions and (B) canonical pathways. The y-axis (−log of the P value) is the probability that each biological function was assigned to the gene set by chance alone. Threshold is indicated by a line and corresponds to P < .05. Analysis and figure generation were performed using IPA with permission (Ingenuity® Systems, www.ingenuity.com).

Figure 6

Biological functions induced by 8117/43 and HSVlacZgC. (A) Selected biological functions and (B) canonical pathways induced by 8117/43 and HSVlacZgC at 2 days (first and third bars in each pathway) and 3 days (second and fourth bars) are shown. Analysis and figure generation were performed using Ingenuity Pathway Analysis with permission (Ingenuity Systems).

Figure 7

Immunohistochemical visualization of Cd11b/c-positive brain macrophages. Three days following (A) HSVlacZgC, (B) 8117/43, or (C) mock injections, animals were perfused, tissue collected, stained with a polyclonal antibody against Cd11b/c (brown), and counterstained with hematoxalin (blue).