Stimulation with a class A CpG oligonucleotide enhances resistance to infection with feline viruses from five different families
© Robert-Tissot et al.; licensee BioMed Central Ltd. 2012
Received: 26 April 2012
Accepted: 30 July 2012
Published: 20 August 2012
Domestic cats are commonly affected by viral pathogens that induce lengthy infections with fatal outcomes. Prevention of viral propagation is of primordial importance in shelters and catteries, where cats from different backgrounds have narrow contacts. Oligonucleotides (ODN) containing cytosine-phosphate-guanosine motifs of class A (CpG-A) are highly potent synthetic inducers of innate antiviral mechanisms. The aim of this study was to test their ability to modulate innate immune responses and prevent viral replication as stand-alone agents in the domestic cat. CpG-A stimulation of feline peripheral blood mononuclear cells (PBMCs) enhanced their proliferation, increased the presence of co-stimulatory molecules on their surface and influenced their gene expression profiles in an antiviral orientation. Incubation of the supernatants of CpG-A stimulated PBMCs with feline cell lines of epithelial and fibroblastic origin induced expression of the antiviral myxovirus resistance (Mx) gene in these target cells, which also showed enhanced resistance to feline viruses from five distinct families, namely Coronaviridae, Herpesviridae, Caliciviridae, Parvoviridae, and Retroviridae. Most importantly, subcutaneous administration of CpG-A in domestic cats systemically increased the expression of Mx, reaching maximal levels within 24 h. Plasma from treated cats could furthermore inhibit viral replication in vitro. Altogether, our data highlight the promising potential of CpG-A to induce a preventive antiviral state in the cat and to protect feline populations against a broad range of virus infections.
Feline viruses are of particularly opportunistic character. In order to adapt to the solitary way of life of ancestral felids, these pathogens have acquired elaborate means to persist within their host population over the course of time. The infection of new hosts upon the rare contact between individuals was evolutionarily assured by very efficient transmission strategies and the induction of latent, chronic and/or asymptomatic infections. Often kept in multi-cat households and placed in catteries or shelters, today’s domestic cat is consequently particularly susceptible to viral infections[1, 2]. The close proximity and high social contact rate of animals with different vaccination states in these stressful environments further increases the risk of infection. Moreover, the strong antigenic variability of several common feline viruses including the feline calicivirus (FCV), the feline coronavirus (FCoV) and the feline leukemia virus (FeLV) support escape from immune responses and lower the efficacy of currently available vaccines. The availability of complementary prophylactic strategies could help protect pet cats in environments with high infectious pressure from long diseases and/or death caused by infections with fatal viruses.
A promising addition to vaccination is the manipulation of innate immunity. Innate pathogen recognition relies on a set of sensory molecules, the Toll-like receptors (TLRs), which enable the immediate reaction of specific immune cells to pathogen “danger signals”, the so-called pathogen-associated molecular patterns (PAMPs). Due to their abundance in all bacterial as well as some viral genomes, oligodeoxynucleotides (ODN) containing unmethylated cytosine–phosphate–guanosine (CpG) motifs are effectively recognized as PAMPs by the vertebrate innate immune system. Response to CpG ODN stimulation is conferred through TLR9, expressed mainly in the intracellular compartments of human B cells and plasmacytoid dendritic cells (pDCs)[6, 7]. Alarmed TLR9 is the initial instigator of gene expression profiles that strongly support antiviral mechanisms: upregulation of costimulatory molecules major histocompatibility complex (MHC) II, B7.1 and B7.2 on the surface of stimulated cells provides them with a stronger antigen presenting potential[8, 9] and production of cytokines such as type I interferon (IFN), interleukin (IL)-12, IFNγ, IL-6 and tumor necrosis factor (TNF)α, contribute to providing an optimal immune environment for the development of innate and adaptive responses against intracellular pathogens[10, 11]. Probably the most important antiviral property of CpG ODN resides in their potential to stimulate the production of high amounts of type I IFN by pDCs. This family of cytokines, which includes IFNα, IFNω and IFNβ, has been shown to considerably enhance natural killer (NK) cell cytotoxicity, promote differentiation, maturation and immunostimulatory functions of monocytes and DCs, induce B cell production of immunoglobulin and T-helper (Th)1 differentiation of T cells[16, 17]. Moreover, upon binding to their ubiquitously distributed receptor, type I IFNs effectively induce the synthesis of various intracellular proteins which interfere with the replication of a broad range of viruses. The myxovirus-resistance protein (Mx) GTPase is a well-studied example of these intracellular antiviral effectors. This enzyme is known to be directly regulated by the type I IFN, and its detection is readily used as marker for upregulation and biological activity of this cytokine family.
