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The Journal of Immunology, 2006, 176: 4940-4949.
Copyright © 2006 by The American Association of Immunologists

CpG Oligodeoxynucleotides Protect Newborn Mice from a Lethal Challenge with the Neurotropic Tacaribe Arenavirus

João A. Pedras-Vasconcelos*, David Goucher*, Montserrat Puig*, Leonardo H. Tonelli{dagger}, Vivian Wang*, Shuichi Ito{ddagger} and Daniela Verthelyi1,*

* Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Federal Drug Administration, Bethesda, MD 20892; {dagger} Section on Neuroendocrine Immunology and Behavior, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892; and {ddagger} Division of Viral Products, Center for Biologics Evaluation and Research, Federal Drug Administration, Rockville, MD 20852


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The innate immune system is key to limiting the early spread of most pathogens and directing the development of Ag-specific immunity. Recently, a number of synthetic molecules that activate the innate immune system by stimulating TLRs have been identified. Among them, synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG ODNs) were shown to activate TLR9-bearing B cells, macrophages, and dendritic cells to induce a strong proinflammatory milieu and a type 1-biased immune response that protects mice from a variety of parasitic, bacterial, and viral infections. Although the protective effect of CpG ODN in adult mice was well established, its effectiveness in neonates, which have lower numbers of dendritic, B, and T cells and tend to favor Th2 responses, was unclear. This study uses the New World arenavirus Tacaribe, a neurotropic pathogen that is lethal in newborn mice, to explore the effectiveness of TLR-mediated innate immune responses. Neonatal BALB/c mice treated with CpG ODN at the time of infection had reduced viral load (p < 0.01) and increased survival (52%, p < 0.001 i.p.; 36%, p < 0.05 intranasally). Protection was achieved in mice treated no later than 3 days postchallenge and appears to be mediated by an increase in Ag-specific Abs (IgG and IgM) and to require inducible NO synthase expression and NO production. To our knowledge, this is the first study assessing the mechanisms by which CpG ODN can protect mice from a neurotropic viral infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The neonatal period is characterized by increased susceptibility to infections, particularly by upper respiratory track pathogens (1, 2). Several factors are thought to contribute to this susceptibility, including lack of pre-existing memory to common pathogens, immature secondary lymphoid organs, impaired neutrophil chemotaxis and degranulation, reduced complement activation (3), and weaker B cell responses, as neonates tend to have lower IgG2a in mice and IgG2 in humans (4). In addition, newborn mice tend to have fewer CD4+ T cells and a Th2 bias as compared with adults (1). Recent reports suggest that the innate immune cell activation via TLR may be impaired in newborns as well (4, 5, 6).

Over the past decade, TLRs and their ligands (natural and synthetic) have emerged as potential targets for immunoprotective therapies through direct stimulation of the immune system. In particular, the activation of TLR7 and TLR8 by guanosine- and uridine-rich ssRNA or synthetic imidazoquinoline-like molecules such as resiquimod (R848) or the activation of TLR9 by bacterial DNA or synthetic oligodeoxynucleotides (ODNs)2 containing unmethylated CpG motifs triggers an immune cascade that results in the proliferation, differentiation, and maturation of multiple immune cells, including B and T lymphocytes, NK cells, monocytes, macrophages, and dendritic cells (7, 8, 9). Together, these cells secrete cytokines and chemokines that create a proinflammatory (IL-1, IL-6, IL-18, and TNF-{alpha}) and Th1-biased (IFN-{gamma} and IL-12) immune milieu.

In murine models, treatment with CpG ODN reduces the severity and time course of infection and facilitates the clearance of bacteria, parasites, and viruses such as herpes simplex virus type 2, Friend retrovirus, and influenza (10, 11, 12). In turn, topical application of imidazolines has been effective against infections such as human papillomavirus and herpes simplex virus (13).

Recent studies demonstrating that resident CNS cells, including microglia, astrocytes, and neurons, express Toll receptors and are capable of generating an innate immune response suggested that TLR ligands might be used to control neurotropic infection (14, 15, 16). However, direct administration of CpG ODN intracerebrally induced acute local inflammation and death, and it was uncertain whether systemic administration of a Toll agonist would activate the immune cells in the CNS in vivo to control an infection.

To assess whether TLR agonists can act as immunoprotective agents against a neurotropic pathogen in neonatal mice, we used a murine model of the Arenaviridae family. The New World arenavirus (Tacaribe serocomplex) is a growing family of enveloped, segmented RNA viruses of increasing medical importance that currently has 23 identified members (17). Four of those viruses (the Junin, Machupo, Guanarito, and Sabia viruses) are causative agents of South American hemorrhagic fevers. Infections occur usually via the respiratory route as a consequence of inadvertent human contact with viruses in rodent feces and urine. Because of their high pathogenic potential, ease of growth in culture, and plastic genomic structure, several arenaviruses have been included in the list of potential biowarfare agents (class 1A) by the Center for Disease Control and Prevention (Atlanta, GA) (18). The Tacaribe virus (TCRV) is a biochemically and serologically close relative of the Junin virus but has a low pathogenic potential for humans and is more easily amenable to laboratory study. Experimentally, it causes lethal meningoencephalitis in mice younger than 1 wk old with death occurring 1–3 wk later, depending on the mouse strain, dose, and route of inoculation (19). In this study we demonstrate that CpG ODN treatment can activate the newborn’s innate immune system to protect it against an otherwise lethal neurotropic infection with the New World arenavirus Tacaribe.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and media

Phosphorothioate CpG ODN (5'-GCTAGACGTTAGCGT-3'; underlined portion represents the active CpG motif) and control ODN (5'-GCTAGAGCTTAGGCT-3') were synthesized at the Center for Biologics Evaluation and Research (CBER; Rockville, MD) core facility. All ODNs had <0.1 endotoxin units of endotoxin per milligram of ODN as assessed by a Limulus amebocyte lysate assay (QCL-1000; BioWhittaker). R848 (resiquimod) was resuspended as per the manufacturer’s instructions (InvivoGen).

