Edouard Cantin

Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase

Small interfering RNAs (siRNA) are potent reagents for directed post-transcriptional gene silencing and a major new genetic tool for investigating mammalian cells. When synthetic siRNAs are used for gene silencing, the costs can be substantial because of variations in siRNA efficacies. An alternative to chemically synthesized siRNAs are siRNAs produced by bacteriophage T7 RNA polymerase. We found that siRNAs synthesized from the T7 RNA polymerase system can trigger a potent induction of interferon α and β in a variety of cell lines. Surprisingly, we also found very potent induction of interferon α and β by short single-stranded RNAs (ssRNAs) transcribed with T3, T7 and Sp6 RNA polymerases. Analyses of the potential mediators of this response revealed that the initiating 5′ triphosphate is required for interferon induction. We describe here an improved method for T7 siRNA synthesis that alleviates the interferon response while maintaining full efficacy of the siRNAs.

Rational design and in vitro and in vivo delivery of Dicer substrate siRNA.

RNA interference is a powerful tool for target-specific knockdown of gene expression. The triggers for this process are duplex small interfering RNAs (siRNAs) of 21–25 nt with 2-bp 3′ overhangs produced in cells by the RNase III family member Dicer. We have observed that short RNAs that are long enough to serve as Dicer substrates (D-siRNA) can often evoke more potent RNA interference than the corresponding 21-nt siRNAs; this is probably a consequence of the physical handoff of the Dicer-produced siRNAs to the RNA-induced silencing complex. Here we describe the design parameters for D-siRNAs and a protocol for in vitro and in vivo intraperitoneal delivery of D-siRNAs and siRNAs to macrophages. siRNA delivery and transfection and analysis of macrophages in vivo can be accomplished within 36 h.

The Immune Response to Herpes Simplex Virus Type 1 Infection in Susceptible Mice Is a Major Cause of Central Nervous System Pathology Resulting in Fatal Encephalitis

ABSTRACT This study was undertaken to investigate possible immune mechanisms in fatal herpes simplex virus type 1 (HSV-1) encephalitis (HSE) after HSV-1 corneal inoculation. Susceptible 129S6 (129) but not resistant C57BL/6 (B6) mice developed intense focal inflammatory brain stem lesions of primarily F4/80+ macrophages and Gr-1+ neutrophils detectable by magnetic resonance imaging as early as day 6 postinfection (p.i.). Depletion of macrophages and neutrophils significantly enhanced the survival of infected 129 mice. Immunodeficient B6 (IL-7R−/− Kitw41/w41) mice lacking adaptive cells (B6-E mice) and transplanted with 129 bone marrow showed significantly accelerated fatal HSE compared to B6-E mice transplanted with B6 marrow or control nontransplanted B6-E mice. In contrast, there was no difference in ocular viral shedding in B6-E mice transplanted with 129 or B6 bone marrow. Acyclovir treatment of 129 mice beginning on day 4 p.i. (24 h after HSV-1 first reaches the brain stem) reduced nervous system viral titers to undetectable levels but did not alter brain stem inflammation or mortality. We conclude that fatal HSE in 129 mice results from widespread damage in the brain stem caused by destructive inflammatory responses initiated early in infection by massive infiltration of innate cells.

Herpes Simplex Virus Type 1 DNA Is Immunostimulatory In Vitro and In Vivo

ABSTRACT Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically immunostimulatory both in vitro and in vivo, tending to promote Th1-like responses. In contrast, CpG dinucleotides in mammalian DNAs are extensively methylated on cytosines and hence immunologically inert. Since the herpes simplex virus (HSV) genome is unmethylated and G+C rich, we predicted that CpG motifs would be highly prevalent in the HSV genome; hence, we examined the immunostimulatory potential of purified HSV DNA in vitro and in vivo. Mouse splenocyte cultures treated with HSV DNA or HSV-derived oligodeoxyribonucleotides (ODNs) showed strong proliferative responses and production of inflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor [TNF], and interleukin-6 [IL-6]) in vitro, whereas splenocytes treated with mammalian CV-1 DNA or non-CpG ODN did not. After immunization with ovalbumin (OVA), only splenocytes from mice immunized with HSV DNA or HSV-ODN as the adjuvants proliferated strongly and produced typical Th1 responses, including CD8+ cytotoxic T-lymphocyte responses, upon restimulation with OVA. Furthermore, HSV-ODN synergized with IFN-γ to induce nitric oxide (NO), IL-6, and TNF production from macrophages. These results demonstrate that HSV DNA and HSV-ODN are immunostimulatory, driving potent Th1 responses both in vitro and in vivo. Considering that HSV DNA has been found to persist in nonneuronal cells, these results fuel speculation that HSV DNA might play a role in pathogenesis, in particular, in diseases like herpes stromal keratitis (HSK) that involve chronic inflammatory responses in the absence of virus or viral antigens.

