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International Immunology Advance Access originally published online on February 7, 2007
International Immunology 2007 19(3):297-304; doi:10.1093/intimm/dxl146
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Immunostimulatory RNA is a potent inducer of antigen-specific cytotoxic and humoral immune response in vivo

Svetlana Hamm1, Antje Heit1, Martina Koffler1, Katharina M. Huster1, Shizuo Akira2, Dirk H. Busch1, Herrmann Wagner1 and Stefan Bauer3

1 Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Trogerstrasse 30, 81675 München, Germany
2 Akira Innate Immunity Project, ERATO, Japan Science and Technology Corporation (JST), Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
3 Institut für Immunologie, Philipps-Universität Marburg, Hans-Meerwein-Strasse 2, 35042 Marburg, Germany

Correspondence to: S. Bauer; E-mail: stefan.bauer{at}staff.uni-marburg.de


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Single-stranded RNA stimulates immune cells and induces the secretion of pro-inflammatory cytokines and type I IFN. As adjuvant RNA can induce a Th2 type of humoral response, however, its potency in the induction of cytotoxic T cells in vivo has not been analyzed. Here we show that immunization with the antigen ovalbumin (OVA) and the adjuvant phosphodiester RNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) induced a Toll-like receptor-7-dependent cytotoxic T cell and humoral response. Staining with SIINFEKL-Kb tetramers demonstrated the induction of antigen-specific T cells that were functional in in vivo cytotoxic T cell assays against SIINFEKL-loaded target cells. In infection experiments with OVA-secreting Listeria monocytogenes, the cytotoxic T cell response strongly reduced the bacterial load in liver and spleen. The RNA-driven humoral response was characterized by OVA-specific antibodies of the IgG1 isotype whereas CpG-DNA induced antigen-specific antibodies of the IgG2a (BALB/c) or IgG2c (C57BL/6) isotype. Furthermore, stimulation with RNA did not induce splenomegaly, a common feature of CpG-DNA-driven immune activation in mice. Taken together, our data confirm that RNA can be used as a safe adjuvant and induces a strong antibody response of the IgG1 isotype. Additionally, we demonstrate that RNA induces an antigen-specific immunity characterized by a potent cytotoxic T cell response to infection.

Keywords: IgG isotype, innate immunity, ovalbumin, Toll-like receptors, vaccination


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The innate immune system recognizes pathogens by means of germ line-encoded pattern recognition receptors (PRRs). PRRs sense various conserved molecular structures of pathogens that are not found in vertebrates (1, 2). A subfamily of PRRs, the Toll-like receptors (TLRs), is important for initiation of an immune response. So far, 13 members (TLR1–13) have been reported that recognize structures from different pathogenic origin such as bacteria, viruses, fungi or protozoan parasites [reviewed in (3)]. One group of TLRs consisting of TLR1, 2, 4, 5, 6 and 11 is located on the cell surface and is responsible for detection of external pathogen structures like lipopeptides (TLR1, 2 and 6) (4, 5), LPS (TLR2 and 4) (6–10), flagellin (TLR5) (11) and profilin (TLR11) (12). The other group is formed by TLR3, 7, 8 and 9, which are localized inside the cell in the endoplasmic reticulum and endosomal–lysosomal compartment where they recognize bacterial or viral nucleic acid. Double-stranded RNA (dsRNA) acts as a viral-associated recognition motif for TLR3 (13), whereas DNA with non-methylated CpG motifs is a pattern for TLR9 (14–16). TLR7 in mice and TLR7 and TLR8 in humans have recently been shown to recognize single-stranded viral and synthetic RNA molecules that are rich in guanosine and/or uridine, provided the RNA is formulated within cationic lipids (17–19).

Stimulation of TLR results in the activation of signaling pathways via MyD88 or TRIF leading to up-regulation of co-stimulatory molecules on antigen-presenting cells (APCs) and production of pro-inflammatory and/or anti-viral cytokines (20). In general, the TLR-mediated signaling via the adaptor molecule MyD88 which is shared by all TLRs except TLR3 induces an antigen-specific Th1 response. Accordingly, in MyD88-deficient mice, antigen-driven immune responses are of a Th2 type suggesting that TLR signals play an important role in directing the immune response towards Th1 (20, 21). Nevertheless, it has been reported that some TLR ligands such as the lipopeptide Pam3Cys induce a predominant Th2 immune response via TLR2 and lead to aggravation of asthma (22).