Distinct classes of ODN have been shown to induce differential immune responses. Class A CpG ODN (CpG-A) consist of CpG motifs in a phosphodiester core, flanked on both ends by phosphorothioate poly (G) sequences. CpG ODN of this class are characterized by their potential to both induce massive type I IFN secretion by pDCs and increase NK cytotoxicity, rendering them ideal candidates as prophylactic enhancers of innate immunity to viral infections. Conversely, class B CpG ODN (CpG-B), which encode multiple CpG motifs on a phosphorothioate backbone, promote monocyte maturation and B cell activation, thus substantially supporting the development of humoral immune responses[22–24]. Both CpG-A and CpG-B have indicated immunostimulatory properties in immune cells of mice, primates and many domestic species in vitro[25–31], while in vivo studies in outbred animals have mainly been carried out with CpG-B[24, 32, 33]. Concerning the cat, specifically synthesized CpG-A molecules were recently shown to induce IFNγ; CpG-B molecules indicated the capability to induce proliferation of feline cells, and CpG-B-adjuvanted allergen indicated potential for immunotherapy in a feline asthma model. Although class C[37, 38] and class P ODN were developed in more recent years in an effort to combine the advantageous effects of both CpG-A and CpG-B and increase immunogenicity, CpG-A remain the strongest inducers of type I IFN described to date.
The potential of CpG ODN as prophylactic stand-alone inducers of innate defense mechanisms has been the subject of only few studies. Most works in this field initially described protection of mice against bacterial[40–44] and parasitic[45–47] infections. More recently, induction of resistance to viral infections was shown also in mouse models for Herpes Simplex Virus, neurotropic arenavirus, foot and mouth disease virus and Vaccinia virus. With exception of the latter, all these studies were carried out with CpG-B. In an outbred species, partial antiviral protection has only been described in two studies so far, in which reduced shedding of herpes and parainfluenza viruses was observed in lambs after administration also of a CpG-B[52, 53]. To our knowledge, prophylactic antiviral potential of CpG-A has not yet been described in outbred animals.
We carried out a series of experiments with the objective to characterize both the immunomodulatory and antiviral properties of CpG-A in the domestic cat. In vitro, the prototype of CpG-A, ODN 2216, strengthened the antiviral qualities of feline immune cells and stimulated their production of soluble molecules that inhibited the replication of five different families of viruses: Coronaviridae, Herpesviridae, Caliciviridae, Parvoviridae, and Retroviridae. In vivo, ODN 2216 induced a systemic antiviral state.
Materials and methods
Animals and ethics statement
Fourteen male castrated SPF cats divided in four different age groups were used in this study: group 1 (c01-c04, 10 weeks), group 2 (c05-c08, 1.5 years), group 3 (c09-c12, 7 years) and group 4 (c13 and c14, 14 years). Cats c13 and c14 from group 4 originated from the same litter; all other individuals were not related to each other. The in vivo experiments were conducted when cats c05-c08 were 3 years of age. All animals were purchased from Liberty Research Inc. (Waverly, NY, USA) and their SPF status was verified as previously described. This study was carried out in strict accordance with regulations of the Swiss law for animal protection (SR 445.1). The Veterinary Office of the Swiss Canton of Zurich officially revised the protocol and approved the study (Permit no. TVB 99/2007 and TVB 100/2007). The animals were housed in groups in an animal-friendly barrier facility under optimal ethological conditions. For blood collections and injections, the cats were sedated with a combination of ketamin and midazolam, and all possible efforts were made to minimize stress and suffering.