mAbs to TCRV were provided by Dr. M. J. Buchmeier of the Scripps Research Foundation (La Jolla, CA) and have been previously described (20). Convalescent sera were generated from 21-day-old mice that had been challenged with TCRV on day 7 of life. Abs to CD45, IL-12 (C15.6 and C17.8), and IL-6 (MP5-20F3 and MP5-32C11) were obtained from BD Biosciences. Abs to glial fibrillary acidic protein (GFAP) and to neuron-specific enolase (NSE) were purchased from Chemicon International. Abs to Diablo were purchased from Alexis. Abs to IFN-{gamma} (RMMG-1 and R4–6A2) were obtained from BioSource International and eBioscience, respectively. The cell growth medium used was RPMI 1640 (Invitrogen Life Technologies) containing 5 or 10% heat-inactivated FCS (R5 or R10 medium), 1.5 mM L-glutamine, and 100 U of penicillin-streptomycin/ml (Invitrogen Life Technologies)

Virus and growth conditions

TCRV strain TRVL 11573 was obtained from American Type Culture Collection (ATCC VR-114) as a suckling mouse brain desiccate. TCRV ampoule contents were resuspended in 1 ml of sterile water, and aliquots were frozen at –70°C. TCRV was expanded in Vero cell monolayers in P75 cell culture flasks (Corning) for 8 days in R10 medium at 37°C. Cell-free supernatant was harvested, aliquoted, and frozen at –70°C until use. The infected Vero cell pellet was resuspended in 2 ml of PBS and homogenized with a PRO200 homogenizer (PRO Scientific) for 15 s on ice. The resulting cell-free virus particles were UV-inactivated for 5 min in a UV Stratalinker 2400 (Stratagene) and stored at –20°C for use in detecting virus-specific Abs.

TCRV levels in Vero culture and brain extracts were determined using the tissue culture ID50 method (TCID50) (21). Briefly, virus-containing suspension was diluted from 10–1 to 10 –11 in 96-well round-bottom plates (Costar) containing R5 medium. Triplicate to quadruplicate dilutions were subsequently overlaid on Vero cell monolayers cultured in 96-well flat-bottom plates (Costar). The virus was allowed to adhere for 1–2 h at 37°C, after which the virus suspension was removed from the plate and fresh R5 medium was added. Cells were monitored for the development of cytopathic effects daily and were scored after 6–8 days of incubation at 37°C. The virus titer was defined as the last dilution showing cytopathic effects in culture in two of three replicate wells.

Animals and infections

BALB/c, C57BL/6, and C57B10.D2 wild-type strains were obtained from the National Cancer Institute (Frederick, MD). C57BL/6 inducible NO synthase (iNOS) 2 KO mice were purchased from The Jackson Laboratory. C57BL/6 TLR9 KO mice were kindly provided by Dr. S. Akira (Osaka University, Osaka, Japan). C57B10 µMT/B cell KO mice were provided by Dr. D. Jankovic (National Institute of Allergy and Infectious Diseases, Bethesda, MD). Mice were housed in sterile microisolator cages in the CBER specific pathogen-free animal facility and bred at 6–12 wk of age. All experiments were approved by the Food and Drug Administration Animal Care and Use Committee.

Neonatal mice were infected with 2000 TCID50 of TCRV i.p., intranasally (i.n.) (10–20 µl), or intracranially (i.c.) (10 µl) 1–3 days after birth, alone or with CpG ODN, control ODN (50 µg/mouse), or R848 (25 µg/mouse) using syringes (Hamilton) and 30-gauge needles (BD Biosciences). Mice that died within 24 h after inoculation were excluded from the study. Uninfected mice that received CpG ODN or saline were used as controls. Administration was performed i.n. by placing a 10-µl drop of TCRV and/or CpG ODN or control ODN on the nostrils. The mice were kept in prone position and allowed to inhale the solution (2~4 min). Delivery and absorption (i.n.) was confirmed by confocal microscopy using fluorescently labeled ODN.

The immunomodulatory treatment was administered at the time of infection (day 0), 3 days prior to infection (day –3), or 3 or 6 days postinfection (day 3 or day 6) as described in Results. In some experiments neonatal mice were treated with the NO synthase inhibitor aminoguanidine (AMG) (40 µg/mouse in 20 µl; Sigma-Aldrich) daily, starting on the day of infection and for a period of 10–12 days. Some mice received convalescent serum transfers (30 µl i.p.) 3 and 8 days postinfection obtained from mice infected on day 7 and bled on day 21 postinfection. The convalescent sera derived from infected mice or from infected mice treated with CpG ODN had similar levels of IgG and IgM Abs to TCRV. Survival for each condition was assessed in 2–5 independent experiments. As previously reported (22), neonatal mice treated with CpG ODN i.p. or i.n. at therapeutic doses experienced no obvious delay in development or weight loss. CpG ODN administered i.c. induced acute encephalitis and death within 24 h. The mice were monitored daily, but, unless stated, infections were allowed to proceed to their natural outcome. Sera were prepared in Microtainer serum separator tubes (BD Biosciences) and kept frozen until used. Brains and spleens were collected under sterile conditions.