Due to the Th1-promoting immune response, TLR agonists have been heavily explored as immunotherapeutics and vaccine adjuvant (23–28). CpG-DNA has proven to be one of the strongest Th1 immune response-inducing adjuvant known in vivo (27). With regard to RNA, immune activation has been demonstrated by various groups (17–19,29, 30). It has recently been reported that naked synthetic phosphorothioate-modified RNA oligonucleotides and ß-galactosidase (29) or micro-particles containing polyuridylic acid and ovalbumin (OVA) (30) induce immune stimulation and specific antibody production. The antibody isotype was primarily of the IgG1 isotype suggesting that RNA in vivo induces a Th2-biased immune response (29, 30). However, induction of a cytotoxic immune response and protection against infection has not been analyzed in detail.

Here we show that injection of phosphodiester RNA and OVA induced OVA-specific cytotoxicity and antibody responses in a TLR7-dependent manner. In comparison with CpG-DNA, immunostimulatory RNA induced lower frequencies of cytotoxic T cells, but these were active in vivo and significantly reduced the bacterial load after Listeria monocytogenes infection. As shown previously, RNA did not induce splenomegaly which is in contrast to the effect of CpG-DNA (29, 31). Further, we observed and confirmed that RNA induced antibodies of the IgG1 isotype indicating a Th2 type of immune response. Overall, our data suggest that RNA is a weaker but safer adjuvant than CpG-DNA and induces an antigen-specific immunity characterized by a potent cytotoxic T cell response.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
C57BL/6 and BALB/c mice were purchased from Harlan (Borchen, Germany). TLR7–/– mice have been described (32). All animals were kept under specific pathogen-free conditions and only female mice were used for the experiments at 8–12 weeks of age.

Reagents
Chicken egg albumin (OVA) was obtained from Sigma–Aldrich (Taufkirchen, Germany). The dominant CD8 T cell epitope of OVA, the peptide SIINFEKL (OVA peptide 257–264), was custom synthesized by Research Genetics (Huntsville, AL, USA). Phosphorothioate-modified CpG-DNA oligonucleotide 1668 with the sequence 5'-TCCATGACGTTCCTGATGCT was synthesized by TIB-MOLBIOL (Berlin, Germany). Cell culture grade RNA oligonucleotide RNA40 with the sequence 5'-GCCCGUCUGUUGUGUGACUC was synthesized by CureVac GmbH (Tübingen, Germany) as phosphodiester RNA with a single thiolated modification on the 3' end. The cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP, abbreviated as DO in figures) was purchased from Roche Diagnostics GmbH (Mannheim, Germany).

Injection of mice
Mice were injected subcutaneously with 100 µl liposomal buffer alone (20 mM HEPES, 150 mM NaCl, pH 7.4), with 200 µg OVA and 30 µg DOTAP in buffer, with 200 µg OVA, 30 µg DOTAP and 100 µg RNA40 in buffer or with 200 µg OVA and 10 nM ODN 1668 in buffer. The injection scheme is presented in Fig. 1(A).


Figure 1
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  		Fig. 1. RNA as adjuvant induces a significant cytotoxic immune response. C57BL/6 mice were injected twice with 3-week interval as indicated (A). Seven days after the second injection, the amount of SIINFEKL-specific, CD8-positive CD62Llow T cells in blood of immunized mice was determined (B). Spleen cells were stimulated with SIINFEKL and CD8-positive T cells were analyzed for cytokine production (C). The cytotoxic capacity of SIINFEKL-specific T cells was assessed by in vivo cytotoxicity (D) and infection experiments with 4 x 105 recombinant Listeria monocytogenes secreting OVA (E). Statistical analysis was performed using a Mann–Whitney test. (C and D). One representative experiment of at least two independent experiments is shown. DOTAP: DO.