Feline PBMC isolation, cell lines, ODNs, cell culture and cell viability assay
Feline PBMCs were isolated from EDTA-anticoagulated whole blood by Ficoll-Hypaque density gradient centrifugation using a standard protocol. Purified cells were counted prior to their utilization in the different experiments using the Sysmex XT 2000iV (Sysmex, Norderstedt, Germany) as described previously, and cultured in RPMI 1640 with Glutamax I (Gibco®, Invitrogen, Basel, Switzerland). Adherent CRFK (ATCC no. CCL-94) and FEA cells were maintained in RPMI 1640 with Glutamax I, while adherent fcwf-4 cells (ATCC no. CRL-2787) were cultured in EMEM (ATCC 30–2003). All media were supplemented with 10% heat-inactivated fetal calf serum (Bioconcept, Allschwil, Switzerland), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco®, Invitrogen).
ODN 2216 and ODN 2243 were obtained from Alexis biochemicals, Enzo Life Sciences AG, Switzerland for in vitro studies and ODN 2216 was synthesized by Microsynth AG, Balgach, Switzerland for the in vivo experiment. Recombinant feline IFNα (rfeIFNα) was obtained from PBL Biomedical, Piscataway, New Jersey, USA. All molecules were solubilized in endotoxin-free PBS. ODN 2243 consists of the same sequence as ODN 2216, with CpG motifs inversed to GpC. For in vitro experiments, both ODNs and rfeIFNα were diluted in RPMI 1640 with Glutamax I supplemented as described above.
Viability of stimulated cells was compared using the trypan blue exclusion test. Briefly, after stimulation for 24 h with increasing concentrations of ODN 2216, ODN 2243 or equivalent volumes of PBS as control, cells were stained with a 0.4% trypan blue solution (Dr Bender and Dr Hobein AG, Zurich, Switzerland) and percentages of viable cells were compared.
PBMCs were treated at a density of 3 × 106 cells/mL with 4 μg/mL ODN 2216 or ODN 2243 or an equivalent volume of endotoxin-free PBS and cultured for 24 h in a 12-well format. During collection of the cells, the adherent cell fraction was removed with 0.05% trypsin-EDTA (Gibco®, Invitrogen). Harvested cells were divided into 3 fractions labeled separately with either anti-feline B7.1 mouse monoclonal IgG (kindly provided by Prof Mary Tompkins, Flow Cytometry and Cell Sorting Laboratory, NC State College of Veterinary Medicine, USA), anti-feline MHCII mouse monoclonal IgG1 (Department of Pathology, Microbiology and Immunology, University of California, Davis, USA) or fluoresceinisothiocyanate (FITC)-conjugated mouse IgG1 as isotype control (BD Bioscience, Allschwil, Swizerland). The fractions were subsequently stained with R-Phycoerythrin (RPE)-conjugated goat anti-mouse IgG1 (BioConcept, Allschwil, Swizerland). Fluorescence data was obtained using the FACSCalibur® instrument (Becton Dickinson, Allschwil, Switzerland) and the CellQuestPro™ software. Gates representing lymphocyte and non-lymphocyte populations were set on the basis of forward versus side scatter, and a total of 50 000 events were acquired in the non-lymphocyte gate. Data was analyzed with the FlowJo software (Tree Star, Olten, Switzerland), whereby an additional gate was set comprising both lymphocyte and non-lymphocyte populations (PBMC gate). MHCII and B7.1 expression levels were determined as mean of fluorescence intensity for each gated cell population. Identical gates were set for all cats in such a way that they comprise the desired cell populations of each animal.
Relative gene expression analysis
PBMCs were stimulated with ODN 2216, ODN 2243 or endotoxin-free PBS at a density of 3 × 106 cells/mL directly after isolation, while CrFK, FEA or fcwf-4 cells were cultured to confluency prior to stimulation. Experiments were carried out in a 96-well format. For stimulation of adherent cells with supernatants (production see below), total cell culture medium was discarded from the wells and the monolayers were further cultured in 100 μL undiluted supernatants for the rest of the experiment. At time points relevant to each experiment, the supernatants were removed and cells were lysed with mRNA lysis buffer (mRNA isolation kit I, Roche Diagnostics, Rotkreuz, Switzerland). mRNA extractions were performed with the mRNA Isolation Kit I and MagNA Pure LC Instrument (Roche Diagnostics) and first strand cDNA was synthesized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Rotkreuz, Switzerland). Real-time quantitative PCR (qPCR) reactions consisted of 5 μL cDNA in a total volume of 25 μL per reaction using the TaqMan® Fast Universal PCR Master Mix (Applied Biosystems). Thermocycling conditions included an initial denaturation of 20 s at 95°C followed by 45 cycles of amplification by melting at 95°C for 3 s and annealing at 60°C for 45 s. Primers and probes for feline genes have been previously described. mRNA expression factors of selected genes, which correspond to ratios of mRNA levels measured in ODN 2216 or ODN 2243 stimulated versus PBS stimulated cells, were calculated and normalized with GeNorm version 3.5, using either both feline β-glucuronidase (GUSB) and tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) (usually) or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) alone (when specified) as reference genes, under conditions validated for the feline species. Generally depicted in the graphs are mean expression factors calculated from duplicate experiments carried out simultaneously. Where results of one cat are illustrated, experiments were conducted with cells of at least 3 individual animals and representative data is shown.