Immunohistochemistry

Brain pathology was assessed at 5 and 10 days postchallenge. Briefly, mice were euthanized and perfused intracardially with cold PBS followed by 4% paraformaldehyde. Brains were then dissected, cryopreserved, and mounted in OCT (Triangle Biochemical Sciences) freezing medium. Serial sagittal sections (15-µm thick) were then collected at ~400 µm intervals spanning an entire hemisphere, allowing for systematic analysis of the cerebellum, upper spinal cord, and brain stem. After blocking in 4% normal goat serum plus 0.01% Triton X-100 (Sigma-Aldrich) in PBS for 1 h at room temperature, the slides were labeled with Abs to TCRV (1/500), GFAP, NSE, CD45, and/or Diablo (all at 1/250) for 1 h at room temperature followed by anti-donkey-Cy5, anti-mouse Cy3, or anti-rabbit Cy5 (at 1/400 for 1 h at room temperature; Chemicon International). Lastly, samples were rinsed with PBS and mounted with a fluorophore stabilizer (Kirkegaard and Perry Laboratories). Controls (not shown) included slides unstained and stained with isotype and species-matched Abs or with secondary Abs alone. Slides were coded and analyzed by a "blinded" reader. Tissues were examined with a Zeiss LSM Pascal laser confocal microscope. Multipass emissions were collected through Cy-3 (bandpass 560–615) and Cy-5 (longpass 650) filters. High-resolution images using the same settings were collected for three separate cerebellar folia, the cerebellar nucleus, and the upper spinal chord of each brain. All acquired images were exported from Zeiss LSM (version 2.8) as full-resolution merged TIFF images for analysis with IMAJIN (www.fda.gov/cber/research/imaging/imageanalysis.html). All images were then batch analyzed and assessed for individual pixel counts. Viral Ag colocalization analysis was confirmed by acquiring Z-stacks of selected areas with the x63 objective.

Quantitative real-time RT-PCR

Total RNA was prepared from individual brains of neonatal mice using TRIzol (Invitrogen Life Technologies) and then purified with RNeasy (Qiagen). For mRNA cytokine detection, total RNA (500 ng/sample) was reverse transcribed into cDNA using an iScript cDNA synthesis Kit (Bio-Rad) as per the manufacturer’s instructions. cDNA samples were treated RNase H (Invitrogen Life Technologies) for 30 min at 37°C and stored at –20°C until used for quantitative RT-PCR.

Quantitative RT-PCR was conducted using the iQ SYBR Green Supermix kit (Bio-Rad) on an iCycler instrument (Bio-Rad) as described (23). Primers used are given in Table I. Values for each target gene were normalized using rat 18S rRNA. Expression values were calculated using the 2–{Delta}{Delta}Ct method (24). To assess the expression of type-1 IFN-inducible genes, total RNA (1 µg/sample) was reverse transcribed into cDNA using the First-Strand cDNA Synthesis Kit (Amersham Biosciences) as per the manufacturer’s instructions. Relative mRNA levels for the Prkr, Irf7, Isgf3{gamma}, Mx2, and Oas1{gamma} were assessed using a mouse multigene-12 RT-PCR profiling kit (SuperArray Bioscience) as per the manufacturer’s instructions. Relative amounts of mRNA for the individual IFN-inducible genes were calculated by first normalizing with the endogenous gene (GADPH) and subsequently calculating the fold increase in respect to the background (control animals) by using Image Gage 4.1 (Fuji Photo Film) software on gel-scanned images.


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  		Table I. Primers used for RT-PCR

 
Cytokine and Ab assays

Cytokine levels were assessed in supernatants (72 h) of splenocytes (5 x 105 cell/well) cultured in R10 medium at 37°C in the presence or absence of heat-killed 105 TCID50 viruses/well. Levels of IFN-{alpha}, IFN-{gamma}, IL-12 p70, and IL-6 were assessed by ELISA as described (25). Briefly, 96-well Immulon 2 HB plates (Thermo Electron) were coated with cytokine-specific Ab and then blocked with PBS plus 1% BSA (Sigma). After washing, the plates were overlaid with the supernatant for 3 h and then washed and treated with the appropriate biotinylated secondary Ab followed by alkaline phosphatase-conjugated avidin (BD Biosciences). Standard curves using recombinant cytokines were generated to quantify the responses. Virus-specific Ab levels were tested in sera using Immulon 1 HB plates coated with UV-irradiated TCRV (at 1/500 in PBS). After blocking, pooled mouse sera (1/50) was added to the wells (2 h) followed by washing and alkaline phosphatase-conjugated Abs. (Southern Biotech Associates). Absorbance was read at 405 nm after 30 min. Pooled sera from surviving adult mice boosted with virus injection i.p. (106 TCID50/mouse) were used as positive control/reference standard. The number of cells secreting cytokines and Abs were determined by ELISPOT as described (26).

Statistical analysis

Differences in survival curves were tested using the Kaplan-Meier method to determine survival fractions and the Mantel-Haenszel log rank test to determine p values. Changes in Ab or cytokine expression were analyzed by parametric or nonparametric ANOVA as appropriate. p values of <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effect of CpG ODN administration on survival of neonatal mice infected with the TCRV

Activation of the innate immune system by administration of CpG ODN or R848 results in the protection from infection in several animal models, leading to their consideration as immunoprotective agents (9, 27, 28). To assess whether R848 and/or CpG ODN can protect neonatal mice from infection, BALB/c mice were infected (i.p.) with TCRV on days 1–3 of life. Untreated mice infected with TCRV developed limb paralysis and died within 18 days of challenge. Treatment with R848 (25 µg/mouse i.p.) did not prevent death; however, 52% of mice treated with CpG ODN (50 µg) i.p. on the day of infection survived into adulthood (Fig. 1A; p < 0.001). The survival rates were similar in mice treated with 25, 50, or 100 µg/mouse (data not shown). The effect was TLR9-dependent, as no protection was evident when mice were treated with ODN lacking an active CpG motif (control ODN; Fig. 1A) or in mice lacking TLR9 expression (Fig. 1B).