 
Ex vivo tetramer staining of primed SIINFEKL-specific, CD8-positive CD62Llow T cells
Seven days after the second injection, ~150 µl blood was analyzed for the presence of SIINFEKL tetramer binding by CD8-positive CD62Llow T cells as described (33). In short, blood cells were depleted of erythrocytes, followed by a staining with 1 µg ml–1 ethidium monoazide (EMA) (Molecular Probes, Karlsruhe, Germany) for 30 min at 4°C for a life/death discrimination. Subsequently, a triple staining with anti-CD8 allophycocyanin (clone CD8{alpha}, Caltag, Hamburg, Germany), anti-CD62L FITC (clone MEL-14, BD Biosciences, Heidelberg, Germany) and MHC SIINFEKL tetramer PE [H-2Kb/SIINFEKL (OVA 257–264)/murine ß2-microglobulin/streptavidin–PE] was performed for 45 min at 4°C. Additionally, an Fc block (CD16/CD32, 2.4G2, BD Biosciences) was used to avoid unspecific antibody binding. At least 1 x 105 CD8-positive events were analyzed on a FACSCalibur flow cytometer.

5,6-Carboxyfluorescein diacetate succinimidyl ester-based in vivo cytotoxicity assay (in vivo kill)
Syngeneic spleen cells (3 x 107 to 4 x 107) per immunized mouse were washed with FCS-free RPMI 1640 medium (PAA, Coelbe, Austria), divided into two groups and stained with 0.5 or 5 mM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) at 37°C. The staining was stopped by adding complete medium. The cells were washed twice with complete medium, and the 5-mM CFSE group was incubated with 1 µM SIINFEKL peptide for 30 min at 37°C. Subsequently, both cell groups were washed three times with PBS, counted and mixed at a 1:1 ratio. The cell mixture was injected into the tail vein of the immunized mice, and after 20 h, blood and spleen were prepared, depleted of erythrocytes and analyzed for CFSE-stained cells by FACS. Percentage of in vivo kill is calculated by using the following formula: % in vivo kill = [(number of unloaded cells (0.5 mM CFSE) – number of SIINFEKL-loaded cells (5 mM CFSE))/number of unloaded cells] x 100.

Intracellular cytokine staining
For the intracellular cytokine staining, BD Cytofix/Cytoperm Plus kit with BD Golgi Plug (BD PharMingen, Heidelberg, Germany) was used. Ten days after the second injection, spleens were removed and depleted of erythrocytes. From each spleen, 107 cells were stimulated for 5 h with 1 µM SIINFEKL. Simultaneously, Golgi Plug was added to block the secretion of newly synthesized cytokines. After the incubation, cells were first stained with 1 µg ml–1 EMA (Molecular Probes) for 30 min at 4°C for a life/death discrimination. Then an extracellular staining with CD8–allophycocyanin/CD62L–PE (clone CD8{alpha}, Caltag/clone MEL-14, BD Biosciences) for 30 min at 4°C followed. Subsequently, cells were permeabilized and fixed according to the manufacturer’s instructions, followed by a staining with anti-IFN-{gamma}–FITC antibody, anti-tumor necrosis factor-{alpha} (TNF-{alpha})–FITC antibody or an FITC-conjugated isotype control antibody (dilution 1:500 for 30 min at 4°C) (all from BD Biosciences).

Antibody ELISA
For antibody detection, serum was obtained from blood of immunized mice 1 week after the second injection. ELISA plates (Nunc, MaxiSorp) were coated overnight with 0.1 mg ml–1 OVA. Sera were diluted 1:300 000 and for detection the following antibodies were used: biotin-SP-conjugated AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch, Hamburg, Germany), biotin anti-mouse IgG1, biotin anti-mouse IgG2a and biotin anti-mouse IgG2ab (IgG2ab equals IgG2c) (BD PharMingen). A streptavidin–HRP (Amersham Biosciences) was used according to the manufacturer's instructions.