A locus on mouse chromosome 6 that determines resistance to herpes simplex virus also influences reactivation, while an unlinked locus augments resistance of female mice

ABSTRACT During studies to determine a role for tumor necrosis factor (TNF) in herpes simplex virus type 1 (HSV-1) infection using TNF receptor null mutant mice, we discovered a genetic locus, closely linked to the TNF p55 receptor (Tnfrsf1a) gene on mouse chromosome 6 (c6), that determines resistance or susceptibility to HSV-1. We named this locus the herpes resistance locus, Hrl, and showed that it also mediates resistance to HSV-2. Hrl has at least two alleles, Hrlr, expressed by resistant strains like C57BL/6 (B6), and Hrls, expressed by susceptible strains like 129S6 (129) and BALB/c. Although Hrl is inherited as an autosomal dominant gene, resistance to HSV-1 is strongly sex biased such that female mice are significantly more resistant than male mice. Analysis of backcrosses between resistant B6 and susceptible 129 mice revealed that a second locus, tentatively named the sex modifier locus, Sml, functions to augment resistance of female mice. Besides determining resistance, Hrl is one of several genes involved in the control of HSV-1 replication in the eye and ganglion. Remarkably, Hrl also affects reactivation of HSV-1, possibly by interaction with some unknown gene(s). We showed that Hrl is distinct from Cmv1, the gene that determines resistance to murine cytomegalovirus, which is encoded in the major NK cell complex just distal of p55 on c6. Hrl has been mapped to a roughly 5-centimorgan interval on c6, and current efforts are focused on obtaining a high-resolution map for Hrl.

Tumor Necrosis Factor (TNF) Protects Resistant C57BL/6 Mice against Herpes Simplex Virus-Induced Encephalitis Independently of Signaling via TNF Receptor 1 or 2

ABSTRACT Tumor necrosis factor (TNF) is a multifunctional cytokine that has a role in induction and regulation of host innate and adaptive immune responses. The importance of TNF antiviral mechanisms is reflected by the diverse strategies adopted by different viruses, particularly members of the herpesvirus family, to block TNF responses. TNF binds and signals through two receptors, Tnfrsf1a (TNF receptor 1 [TNFR1], or p55) and Tnfrsf1b (TNFR2, or p75). We report here that herpes simplex virus 1 (HSV-1) infection of TNF−/− mice on the resistant C57BL/6 genetic background results in significantly increased susceptibility (P < 0.0001, log rank test) to fatal HSV encephalitis (HSE) and prolonged persistence of elevated levels of virus in neural tissues. In contrast, although virus titers in neural tissues of p55−/−N13 mice were elevated to levels comparable to what was found for the TNF−/− mice, the p55−/−N13 mice were as resistant as control C57BL/6 mice (P > 0.05). The incidence of fatal HSE was significantly increased by in vivo neutralization of TNF using soluble TNFR1 (sTNFR1) or depletion of macrophages in C57BL/6 mice (P = 0.0038 and P = 0.0071, respectively). Strikingly, in vivo neutralization of TNF in HSV-1-infected p55−/− p75−/− mice by use of three independent approaches (treatment with soluble p55 receptor, anti-TNF monoclonal antibody, or in vivo small interfering RNA against TNF) resulted in significantly increased mortality rates (P = 0.005), comparable in magnitude to those for C57BL/6 mice treated with sTNFR1 (P = 0.0018). Overall, these results indicate that while TNF is required for resistance to fatal HSE, both p55 and p75 receptors are dispensable. Precisely how TNF mediates protection against HSV-1 mortality in p55−/− p75−/− mice remains to be determined.

Passively Administered Pooled Human Immunoglobulins Exert IL-10 Dependent Anti-Inflammatory Effects that Protect against Fatal HSV Encephalitis