Production of supernatants
For each cat, PBMCs were resuspended in supplemented RPMI 1640 with Glutamax I (Gibco®, Invitrogen) at a concentration of 106 cells/mL in 6-well plates and stimulated immediately after isolation with 4 μg/mL ODN 2216, 4 μg/mL ODN 2243 or an equivalent volume of endotoxin-free PBS. After 24 h incubation, supernatants were harvested by centrifugation of the cultures twice at 2000 × g for 10 min, aliquoted and stored at −20°C. Large supernatant quantities were produced with PBMCs purified after one blood collection, enabling the utilization of the same supernatants for virus inhibition experiments concerning VSV, FCV, FCoV, FHV and FPV. New supernatant batches were produced for use in FeLV inhibition assays. Supernatants derived from PBMCs stimulated with ODN 2216, ODN 2243 and endotoxin-free PBS are referred to as Sup2216, Sup 2243 and Sup Neg in the text and in the figures.
CrFK and fcwf-4 grown to confluency in 12-well plates were stimulated with 600 μL of PBMC supernatants produced as explained above. At the time points indicated, cells were harvested and counted. 106 cells of either cell line were resuspended in 30 μL sample buffer (0.5 M Tris(hydroxymethyl)aminomethane, 5% SDS, 10% β-mercaptoethanol, 40% glycerol, and 0.05% bromphenol blue) and boiled at 95°C for 5 min. SDS-PAGE separation and submersed immunoblotting procedures were carried out as previously described. The Spectra Multicolor Broad Range Protein Ladder (Fermentas GmbH, St. Leon-Rot, Germany) served as molecular weight standard marker for each blot. For protein visualization, membranes were first cut immediately below the 80kB marker band. The top and bottom membrane fractions were incubated with murine anti-human Mx MAb M143 (generously provided by Dr J. Pavlovic, Institute for Virology, University of Zürich, Switzerland) and murine anti-β-actin monoclonal antibody as a loading control (Sigma Aldrich GMbH, Buchs, Switzerland) respectively. Both fractions were subsequently incubated with a peroxidase-labelled goat anti-mouse IgG (Jackson Immunoresearch, Newmarket, Suffolk, UK). Bands were digitalized using the Chemigenius 2 BioImaging System (Syngene, Cambridge, UK).
Viruses and viral inhibition assays
FeLV-A Glasgow-1 strain (a generous gift from Prof. M. Hosie and O. Jarret, University of Glasgow, Great Britain) was titrated on FEA cells, and the lowest stock dilution leading to productive infection of the cells after 48 h was used for inhibition assays. Experiments were carried out in 96-well plates and cells were treated with 100 μL of supernatants or relevant controls immediately prior to inoculation. Every second day thereafter, 50 μL culture medium was replaced by the same volume of fresh supernatant. At appropriate time points, cells and supernatants were harvested and total nucleic acid was extracted from both the cells and supernatants using the MagNA Pure LC DNA Isolation Kit I and MagNA Pure LC Instrument (Roche Diagnostics). Viral replication in supernatants and proviral loads in cells were measured by real-time RT-PCR and real-time PCR respectively, with assays previously described. The time course experiments were conducted with supernatants derived from PBMCs of three cats and the measurements on day 8 post inoculation were carried out with material derived from two additional cats. In order to facilitate interpretation of the figures illustrating measurements of viral RNA loads, 45 cycles-cycle threshold (Ct) values were calculated and means of duplicate wells are depicted.