Listeria monocytogenes infection
Five weeks after the second injection, immunized mice were injected in the tail vein with 4 x 105 recombinant L. monocytogenes secreting OVA (L. monocytogenes-OVA, kindly provided by H. Shen (University of Pennsylvania School of Medicine, Philadelphia, PA, USA) (34). Listeria monocytogenes were grown in brain–heart infusion (BHI) medium (Oxoid GmbH, Wesel, Germany) to the density of OD600 = 0.1, which equals 108 bacteria ml–1 for this strain. The amount of injected bacteria was controlled by plating the bacteria on BHI plates and colony numbers were determined after overnight incubation at 37°C. Three days after the infection, spleen and liver were removed, homogenized, lysed in 0.1% Triton solution and plated on BHI plates in three different dilutions as follows: 1:5000, 1:50 000 and 1:500 000. The plates were incubated at 37°C overnight and colonies were counted.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Phosphodiester RNA as adjuvant induces a potent cytotoxic immune response
To characterize the adjuvant function of RNA, we immunized mice with chicken OVA and RNA complexed to the cationic lipid DOTAP (DO). After a boost injection, we analyzed the induction of SIINFEKL-specific CD8+ T cells, their cytokine profile and assessed their cytotoxic activity in vivo by infection experiments (Fig. 1A). Injections with DOTAP/OVA containing no RNA (OVA + DO) or solely buffer (control) served as negative controls. Immunizations with CpG-DNA 1668 were performed in parallel to compare the adjuvant capacity of RNA.

One week after the second injection, ~4.7% of all CD8-positive cells in the blood of RNA-immunized mice were activated (CD62Llow) SIINFEKL-specific T cells (Fig. 1B). This number was significantly higher than the amount of SIINFEKL-specific cells in both negative control groups (P < 0.05). CpG-DNA is a strong inducer of a cytotoxic T cell response (27) and a high percentage of CD8-positive cells (24%) were SIINFEKL specific. The antigen-specific cytotoxic T cells in RNA-treated and CpG-DNA-treated mice produced IFN-{gamma} and TNF-{alpha} after stimulation with SIINFEKL peptide indicating their functionality (Fig. 1C). The cytotoxic potential of the CD8+ T cells was tested using CFSE-stained SIINFEKL peptide-loaded syngeneic cells that were injected into immunized mice. On average, 20% of SIINFEKL-loaded cells were killed in RNA-treated mice 20 h after injection of the cells, whereas no cytotoxicity could be observed in both negative control groups (Fig. 1D) (P < 0.01). With CpG-DNA as adjuvant, almost all SIINFEKL peptide-loaded cells were eliminated.

Next, we analyzed the adjuvant ability of RNA in inducing immune protection against L. monocytogenes infection. Listeria monocytogenes is an intracellular pathogen whose elimination requires a Th1 immune reaction. CpG-DNA, as a very strong Th1 adjuvant, provides an optimal protection against L. monocytogenes (35–37). Five weeks after the second injection, immunized mice were infected with 4 x 104 OVA-secreting bacteria, an amount corresponding ~2x LD50. Three days after infection, spleen and liver of infected mice were analyzed for bacterial load. RNA-driven vaccination reduced the bacterial load in these organs by 90% whereas in both control groups no protective effect was observed (Fig. 1E). Similar results were obtained by injections with 1 x 104 Listeria (data not shown). In comparison, CpG-treated animals cleared the Listeria infection efficiently.

Overall, immunizations utilizing RNA induce a strong cytotoxic response leading to the reduction of SIINFEKL-presenting cells in vivo and the reduction of bacterial burden in a L. monocytogenes infection model.

Phosphodiester RNA as adjuvant induces OVA-specific antibodies of Th2 type
To investigate the humoral immune response induced by the RNA–OVA mixture, we analyzed the production of OVA-specific antibodies in the sera of immunized mice 1 week after the second injection (Fig. 1A). C57BL/6 and BALB/c mice treated with RNA or CpG-DNA as adjuvant produced higher amounts of OVA-specific IgG antibody than control mice although injections of OVA/DOTAP induced some OVA-specific IgG (Fig. 2). As anticipated, CpG-DNA induced mainly antibodies of the IgG2c isotype in C57BL/6 mice [IgG2c substitutes the IgG2a isotype in C57BL/6 mice (38)] or IgG2a isotype in BALB/c mice which is the result of a Th1-driven immune response. It is of note that the BALB/c mouse strain tends to react towards infection with a Th2 response, a circumstance that can explain the relatively high amount of IgG1, the isotype typically associated with a Th2 immune response, in the sera of CpG-DNA-treated BALB/c mice. Interestingly, RNA predominantly induced IgG1 antibodies in both mouse strains.