Figure 1
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  		FIGURE 1. CpG ODNs (concurrent or 3 days postchallenge) protect neonatal mice from lethal TCRV infection in a TLR9-dependent manner. Neonatal BALB/c (A, C, and D) or TLR9 KO (B) mice (1–3 days old) were infected with TCRV (2000 TCID50 of strain TR11573 in 10 µl) i.p. (A, B, and D) or i.n. (C). A, Infected mice were treated i.p. with CpG ODN, control ODN lacking the CpG motif (50 µg/mouse), or R848 (resiquimod; 25 µg/mouse i.p.) at the time of infection. B, TLR9 KO mice treated with CpG ODN i.p. on the day of the challenge. C, Mice were challenged and treated (i.n.) on the day of the infection. D, Mice treated (i.p.) 3 days after the virus challenge (i.p.). The mice were monitored daily, but TCRV infection was allowed to proceed to its natural outcome. Note the improved survival in mice treated with CpG ODN (i.p. concurrent: ***, p < 0.001(A); i.p. postchallenge: **, p < 0.01 (D); i.n. concurrent: **, p < 0.01(C)) compared with untreated mice. CpG ODN alone did not modify survival of the neonates. Results show data from 3 to 5 experiments. For statistical analysis, the Kaplan-Meier method was used to determine survival fractions, and the Mantel-Haenszel log rank test was used to determine p values. post-inf, postinfection;

 
Because New World arenaviruses are often transmitted to people via the respiratory route, we next assessed whether CpG ODN could also improve the survival of mice infected by this route. Mice infected with the TCRV i.n. died within 15 days of challenge. Intranasal administration of CpG ODN (50 µg) immediately following infection resulted in 36% survival (Fig. 1C; p < 0.01). Although i.p. administration of CpG protected mice from an i.n. challenge (50% survival), i.n. administration of CpG did not protect the mice from an i.p. TCRV infection (not shown).

CpG ODN protects mice from an ongoing Tacaribe infection

Several studies suggest that the optimal time for the administration of CpG ODN varies depending on the pathogen. Empirical data suggest that, in infections with rapidly dividing pathogens, optimal protection is evident when the CpG ODNs are administered 3–6 days before infection (29). In contrast, for infections that have slower kinetics such as leishmaniasis, CpG ODN can induce a protective response even when administered weeks after infection (30). We next assessed the optimal time for CpG ODN treatment of mice infected with the TCRV. BALB/c mice were challenged with the TCRV i.p. on day 4 of life and treated with CpG ODN i.p. on days 1, 4, 7, or 10 of life. Untreated mice served as controls. The protective effect of CpG ODN was best when the treatment was administered at the time of challenge (Fig. 1A). Mice treated 3 days after the challenge (Fig. 1D) were protected to a lesser, but still significant, degree (30% survival; p < 0.01). However, no protection was evident in mice that were treated with CpG ODN either 3 days before or 6 days postchallenge (not shown).

Effect of CpG ODN on intracranial Tacaribe infections

The TCRV is a neurotropic virus. In untreated mice it rapidly migrates to the brain where it replicates. We reasoned that the lack of protection observed when the CpG ODNs were administered >3 days after infection might reflect an inability of the CpG ODN to control the infection once the virus reached the CNS. To test whether the protective effect of CpG ODN was restricted to acting in the periphery, reducing the viral load or even preventing the virus from reaching the brain, neonates were infected i.c. (10 µl/mouse) with TCRV and treated with CpG ODN i.p. Mice infected i.c. died sooner than animals infected i.p. or i.n., as 100% of the mice were dead by day 12 (Fig. 2A). Of note, treatment with CpG ODN i.p. resulted in a 20% survival rate (p < 0.01). The protective effect of CpG ODN was still evident in mice treated 3 days after i.c. infection (23% survival; p < 0.01) (Fig. 2B).


Figure 2
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  		FIGURE 2. CpG ODNs protect neonatal mice from lethal intracranial (i.c.) TCRV infection. BALB/c neonates (1–3 days old) were infected i.c. with TCRV and treated with CpG ODN i.p., either concurrently (A; **, p < 0.01) or 3 days postchallenge (B; **, p < 0.01), as described in the Fig. 1 legend. Control mice received CpG ODN and control ODN lacking the CpG motif either i.p. or i.c. Results show data from 3 to 5 independent experiments. Statistical analysis of survival rates was determined by the Kaplan-Meier/Mantel-Haenszel log rank test. post-inf, postinfection.

 
Effect of CpG ODN on viral load

To examine the mechanism by which CpG ODN increased survival, neonatal mice were infected with TCRV i.p. and then sacrificed at specific time points after infection. Viral loads in spleen and brain were assessed by TCID50 on days 1 (3 h after challenge), 3, 7, and 10 after infection. No live viruses were recovered from spleen or sera. However live viruses were cultured from brain tissue 7 days after infection, regardless of the route of infection. As shown in Fig. 3A, on day 7 untreated mice had viral titers of 106 TCID50. The increased survival observed in the CpG-treated mice was associated with a decreased, yet detectable, viral load (104 TCID50) in the brains of those mice. Importantly, no live virus could be isolated from the brains of CpG-treated mice past weaning age (not shown), indicating that the surviving mice cleared the virus.


Figure 3
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  		FIGURE 3. CpG ODNs decrease the viral load in the brains of TCRV-infected neonatal mice. A, Viral loads in the brains of BALB/c neonates (three per group) were determined by the TCID50 method 3 h and 4, 7, and 10 days post i.p. infection (post-inf). Results are shown as means of viral load ± SD. B, Enumeration of viral Ag levels (pixel counts) in brain sections from i.p. infected TCRV, TCRV plus CpG ODNs (TCRV + CpG), and uninfected control BALB/c neonates (3–4 mice/group) 5 or 10 days postinfection. Pixel counts (mean ± SD of >25–35 images/mouse brain) were performed using IMAJIN software. Statistical analysis was done using Student’s t test comparing TCRV to TCRV plus CpG at each specific time point (A) and one-way ANOVA (B) (*, p < 0.05; **, p < 0.01).