In vivo experiment
Prototype CpG-A, ODN 2216, induce proliferation of primary feline immune cells and enhance their expression of costimulatory surface molecules
Proliferation of PBMCs in response to treatment with CpG ODN gives strong indications about the biological activity of the stimulatory molecule and has been used to screen ODN in many species. In relation to this, the potential of CpG-A to induce proliferation of feline PBMCs were assessed by measurement of 3 H-thymidine incorporation after stimulation. Although considerable variability was observed between individuals, a 2-fold increase in proliferation was observed after stimulation with ODN 2216 when compared to stimulation with inactive control ODN 2243 (p = 0.0281, Figure1A). The cells of three cats (c08, c09, c12) could be stimulated to particularly high proliferative rates by ODN 2216 (values above the mean of eight cats illustrated in Figure1A) in the following order: c08 > c12 > c09.
Another characteristic feature of stimulatory ODN is their ability to enhance interactions between various immune cell populations by upregulation of cell surface costimulatory molecules. With the objective to test whether ODN 2216 could exert such properties in feline immune cells, the expression of B7.1 and MHCII was measured by flow cytometry in stimulated PBMCs of the same eight cats as above. The expression of both co-stimulatory molecules was evaluated in gates defined to contain a PBMC population, a lymphocyte population and a non-lymphocyte population of cells. The observed effects varied considerably between the cells of individual cats, ranging from no alterations to an increase of 400% stained cells in some cellular subpopulations after ODN 2216 stimulation. Also, in some animals, stimulation with the control ODN 2243 indicated similar staining patterns as PBS, whereas in other cats the induction of effects comparable to those of ODN 2216 could be observed. The response of cells originating from a particular cat to stimulation with ODN 2243 did not however correlate with the influence of ODN 2216 on the expression of either surface molecule on these same cells. Overall, an increased expression of both B7.1 and MHCII was measured in gated PBMCs upon a 24 h stimulation with ODN 2216 when overlaid with the expression of these molecules after ODN 2243 or PBS stimulation (Figure1B and C). Considering all eight cats, lymphocytic cells expressed significantly higher levels of B7.1 and MHCII when exposed to ODN 2216 than after treatment with PBS (p = 0.0195 and p = 0.039 respectively, Figure1D and F). Feline MHCII expression on lymphocytes also seemed to be affected by ODN 2216 and ODN 2243 in a similar manner (Figure1F). For non-lymphocytic cells, ODN 2216 more specifically affected the expression of B7.1 molecules, inducing significant upregulation of surface levels of this molecule when compared to stimulation with ODN 2243 (p = 0.0391) and PBS (p = 0.0039) for 24 h (Figure1E). In contrast, the expression of MHCII was not significantly altered in cells gated as non-lymphocytes at this time point after stimulation (Figure1G). Finally, the cells of the three cats (c08, c09 and c12) that exhibited the highest proliferation rates in response to ODN 2216 (Figure1A) also indicated the strongest increase in expression of both cell surface molecules in all cellular subpopulations tested.
ODN 2216 influence type I IFN and proinflammatory gene expression in primary feline immune cells
ODN 2216 broadly influence the gene expression profile in primary feline immune cells of adult cats
In an effort to assess both the breadth of the effects conferred by treatment with a CpG-A and possible variability in the responses obtained in individual cats, the mRNA expression of ten genes relevant to early immune responses was measured in ODN 2216 stimulated PBMCs of fourteen cats divided in four different age groups (group 1: 10 weeks (n = 4), group 2: 1.5 years (n = 4), group 3: 7 years (n = 4), group 4: 14 years (n = 2)). Only slight individual variability in gene induction was observed after 24 h stimulation of immune cells from adult cats ranging between 1.5 and 14 years of age (Figure2B-D). Overall, the mRNA expression profile measured in stimulated PBMCs of these cats corroborated their induction of strong antiviral immune responses. The expression of type I IFN mRNA, including IFNα, IFNβ and IFNω was substantially increased in the immune cells subjected to ODN 2216 stimulation from every adult cat, with minimal inductions of 490, 60 and 1600-fold respectively observed in the older animals of group 4 (Figure2D). Moreover, all individual IFNα subtypes tested were induced at similar levels in the cells of four adult cats from group 2 (Figure2E). Increased levels of proinflammatory cytokine mRNA were also measured in most individuals of groups 2–4, with IL-6 more systematically increased than TNFα. The cells from these cats indicated a typical Th1 orientation after stimulation, with enhanced transcription of IL-12 in 9/10 and IFNγ in 6/10 animals, together with absent or only low induction of IL-4. Stimulation of PBMCs with ODN 2216 also created an optimal environment for NK cell activity, as indicated by the increases in mRNA expression of the NK cell stimulator IL-15 by up to 20-fold and of the NK cell effector Granzyme B by up to 7-fold. The cells of those cats (c08, c09, c12) that had most effectively proliferated (Figure1A) and/or exhibited the strongest expression of co-stimulatory molecules (Figure1B-E) following ODN 2216 stimulation also consistently expressed the highest mRNA levels of IFNα, IFNω, IL-6, Il-12, IL-15 and Granzyme B. PBMCs of cat c08 (group 2, Figure1B) were also by far most responsive to stimulation with ODN 2216. When cells from kittens of group 1 were stimulated, higher individual variability was observed than in adult animals (Figure2A). The PBMCs of only 2 out of 4 cats from this group could be stimulated with ODN 2216 to increase mRNA expression of the tested genes. In both individuals, not only the mRNA expression of type I IFN genes was enhanced at much lower levels than observed in adult cats but the overall immune response favored a Th2 direction, characterized by upregulated IL-4 and downregulated IL-12 mRNA levels (Figure2A).