Figure 2
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  		Fig. 2. RNA induces OVA-specific antibodies of Th2 type. One week after the second injection, the presence and isotypes of OVA-specific antibodies were analyzed in the sera of immunized C57BL/6 (A) (n = 12) and BALB/c (B) (n = 6) mice. One representative experiment of at least two independent experiments is shown. Statistical analysis was performed using a Mann–Whitney test.

 
Overall, vaccination with RNA and CpG-DNA as adjuvant induces comparable amounts of OVA-specific IgG antibodies, the various composition of antibody isotypes, however, suggests that RNA in contrast to CpG-DNA does not induce a Th1-based humoral immune response.

The adjuvant function of phosphodiester RNA is TLR7 dependent
Single-stranded RNA (ssRNA) stimulates the immune system through TLR7 in mice and through TLR 7 and 8 in humans (17–19). To investigate, if the adjuvant activity of ssRNA is TLR7 dependent in vivo, we immunized TLR7-deficient C57BL/6 mice with RNA–OVA and control mixtures. CpG-DNA as adjuvant induced a high number of SIINFEKL-specific CD62LlowCD8+ T cells and an efficient cytotoxicity against SIINFEKL-loaded syngeneic cells in TLR7–/– mice (Fig. 3A). In contrast, ssRNA did not induce SIINFEKL-specific cytotoxic cells and subsequently did not lead to SIINFEKL-specific cytotoxicity in these mice. Similar result could be observed for the humoral response against OVA. Whereas CpG-DNA induced the production of OVA-specific antibodies in TLR7-deficient mice, RNA failed to induce OVA-specific antibodies (Fig. 3B). Thus, the presence of TLR7 seems to be necessary for ssRNA-driven adjuvant function.


Figure 3
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  		Fig. 3. The adjuvant function of RNA is TLR7 dependent. TLR7-deficient and wild-type mice were immunized and 1 week after the second injection percentage of SIINFEKEL-specific CD8+ T cells and their cytotoxic potential in vivo was analyzed (A) (n = 3 for OVA + DO and OVA + CpG-DNA and n = 6 for OVA + DO + RNA). Similarly, the presence and isotypes of OVA-specific antibodies were analyzed in the sera of immunized mice (B). One representative experiment of at least two independent experiments is shown.

 
Immunostimulatory phosphodiester RNA does not induce splenomegaly
Although CpG-DNA is a strong Th1-promoting adjuvant, side effects such as splenomegaly and alteration of mouse lymphoid morphology have been reported and may jeopardize its application (31, 39). We analyzed the spleen weight of CpG-DNA- and RNA-treated mice 1 week after the second injection and observed that CpG-DNA-treated mice had a 50% larger spleen compared with mice from negative control groups (Fig. 4). Interestingly, mice injected twice with RNA as adjuvant showed no noticeable changes in the spleen weight, demonstrating that phosphodiester RNA does not induce a strong systemic immune response.


Figure 4
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  		Fig. 4. RNA does not induce splenomegaly. To detect spleen enlargement, spleens of immunized mice (n = 9 in each group) were weighed 1 week after the second injection. Control groups were injected with PBS. Statistical analysis was performed using a Mann–Whitney test.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The innate immune system senses nucleic acid via TLRs and leads to the stimulation of APCs. TLR3, TLR7, TLR8 and TLR9 are located within the endosomal compartment and recognize ssRNA (TLR7/8), dsRNA/mRNA (TLR3) and ssCpG-DNA (TLR9) (3). Overall, nucleic acids such as CpG-DNA, ssRNA and mRNA are characterized by a strong immunostimulatory capacity and therefore are good candidates for a new class of adjuvant (18, 29, 30, 40–42). Accordingly, CpG-DNA induces the production of Th1-biased cytokines like IL-12 and IFN-{alpha} in vitro (27) and has been proven as adjuvant in tumor therapy and infection models (35, 43, 44). Currently, human trials for the use of CpG-DNA in tumor therapy are ongoing and seem promising (45). Concerning RNA, the induction of cytokine in vivo and in vitro has been reported, although the potential in inducing a cytotoxic T cell response in vivo and protection during infection has not been addressed in detail (29, 30, 46). Scheel et al. (29) demonstrate that naked RNA oligonucleotides and ß-galactosidase induce immune stimulation and antigen-specific antibodies of the IgG1 isotype. While Westwood et al. (30) confirm immune stimulation and specific antibody production by injecting antigen (OVA) and RNA (polyuridylic acid), the activity of the vaccine strictly relies on complexation within micro-particles.