 
To better understand the progression of the infection, direct visualization of TCRV Ags in brain sections was performed by immunohistochemistry using an Ab specific for the viral surface glycoprotein. Viral Ag was detectable by day 5 (Fig. 4, C and D) in the upper spinal chord and cerebellum and was most evident in the subarachnoid space (Fig. 4C). Viral Ag was quantitated using 25–35 images per mouse brain. Similar levels of virus Ags and distribution were evident at day 5 in treated and untreated animals (Figs. 3B and 4, C and D). By day 10, however, untreated animals showed greater levels of virus Ag, whereas those treated with CpG ODN did not (Figs. 3B and 4, E and F). In terms of cell tropism, on day 5 TCRV localized primarily in GFAP+ cells but was also evident in NSE+ neurons and CD45dim microglia (Fig. 4, G–I). CpG ODN treatment did not change the cellular distribution of the TCRV (not shown). Ten days after infection the brains of untreated mice showed clear signs of disorganization of the parenchyma, astrocytosis and apoptosis (Diablo+ cells), whereas those from treated animals showed only mild viral infiltration, and their astrocytes and neurons were Diablo– (Fig. 4, E, F, J, and K).


Figure 4
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  		FIGURE 4. CpG treatment decreases levels of TCRV Ag detectable in infected neonatal mouse brains. Confocal microscopy images of the cerebellum of mice 5 or 10 days postinfection. TCRV Ag is shown in red, and GFAP is shown in green. Yellow denotes colocalization of TCRV with GFAP, indicating that the virus infects astrocytes. A, H&E-stained image of a neonatal (5 days old) mouse brain; boxes indicate the five sites where the virus was quantified. B, Cerebellar folia of age-matched uninfected mouse at day 6. C and D, Analogous sections from day 5 post-TCRV infection in untreated (TCRV) (C) or treated (TCRV plus CpG) mice (D). E and F, Day 10 postinfection from TCRV (E) and TCRV plus CpG (F) mice. G–I, Colocalization of TCRV Ag (red) with GFAP (green) (G), CD45 (blue) (H), or NSE (green) (I) 5 days postinfection using confocal microscopy and IMAJIN software. Colocalization was confirmed by Z-stack analysis. J and K, TCRV Ag (red), GFAP (green) and Diablo (second mitochondria-derived activator of caspase (SMAC); blue) in the brains of untreated (J) and CpG ODN-treated (K) mice 10 days postinfection. Note that Tacaribe-infected astrocytes undergoing apoptosis appear white, whereas other infected apoptotic cell types appear magenta.

 
Role of TNF-{alpha} and IFN-{gamma} in CpG ODN-mediated immunoprotection from Tacaribe meningoencephalitis

CpG ODN-mediated immunoprotection against other intracellular pathogens is mediated by the proinflammatory and type 1 adaptive immune response it fosters (7). Because IFNs are known to inhibit replication of other arenaviruses (31), we next investigated whether CpG ODN treatment enhanced the IFN-{gamma} response to the virus. Spleen cells from treated and untreated mice were collected 4 and 7 days postinfection and restimulated in vitro with heat-inactivated TCRV. As shown in Fig. 5A, 4 days after challenge splenocytes from TCRV-infected mice produced high levels of IFN-{gamma} in response to TCRV Ags regardless of treatment. By day 7 after infection, however, higher IFN-{gamma} levels were produced by splenocytes from untreated mice than from mice treated with CpG ODN (p < 0.05; Fig. 5A).


Figure 5
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  		FIGURE 5. CpG-mediated protection is not associated with increased type 1 cytokine production. A, IFN-{gamma} levels were measured by ELISA in supernatants of splenocytes from infected BALB/c mice collected 4 or 7 days postinfection and restimulated in vitro with 105 TCID50 heat-inactivated TCRV per well for 72 h. Results are means ± SD of two representative experiments. B and C, mRNA levels in brain of infected (i.n.) C57BL6/J mice (2–3 mice/group) 5 and 13 days postinfection (post-Inf.). mRNA levels for IFN-{gamma} and TNF-{alpha} were assessed by real-time quantitative PCR using the iQ SYBR Green/iCycler system. D, mRNA for type1 IFN-inducible genes were assessed using a profiling kit from SuperArray Bioscience (one of five mice tested in two independent experiments). Statistical analysis was done by Student’s t test (*, p < 0.05; **, p < 0.01).

 
Because the in vitro response of splenocytes to a neurotropic virus may not reflect the immune milieu at the site of infection, the expressions of IFN-{gamma}, TNF-{alpha}, and type 1 IFN-inducible genes were assessed in the brain 5 and/or 13 days after challenge. Results indicate that infected mice had significantly increased levels of IFN-{gamma} and TNF-{alpha} mRNA compared with uninfected controls (Fig. 5, B and C). Further, although IFN{alpha} was not readily detectable, mRNA for several type 1 IFN-inducible genes (Prkr, Irf7, Isgf3{gamma}, Mx2, and Oas1{gamma}) were increased 5 days after infection, suggesting that the virus does induce type 1 IFN expression (Fig 5D). Of note, the mRNA levels for mice treated with CpG ODN tended to be lower than those for the untreated ones, particularly at day 13, likely reflecting the comparatively lower viral load. This suggested that the increased survival and reduced viral load of CpG ODN-treated mice were unlikely to be mediated by enhanced type1 cytokine production

Increased virus-specific Ab levels are seen in infected animals that are CpG treated

Previous studies had indicated that Abs play an important role in protection from arenaviruses (18, 32). Because B cells express TLR9 and are activated directly by CpG ODN, we hypothesized that the improved survival rate could result from increased Ag-specific Ab levels. As shown in Fig. 6A, Ag-specific Abs, mainly IgM, were evident 10 days after infection in untreated TCRV-infected mice. In contrast, mice treated with CpG ODN developed high levels of IgM and IgG Ag-specific Abs by day 5 (Fig. 6, A and B). The increased expression of IgG Abs included both IgG1 and IgG2a subisotypes (not shown). Of note, no shift in the ratio of IgG1:IgG2a was evident in treated mice, further supporting the notion that CpG ODN does not induce a Th1 shift in this disease model.