In order to determine whether a discrepancy in expression of TLR9 between the PBMCs of adult animals and kittens could play a role in these observations, mRNA levels of this gene were measured in immune cells of each cat. Although basal TLR9 expression was similar in the PBMCs of the cats of all age groups (Figure2F), ODN 2216 stimulation increased TLR9 transcription in the cells of kittens, but decreased transcription of this gene in the cells of adult cats so that differences in the mRNA levels of this receptor were significantly higher in young cats (group 1) than in adult cats of groups 2 and 3 (p = 0.0286) (Figure2G).
ODN 2216 induce the production of soluble molecules that activate intracellular antiviral mechanisms in feline target cells
ODN 2216 inhibit replication of common feline viruses in vitro
Means of viral inhibition rates measured in fcwf-4 cells after treatment with supernatants derived from stimulated PBMCs of 8 adult cats
The differential inhibition of the viruses tested by the same supernatants is reflected by the distinct viral inhibition ratios observed (Figure5B-E and Table1). Sensitivity of each virus to rfeIFNα correlated with sensitivity to the Sup 2216 and induction of Mx in target cells by the supernatants highly correlated with the inhibition of all viruses (Figure5F-I). Finally, Sup 2216 derived from PBMCs of cats c08, whose cells had indicated strong responsiveness to stimulation with ODN 2216 in previous experiments, most efficiently inhibited the replication of all viruses.
Similar results were notably obtained when the supernatants of PBMCs derived from all cats were incubated with CrFK cells prior to their inoculation with all the above-mentioned viruses (data not shown). This cell line has previously indicated less sensitivity to the antiviral effects of type I IFN and generally 10-fold higher amounts of rfeIFNα were found in titration experiments to be required for the inhibition of all five viruses. Concordantly, average fold viral inhibition in CrFK cells by the Sup 2216 was approximately half that observed in fcwf-4 cells.
ODN 2216 inhibits replication of a retrovirus in vitro
ODN 2216 induces an antiviral state in the domestic cat in vivo
In the present study, we conducted both in vitro and in vivo experiments that characterize immunomodulatory and antiviral effects of a CpG-A molecule in the domestic cat. We show that ODN 2216, the first described CpG-A, can upregulate the expression of a series of genes in feline immune cells that play important roles in early responses to viruses. Soluble molecules produced by feline PBMCs upon ODN 2216 stimulation significantly increased resistance of various feline target cell lines to propagation of viruses from five different families, namely FCV, FPV, FHV, FCoV and FeLV. The observed repression of viral replication highly correlated with the mRNA expression of type I IFN genes in stimulated PBMCs as well as the induction, prior to inoculation, of antiviral mechanisms in the target cells. Furthermore, a single administration of ODN 2216 significantly increased the expression of Mx in the blood of treated cats. Plasma from these animals could also inhibit replication of FCV in vitro. Our data underline the prophylactic potential of ODN 2216 in an outbred species as a stand-alone agent and against a large range of viral pathogens simultaneously. Cats may highly benefit from such a molecule when placed in environments with strong infectious pressure such as catteries, shelters or pet shows.