Here we show that OVA and ssRNA complexed to the cationic lipid DOTAP induce OVA-specific cytotoxic T cells that produce IFN-{gamma} and TNF-{alpha}. These antigen-specific cytotoxic T cells were active in in vivo killing SIINFEKL-loaded syngeneic cells and lead to protection against a L. monocytogenes (expressing OVA) infection. Concerning the humoral response, we observed that RNA does not induce a typical Th1 Ig isotype profile as reported for CpG-DNA. Accordingly, ssRNA did not induce OVA-specific IgG2c in C57BL/6 [IgG2a is absent in C57BL/6, (38)] and IgG2a in BALB/c mice, but promoted a predominantly IgG1-driven humoral response. Although we did not detect OVA-specific IgE in undiluted blood samples (data not shown), our observation is in concordance with recent reports demonstrating that RNA was a potent inducer of antigen-specific antibodies of the IgG1 isotype that is also characteristic for a Th2-type immune response (29, 30). The adjuvant effect of ssRNA is entirely mediated by TLR7 since TLR7-deficient mice show no induction of OVA-specific cytotoxic T cells and antibodies. Overall, the antigen-specific immunity induced by RNA in vivo via TLR7 seems to be characterized by a potent cytotoxic T cell response to infection and a strong antibody response reflecting a Th2 immune response. The Th2 phenotype in regard to antibody isotype is surprising since TLR7-driven signaling strictly relies on MyD88 which is essential for a Th1-driven immune response (21). It is possible that signal strength may influence the outcome of the type of immune response. Since we used phosphodiester RNA molecules that have a relatively short half-life in vivo, it can be envisaged that the ligand is only available for a short time limiting the duration of cellular activation. Accordingly, it has been reported that low concentrations of the synthetic TLR2 ligand Pam3Cys induce a predominant Th2-biased immune response with high levels of IL-13 and IgG1 and aggravation of asthma (22). As well Porphyromonas gingivalis LPS, which signals through TLR2 (6), induces a Th2 response characterized by significant levels of IL-13, IL-5 and IL-10, but lower levels of IFN-{gamma} (26). Therefore, evidence accumulates that TLRs influence Th2-driven immune responses that may be driven by low-level signaling via MyD88-dependent or -independent mechanism.

Repeated injection with CpG-DNA can lead to adverse effects through over-stimulation of the immune system (31, 39). In our experiment, we observed a spleen enlargement in CpG-DNA-treated mice but not in animals treated with RNA as adjuvant which confirms the finding by Scheel et al. (29). Therefore, RNA seems to have lesser side effects than CpG-DNA in mice although its adjuvant capacity is weaker compared with CpG-DNA. Of note, this conclusion does not necessarily apply to humans since the expression pattern of TLR7, TLR8 and TLR9 varies in both species. In humans, TLR7 and TLR8 are expressed on various APCs, whereas TLR9 is basically only expressed on B cells and plasmacytoid dendritic cells (47, 48). In contrast, TLR8 is presumably non-functional in mice and TLR9 has a much broader expression pattern compared with humans (15, 49). Therefore, it is possible that ssRNA is a stronger adjuvant in humans than in mice and may even be superior to CpG-DNA in humans.