HSV-1 is the leading cause of sporadic encephalitis in humans. HSV infection of susceptible 129S6 mice results in fatal encephalitis (HSE) caused by massive inflammatory brainstem lesions comprising monocytes and neutrophils. During infection with pathogenic microorganisms or autoimmune disease, IgGs induce proinflammatory responses and recruit innate effector cells. In contrast, high dose intravenous immunoglobulins (IVIG) are an effective treatment for various autoimmune and inflammatory diseases because of potent anti-inflammatory effects stemming in part from sialylated IgGs (sIgG) present at 1–3% in IVIG. We investigated the ability of IVIG to prevent fatal HSE when given 24 h post infection. We discovered a novel anti-inflammatory pathway mediated by low-dose IVIG that protected 129S6 mice from fatal HSE by modulating CNS inflammation independently of HSV specific antibodies or sIgG. IVIG suppressed CNS infiltration by pathogenic CD11b+ Ly6Chigh monocytes and inhibited their spontaneous degranulation in vitro. FcγRIIb expression was required for IVIG mediated suppression of CNS infiltration by CD45+ Ly6Clow monocytes but not for inhibiting development of Ly6Chigh monocytes. IVIG increased accumulation of T cells in the CNS, and the non-sIgG fraction induced a dramatic expansion of FoxP3+ CD4+ T regulatory cells (Tregs) and FoxP3− ICOS+ CD4+ T cells in peripheral lymphoid organs. Tregs purified from HSV infected IVIG treated, but not control, mice protected adoptively transferred mice from fatal HSE. IL-10, produced by the ICOS+ CD4+ T cells that accumulated in the CNS of IVIG treated, but not control mice, was essential for induction of protective anti-inflammatory responses. Our results significantly enhance understanding of IVIGs anti-inflammatory and immunomodulatory capabilities by revealing a novel sIgG independent anti-inflammatory pathway responsible for induction of regulatory T cells that secrete the immunosuppressive cytokine IL-10 and further reveal the therapeutic potential of IVIG for treating viral induced inflammatory diseases.

Evaluation of antiviral immunity using vaccinia virus recombinants expressing cloned genes for herpes simplex virus type 1 glycoproteins.

Immunization of mice with vaccinia virus recombinants expressing the glycoproteins B or D of herpes simplex virus type 1 (HSV-1) induced humoral antibody as well as multiple aspects of HSV-1-specific T lymphocyte-mediated responses. However, vaccinated mice were not completely resistant to HSV-1 challenge and were unable to eliminate an epithelial infection rapidly. Evidence is presented which indicates that immunization with either vaccinia virus recombinant, while inducing the necessary protective populations of CD4+ T lymphocytes, fails to induce the complementing CD8+ cytotoxic T lymphocytes necessary for high levels of protection against a primary HSV-1 infection. These findings are discussed with relevance to the future development of anti-HSV vaccines.

A recombinant vaccinia virus expressing herpes simplex virus type 1 glycoprotein B induces cytotoxic T lymphocytes in mice.

Spleen cells from BALB/c (H-2d) mice vaccinated with vgB11, a recombinant vaccinia virus which expresses glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1), lysed EMT6 (H-2d) target cells infected with vgB11 or with HSV-1 but did not lyse uninfected EMT6 cells or infected L-929 (H-2k) target cells. Unlabelled target cell competition of lysis showed that only syngeneic cells infected with vgB11 or HSV-1 inhibited lysis of radiolabelled HSV-1-infected targets. These results demonstrate that vgB11 induces H-2-restricted anti-HSV-1 cytotoxic T lymphocytes and that gB is the target antigen.

Establishment of HSV1 latency in immunodeficient mice facilitates efficient in vivo reactivation.

The establishment of latent infections in sensory neurons is a remarkably effective immune evasion strategy that accounts for the widespread dissemination of life long Herpes Simplex Virus type 1 (HSV1) infections in humans. Periodic reactivation of latent virus results in asymptomatic shedding and transmission of HSV1 or recurrent disease that is usually mild but can be severe. An in-depth understanding of the mechanisms regulating the maintenance of latency and reactivation are essential for developing new approaches to block reactivation. However, the lack of a reliable mouse model that supports efficient in vivo reactivation (IVR) resulting in production of infectious HSV1 and/or disease has hampered progress. Since HSV1 reactivation is enhanced in immunosuppressed hosts, we exploited the antiviral and immunomodulatory activities of IVIG (intravenous immunoglobulins) to promote survival of latently infected immunodeficient Rag mice. Latently infected Rag mice derived by high dose (HD), but not low dose (LD), HSV1 inoculation exhibited spontaneous reactivation. Following hyperthermia stress (HS), the majority of HD inoculated mice developed HSV1 encephalitis (HSE) rapidly and synchronously, whereas for LD inoculated mice reactivated HSV1 persisted only transiently in trigeminal ganglia (Tg). T cells, but not B cells, were required to suppress spontaneous reactivation in HD inoculated latently infected mice. Transfer of HSV1 memory but not OVA specific or naïve T cells prior to HS blocked IVR, revealing the utility of this powerful Rag latency model for studying immune mechanisms involved in control of reactivation. Crossing Rag mice to various knockout strains and infecting them with wild type or mutant HSV1 strains is expected to provide novel insights into the role of specific cellular and viral genes in reactivation, thereby facilitating identification of new targets with the potential to block reactivation.