Cells of the feline immune system were strongly influenced when cultured in the presence of ODN 2216. First, feline PBMCs significantly proliferated in presence of ODN 2216, indicating a direct and /or indirect stimulation of one or more immune cell subpopulations by this molecule. Although in contrast to other classes of ODN CpG-A does not generally induce lymphocyte proliferation in mice, similar effects have previously been observed in ovine cells, where higher concentrations of CpG-A could induce minimal levels of proliferation. Additionally, ODN 2216 increased the expression of cell surface co-stimulatory molecules in PBMCs, reinforcing possible interactions between various immune cell populations in subsequent specific responses. While B7.1 molecules were upregulated on both lymphocytic and non-lymphocytic cells after ODN 2216 stimulation, MHCII expression could only be increased on lymphocytic cells. This observation may be linked to the time point of our measurements, or to the necessity of transport to the cell surface of MHCII molecules coupled with antigen for presentation to other immune cells. Finally, the transcription of a series of markers of innate immune responses was considerably influenced in feline PBMCs by stimulation with ODN 2216. The most astonishing effects of this molecule thereby remain the potent induction of IFNα and IFNω, with mRNA expression of these genes increased by up to 12 000 and 35 000-fold respectively in PBMCs of certain animals. In line with observations published recently, the induction of IFNγ by this CpG ODN remained moderate; nevertheless, the measurements carried out 24 h after stimulation of purified feline cells support induction of Th1-oriented immune responses and enhanced NK cell activity, both highly desired in the contexts of viral infection.
Variability in the immunomodulatory and antiviral effects of ODN 2216 on feline cells was mainly related to the age of the cats. Among middle-aged adult cats, consistency in the stimulatory potential of this molecule was reflected through the highly similar patterns in gene expression profiles induced in immune cells after stimulation, as well as through the moderate deviations in those experiments aiming to characterize the immunomodulatory properties of ODN 2216 (Figure1). The cells of several animals however, in particular cat c08, but also c09 and c12, indicated particularly strong responsiveness to stimulation throughout the study. Such observations are most likely linked to the genetic variability of individuals of an outbred species, as inherited factors are known to play an important role in the magnitude of innate immune responses. With respect to this observation, it should be noted that the SPF origin and maintenance in a barrier facility of the animals included in this study may slightly lower the variability in strength and breadth of innate immune responses, and studies with cells obtained from field cats would give further information on divergence in response to stimulation of the innate immune cells of individual animals. The immune cells of cats of 14 years of age seemed to respond slightly less well to ODN 2216 than those of younger adult animals, while PBMCs of kittens indicated much more reticence to stimulation, with either limited or absent upregulation of both type I IFN and other genes measured after incubation with ODN 2216. Stimulated PBMCs from this group of young animals moreover indicated a tendency to develop an immunologic environment with a Th2 orientation, including higher production of IL-4 and impaired induction of IL-12 compared to cells from adult cats. Although the kittens included in this study were already of 10 weeks of age, these observations strongly corroborate the immature IFN system of neonatal mice, the impaired immune cell activation via TLR9 in human neonatal mononuclear cells[71, 72] and the bias towards Th2 rather than Th1 responses in human fetuses and neonates[73, 74]. Furthermore, concordantly to findings in human neonatal blood[75, 76], basal TLR9 transcription in kittens indicated levels similar to those of adults. However, ODN 2216 stimulation increased mRNA expression of this gene only in the young animals (Figure2C). These results open new perspectives on possible explanations for the qualitative discrepancy between innate immune responses in newborns and adults. The availability of specific antibodies to feline TLR9 would greatly support further investigations in this direction.
The experiments conducted both in vitro and in vivo in this study underline the feasibility of utilizing Mx as a marker for induction of resistance to viral infections in the domestic cat. Admittedly, both the supernatants of PBMCs stimulated with CpG-A and serum of treated cats contain a mixture of molecules that could play a role in the observed antiviral effects. However, the viral inhibition in our in vitro assays was similar to that obtained after treatment of the cells with rfeIFNα and highly correlated with the induction of Mx transcription in the cell lines incubated with the supernatants. After ODN 2216 injection in vivo, Mx expression in plasma also strongly correlated with its antiviral potential. It is however important to note that Mx mRNA and protein cannot be mechanistically linked to inhibition of the viruses tested. Although the expression of other antiviral molecules such as 2'5'oligoadenylate synthetase (OAS) and the RNA-dependent protein kinase (PKR) was not measured, their induction has been reported following CpG ODN stimulation of PBMCs in other species[77, 78] and differential interplay between several effector mechanisms most likely leads to the inhibition of individual viruses.