Figure 6
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  		FIGURE 6. CpG-induced protection of newborn mice infected with TCRV is associated with increased virus-specific Abs and requires presence of mature B cells. A and B, IgM (A) and IgG (B) TCRV-specific abs in BALB/c sera were determined by ELISA. Uninfected animals served as controls. Differences in Ab levels were tested by Kruskal-Wallis ANOVA on ranks followed by Dunn’s test. C, Neonatal BALB/c mice challenged with TCRV were treated on days 3 and 8 postinfection (post-inf.) with antisera ({alpha}sera; 30 µl i.p.) from 3-wk-old naive (control {alpha}sera) or convalescent mice that had been left untreated (TCRV {alpha}sera) or treated with CpG ODN (T+C {alpha}sera) at the time of infection. Differences in survival were tested using the Mantel-Haenszel log rank test. Results are pooled data from two independent experiments (*, p < 0.05). D and E, B cell-deficient C57BL/10 µMt KO mice (1–3 days old) were infected (i.p.) and treated as described in the Fig. 1 legend. Results are pooled data from triplicate experiments. E, Viral load in brain was assessed 14 days postinfection. Results are means ± SD of three mice in each of two independent experiments. F, Survival of µMt KO mice treated on days 3 and 8 postinfection with naive (control {alpha}sera) or convalescent sera from untreated (TCRV {alpha}sera) or CpG ODN-treated (T+C {alpha}sera) BALB/c mice. Results are pooled data from two independent experiments (*, p < 0.05).

 
To establish whether early expression of Abs to TCRV was protective, infected newborn mice were treated with sera from convalescent or naive mice on days 3 and 8 postchallenge. As shown in Fig. 6C, the early administration of sera from older mice improved survival. Survival was optimal in mice that received sera from convalescent mice that had been treated with CpG ODN at the time of infection (89% survival; p < 0.05), compared with 56% survival in mice that received convalescent sera from mice not treated with CpG ODN and 29% in those that received sera from age-matched mice never exposed to the virus. Although the mice that received sera from CpG ODN-treated convalescent mice had better survival rates than those that received sera from untreated convalescent mice, the difference did not reach statistical significance.

To establish whether the increase in anti-TCRV Abs in CpG ODN-treated mice was a reflection of the CpG ODN-induced polyclonal B cell activation (33), the number of splenocytes secreting Abs to TCRV was determined by ELISPOT 10 days after infection. Treatment with CpG ODNs increased the number of cells secreting Abs to TCRV by >2-fold (110%), compared with infected mice that did not receive treatment. In contrast, only a 20–30% increase was evident in the number of cells secreting Abs to other specificities (ssDNA) or in the overall number of Ig-secreting cells ex vivo by the same mice, indicating that the administration of CpG ODN at the time of infection selectively increased the number of anti-TCRV-secreting cells (data not shown). These results suggested that CpG ODN promoted an early Ab response to the virus that could play an important role in CpG ODN-mediated protection against the TCRV.

To confirm that the Ab response induced by CpG ODN treatment is required for CpG ODN-mediated immunoprotection, newborn µMT KO mice, which lack developmentally mature B cells, were infected with TCRV (34, 35). These mice died despite CpG ODN treatment but could be rescued when treated with sera from convalescent mice (Fig. 6F) as had been observed in BALB/c mice (Fig. 6C). Of note, however, despite the lack of survival, µMT KO mice treated with CpG ODN tended to live longer and had lower viral loads than untreated controls (Fig. 6, D and E), indicating that the humoral response, although necessary, was likely not the only mechanism responsible for the improved survival mediated by CpG ODN.

NO production by iNOS is required for the immunoprotective effect of CpG ODN

CpG ODNs are known to induce the expression of iNOS in vitro in microglial cells (36), and NO production has been shown to be critical for CpG ODN-mediated protection against Listeria monocytogenes (37). Because previous studies showed that iNOS also plays a major role in the survival of mice infected with the arenavirus Junin (38), we next determined whether iNOS activation plays a role in CpG ODN-mediated protection against the TCRV. BALB/c mice were treated daily with AMG (40 µg/mouse/day), an iNOS blocker. By itself, AMG had no deleterious impact on neonatal health, nor did it modify TCRV viral titers (Fig. 7, A and B). When administered to CpG ODN-treated mice challenged with Tacaribe, however, AMG impeded the rescue of BALB/c mice (Fig. 7A). Unlike µMT KO mice, no reduction in brain viral load was evident in CpG ODN-treated BALB/c mice that received AMG (Fig. 7B). Infection of mice lacking a functional iNOS (iNOS KO) confirmed that iNOS is needed for CpG ODN-mediated protection (Fig. 7C). Further, as with AMG-treated BALB/c mice, CpG ODN treatment did not reduce the viral load in iNOS KO mice. We next assessed whether the lack of protection in iNOS KO mice could be due to a defect in their Ab response to TCRV. As shown in Fig. 7E, 10 days post infection the level of IgM Abs to TCRV in iNOS KO mice was similar to that found in C57BL/6 mice; however, the CpG ODN-induced increase in IgG anti-TCRV Abs was not present, suggesting that iNOS treated mice may have a defect in their CpG ODN-mediated Ab response that was responsible for their lack of clinical effect. We reasoned that, if the lack of survival was secondary to a defect in IgG Abs to TCRV, passive transfer of convalescent sera would rescue these animals. However, unlike BALB/c and µMT KO mice, transfer of convalescent sera did not rescue iNOS KO mice (Fig. 7F), indicating that a functional iNOS, in addition to an enhanced Ab response, is required for survival.


Figure 7
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  		FIGURE 7. CpG-induced protection of newborn mice infected with TCRV requires NO production in vivo. A, Neonatal BALB/c mice were infected on day 1 and treated with CpG ODN alone or with CpG ODN plus daily administration of AMG (40 mg/mouse i.p./day). Control mice included uninfected mice treated with AMG, TCRV-infected mice, and TCRV-infected mice treated with AMG. B, Viral titers (5–6 mice per group) were determined on day 9 postinfection (post-inf.) as in Fig. 3. Results are means ± SD (*, p < 0.05). C, Survival of iNOS-deficient C57BL/6, iNOS KO, or wild-type (WT) mice infected (i.p.) with TCRV or TCRV plus CpG (TCRV + CpG). Results show data from three to five experiments (***, p < 0.001). D, Viral load in the brain of iNOS KO mice 10 days postinfection. Results are means ± SD. E, IgM and IgG Abs to TCRV in sera from B6 wild-type or iNOS KO mice. Results are means ± SEM of three independent experiments (each with three animals per group). F, Survival of iNOS KO-mice treated on days 3 and 8 postinfection with naive or convalescent sera from 7-day-old BALB/c mice left untreated or treated with CpG ODN at the time of the infection (same antisera ({alpha}sera) used in Fig. 6). Results are pooled data from two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Vaccine development remains the most effective and efficient manner to reduce susceptibility to infection by pathogens with high prevalence in the population. However, newly developed immunomodulators provide an alternative approach to limit the spread and pathogenicity of emerging or sporadic pathogens against which vaccine development is not a practical alternative. Accumulating evidence suggests that immunomodulators might be effective even in populations where vaccines have frequently failed, such as the newborn, the elderly or the immunocompromised (39, 40). In this study we report that treatment with CpG ODN protects 20–50% of neonatal mice from a lethal challenge with the neurotropic New World arenavirus Tacaribe. Protection was attained when the mice were treated (i.p. or i.n.) at the time or up to 3 days postchallenge. The protective effect was TLR9 dependent and associated with reduced type-1 IFN-inducible genes, IFN-{gamma} and TNF-{alpha} expression in the brain, and increased virus-specific IgM and IgG Abs in sera. Protection also required the expression of iNOS. To our knowledge, this is the first study assessing the mechanisms by which CpG ODN can protect neonatal mice from an otherwise lethal neurotropic virus infection.

Numerous studies in adult mice had shown that the activation of the innate immune system and the fostering of a strong Th1–type response by CpG ODN leads to reduced susceptibility to pathogens as diverse as Listeria, Francisella, Klebsiella, Anthrax, Mycobacterium, Plasmodium, or Leishmania (30, 41, 42, 43). In addition, recent studies show that systemic administration of CpG ODN improved recovery of mice infected with the Friend leukemia virus and senescent mice infected with the influenza virus (12, 44). Protection in all cases was linked to induction of a Th1 response.

In newborn mice infected with the TCRV, the lethal meningoencephalitis they develop appears to be T cell-mediated as demonstrated by the survival of thymectomized and nudenu/nu mice (45). Further, preliminary studies from our laboratory have shown that mice lacking IFN-{gamma} (IFN-{gamma} KO) or treated with anti-TNF-{alpha} Abs have improved survival (J. Pedras-Vasconcelos and D. Verthelyi, unpublished observations). These findings are in agreement with studies from Riviere et al. (46) showing that the severity of the choriomeningitis in suckling mice correlated with the IFN production. In this context, it was a concern that CpG ODN treatment might be detrimental to the disease progression. However, as shown in Fig. 5, CpG ODN treatment was associated with relatively lower in situ and systemic levels of TNF-{alpha}, IFN-{gamma}, and type 1 IFN-inducible genes compared with untreated infected animals.

Although most of the early studies that assessed the immunoprotective effects of CpG ODN used the i.p. route, recent studies have favored compartmentalized delivery of CpG ODN in the prevention of a localized infection (47). In our studies i.p. administration of CpG ODN achieved better protection than the i.n. route. Moreover, CpG ODN administered i.p. protected mice against i.p., i.n., and even i.c. challenges, whereas the i.n. route did not improve the survival of mice challenged i.p.

Regardless of the route of infection, TCRV circulates at very low concentrations in the periphery and only begins to replicate in earnest when it reaches the spinal chord and the brain between days 4 and 5 (19). Confocal images clearly show that on day 5 postinfection viral Ags are present in astrocytes and neurons and, to a lesser extent, in CD45dim microglia, and appear to be located in the areas adjacent to the meninges. Treated and untreated mice have similar viral loads in the brain with comparable cellular distribution. By day 10, however, the viral infection in the untreated mice has progressed as shown by higher viral load and a change in the distribution of the viral Ag, which can be seen penetrating a disorganized parenchyma. Furthermore, at this time infected and surrounding astrocytes appear engorged, and staining for the cytoplasmic Diablo protein, an early marker for apoptosis, is evident in local astrocytes and neurons (Fig. 6, and data not shown) (48). The similar viral progression in treated and untreated mice early in infection suggests that the antiviral effect does not take place in the periphery by reducing the virus titer that reaches the brain, but rather is the result of the immune response that takes place in the brain.