In comparison to ODN of other groups, CpG-A have been used in much fewer studies concerning innate antiviral responses. This is most likely the result of the more time consuming and costly manufacturing process of these molecules linked to the synthesis of the flanking poly (G) strings necessary for optimal immune effects. Also, CpG-A exhibit only weak stimulation of B cells, which produce antibodies that are valuable in later antiviral responses. Stability of CpG-A in vivo has also been questioned due to the only partial phosphorothioate backbone of these molecules. This can be partially compensated however, by the formation, through association of their poly (G) stretches, of highly ordered G-tetrad structures with enhanced stability. Their smaller content in synthetic backbones could moreover help reduce possible long-term side effects[79, 80]. Most importantly, CpG-A remain the most potent inducers of type I IFN, themselves the most biologically active antiviral molecules known to date. With regard to this, a recombinant feline type I IFN marketed in both Japan (Intercat®) and Europe (Virbagen Omega®) has made its way into therapeutic protocols for FCV, FHV, FeLV and canine parvovirus infections[67, 81–86] and has demonstrated preventive capacities in a cattery developing an outbreak of FPV. However, when compared to direct initiation of antiviral mechanisms by a recombinant IFNα protein, administration of CpG ODN holds the advantage of inducing the production of all type I IFN and their subtypes, which have been shown to possess differential antiviral properties and kinetics. Our data demonstrate that five viral species belonging to the Calicivirus, Herpesvirus, Parvovirus, Coronavirus and Retrovirus families were highly sensitive to the immunologic effects of ODN 2216 in feline cells. Strength and breadth of antiviral defense mechanisms induced in vivo by this molecule have the potential to outweigh those observed with recombinant IFN.
In frame with the results of our in vitro experiments, the administration of ODN 2216 in vivo could promote an antiviral state in the domestic cat in the absence of adverse reactions. Just as stimulated PBMCs exhibited high mRNA expression of type I IFN and only a marginal increase in mRNA levels of the proinflammatory cytokines IL-6 and TNFα, treated cats showed significantly increased Mx expression in their blood and lacked flu-like symptoms generally linked with administration of immunostimulatory molecules in vivo. Additionally, although our in vivo studies included a limited number of cats, the strength of responses to ODN 2216 stimulation in vitro and in vivo for individual animals strongly correlated, indicating that individual sensitivity to stimulation most likely can be predicted with in vitro experiments. Our data further show that a single subcutaneous injection of ODN 2216 sufficed to induce the systemic presence of antiviral molecules for 24 to 36 h in the plasma of treated cats; during this time, an increase of Mx expression of at least 4-fold in the blood of treated cats was indicative of significant inhibition of viral replication in vitro. These observations support possible resistance to viral infection by ODN 2216 for several days, a phenomenon described already in mouse models.
Altogether, this study underlines the strong potential for ODN 2216 in the prevention of a large variety of viral diseases. CpG-A molecules should thus not be left aside when stimulating innate immunity for prophylactic purposes. Future studies should aim at optimizing the kinetics of these molecules in vivo, while addressing safety, stability and manufacturing issues. Combinations with adjuvants or other TLR agonists as well as optimized administration protocols should support research in this direction. Field studies with larger cohorts of animals will provide further insights on the utilization of CpG-A molecules for the prevention of viral infections in a clinical setting.
This project was supported by the ProMedica Foundation, Chur, Switzerland as well as by the Research Commission and the Young Academic Support Committee of the University of Zürich.
The authors would like to thank Prof. Thomas Lutz from University of Zürich, Switzerland for access to facilities enabling work with radioactive isotopes and acknowledge the excellent technical assistance offered by Theres Meili-Prodan and Beat Grenacher. Sincere thanks are also addressed to Anouk Robert-Tissot for professional support in the generation of the figures. The laboratory work was performed using the logistics of the Center for Clinical Studies at the Vetsuisse Faculty of the University of Zurich.
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