Previous studies had shown that CpG ODN-activated I-{kappa}B kinase and JNK in astrocytes induce cytokine and chemokine production. Microglial cells treated with CpG ODN up-regulate B7-1, B7-2, and CD40 and secrete increased levels of TNF-{alpha}, IL-12, and NO (15, 16, 49). It is unclear at this time whether in vivo CpG ODN crosses the blood brain barrier to act directly on local glial cells, induces the activation of immune cells that migrate to the brain (although CD45bright cell are not evident until late in the infection), or causes a systemic change in the immune milieu (cytokine, chemokine, NO, etc.) that modifies the local environment or response to the virus.

Our studies suggest that CpG ODN mediates protection by accelerating the development of virus-specific Abs. This hypothesis is in line with the previously reported treatment of hemorrhagic fever by transfer of convalescent sera (45). The mechanism of action of the Abs is not entirely clear. One possibility is that they act in the periphery by reducing the viral load that reaches the brain; however, our studies show similar viral loads in brain on day 4 or 5 after infection in treated and untreated animals. More likely, the Abs are required for viral clearance as described for Sindbis virus (reviewed in Ref.50) and the mouse hepatitis virus (34), two virus models where the Abs help control virus replication in CNS. Of note, sera is known to exert immunoregulatory effects independent from the presence of Ag-specific Abs, such as the Fc receptor-dependent induction of anti-inflammatory cytokines (51, 52). The increased survival of infected wild-type and µMt KO neonatal mice following treatment with sera from naive mice would support this notion (Fig. 6, C and F, respectively).

Although Abs clearly can protect mice from infection, their absence does not accelerate disease. Indeed, µMT KO mice, which cannot mount an Ab response to the virus, do not succumb more rapidly to Tacaribe infections. This finding suggests that in the natural history of Tacaribe infections in wild-type (BALB/c or C57BL/6) neonate mice, the development of Abs occurs too late to modify disease outcome. Therefore, it is only when the development of Abs is accelerated (by CpG ODN treatment or by transfer of immune sera) that the Abs can affect disease outcome.

In addition to accelerating the development of Abs, CpG ODN may induce a qualitative change in the Abs generated. Mice that received convalescent sera from animals treated with CpG ODN at the time of infection consistently tended to have higher survival rates relative to those that received sera from infected but untreated mice. The observation that the IgG and IgM levels of anti-TCRV Abs transferred were similar suggests a "qualitative" difference in the Abs generated in the CpG ODN-treated mice. Although theoretically possible, it is unlikely that the difference in survival is due to the presence of CpG ODN in the sera, because the convalescent sera were obtained 15 days after the administration of CpG ODN to the serum donor. Further studies will be necessary to determine whether mice treated with CpG ODN generate Abs that have higher affinity than untreated ones. Assessment of IgG1 and IgG2a Abs to TCRV did not suggest a shift in the IgG subisotype generated (data not shown).

iNOS KO mice could not be rescued by CpG ODN treatment, and the inhibition of iNOS using AMG in infected BALB/c mice negated the protective effect of CpG-ODN. This finding is in accordance with previous studies showing that iNOS plays an important role in the clearance of several pathogens, including Leishmania and Klebsiella (53), and is required for CpG ODN-mediated protection from Listeria (54). The assessment of Ab levels in iNOS KO mice and AMG-treated mice (not shown) suggested that these mice make lower levels of virus-specific IgG Abs. However, the serum transfer studies show that iNOS KO mice cannot be rescued by exogenous Abs, underscoring the role of iNOS in survival. In differentiating the contribution of iNOS and Abs to the CpG ODN-mediated protection, it is important to underscore that µMT mice have normal levels of iNOS mRNA expression and function (data not shown). Studies by Creon (55) and Karupiah et al. (56) suggested that iNOS inhibits viral replication by S-nitrosylation of cysteine residues in essential viral proteins. An alternative mechanism is suggested by studies in Junin arenavirus and Sindbis alphavirus (57). In both models, inhibition of iNOS did not impact viral load but increased virus-induced pathology (38). In the arenavirus study, decreased mortality was associated with increased astrocytosis, whereas in mice infected with encephalomyelitic Sindbis alphavirus, specific inhibition of iNOS with NG-nitro-L-arginine methyl ester decreased the survival of infected neurons (57). Lastly, NO could act by decreasing the IFN-{gamma} production and indirectly promoting anti-inflammatory responses, as suggested by studies of herpes simplex virus type 1 (58) and the influenza virus (59). Of note, although iNOS expression in infected areas was confirmed by immunohistochemistry, its local expression was only evident 10 days after infection on CD45bright infiltrating monocytes (data not shown). The role of local vs systemic iNOS expression is yet unclear and will be the focus of future studies.

In summary, the studies presented show that activation of the innate immune system via TLR9 in neonates accelerates the host’s Ab response to a neurotropic virus, improving the survival of the challenged animals. Importantly, neonatal exposure to CpG ODN appears to be safe, because no changes in growth rate or adverse events were observed in this or other studies (60). These findings support the development of CpG ODNs as immunoprotective agents in humans.


   Acknowledgments
 
We thank Dr. M. J. Buchmeier for providing the Abs to TCRV, Drs. Dragana Jankovic and Shizuo Akira for access to the µMT and TLR9 KO mice, respectively, Chad Gonsolli for technical assistance, and Dr. E. Romano for advice in the statistical evaluation of the data. We thank Drs. Christian Sauder and Dennis Klinman for careful review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest. The assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Food and Drug Administration.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Daniela Verthelyi, Building 29A, Room 3B19, 8800 Rockville Pike, Bethesda, MD 20892. E-mail address: Verthelyi{at}cber.fda.gov Back

2 Abbreviations used in this paper: ODN, oligodeoxynucleotide; AMG, aminoguanidine; GFAP, glial fibrillary acidic protein; i.c., intracranially; i.n., intranasally; iNOS, inducible NO synthase; KO, knockout; NSE, neuron-specific enolase; TCID50, tissue culture ID50; TCRV, Tacaribe virus. Back

Received for publication April 28, 2005. Accepted for publication February 3, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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