One disadvantage of RNA used as adjuvant may be the necessity of complexation to cationic lipids for efficient immune stimulation. Although Scheel et al. (29) report that naked phosphorothioate-modified RNA oligonucleotides induces immune stimulation in vivo, Westwood et al. (30) demonstrate that encapsulation of RNA and antigen is critical for immune stimulation. Of note, the RNA utilized in our study and by Westwood et al. (30) was phosphodiester RNA which presumably needs protection due to the short half-life. Naked phosphorothioate-modified RNA may circumvent the need for complexation and future developments could focus on RNA modifications that further enhance half-life and RNA uptake allowing the use of lower RNA concentrations for immunostimulation.

Overall, we have confirmed that RNA is a potent adjuvant in vivo. Our study extends recent work on RNA as adjuvant (29, 30) by demonstrating that RNA induces cytotoxic T cells in a TLR7-dependent manner. The cytotoxic T cell response is potent and reduces bacterial load in an infection model with the intracellular pathogen L. monocytogenes. Future investigation will focus on using ssRNA for tumor therapy and adjuvant application in humans.


   Acknowledgements
 
We would like to thank Philipp Yu for critically reading the manuscript. This work was supported by Sonderforschungsbereich 576 (S.B. and D.H.B.) and Transregio-SFB22 (S.B.). K.M.H. is supported by Hochschul- und Wissenschaftsprogramm II.


   Abbreviations
 
APC, antigen-presenting cell
BHI, brain–heart infusion
CFSE, 5,6-carboxyfluorescein diacetate succinimidyl ester
DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate
dsRNA, double-stranded RNA
EMA, ethidium monoazide
OVA, ovalbumin
PRR, pattern recognition receptor
ssRNA, single-stranded RNA
TLR, Toll-like receptor
TNF-{alpha}, tumor necrosis factor-{alpha}

   Notes
 
Transmitting editor: R. Medzhitov

Received 6 October 2006, accepted 21 December 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Medzhitov R and Janeway C Jr. (2000) Innate immune recognition: mechanisms and pathways. Immunol. Rev. 173:89.[CrossRef][Web of Science][Medline]
  2. Medzhitov R and Janeway C Jr. (2000) Innate immunity. N. Engl. J. Med. 343:338.[Free Full Text]
  3. Takeda K and Akira S. (2005) Toll-like receptors in innate immunity. Int. Immunol. 17:1.[Abstract/Free Full Text]
  4. Takeuchi O, Kawai T, Muhlradt PF, et al. (2001) Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int. Immunol. 13:933.[Abstract/Free Full Text]
  5. Takeuchi O, Sato S, Horiuchi T, et al. (2002) Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J. Immunol. 169:10.[Abstract/Free Full Text]
  6. Hirschfeld M, Weis JJ, Toshchakov V, et al. (2001) Signaling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect. Immun. 69:1477.[Abstract/Free Full Text]
  7. Hoshino K, Takeuchi O, Kawai T, et al. (1999) Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162:3749.[Abstract/Free Full Text]
  8. Poltorak A, He X, Smirnova I, et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085.[Abstract/Free Full Text]
  9. Qureshi ST, Lariviere L, Leveque G, et al. (1999) Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J. Exp. Med. 189:615.[Abstract/Free Full Text]
  10. Werts C, Tapping RI, Mathison JC, et al. (2001) Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism. Nat. Immunol. 2:346.[CrossRef][Web of Science][Medline]
  11. Hayashi F, Smith KD, Ozinsky A, et al. (2001) The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410:1099.[CrossRef][Medline]
  12. Yarovinsky F, Zhang D, Andersen JF, et al. (2005) TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science 308:1626.[Abstract/Free Full Text]
  13. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. (2001) Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413:732.[CrossRef][Medline]
  14. Bauer S, Kirschning CJ, Hacker H, et al. (2001) Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl Acad. Sci. USA 98:9237.[Abstract/Free Full Text]
  15. Hemmi H, Takeuchi O, Kawai T, et al. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408:740.[CrossRef][Medline]
  16. Takeshita F, Leifer CA, Gursel I, et al. (2001) Cutting edge: role of Toll-like receptor 9 in CpG DNA-induced activation of human cells. J. Immunol. 167:3555.[Abstract/Free Full Text]
  17. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. (2004) Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303:1529.[Abstract/Free Full Text]
  18. Heil F, Hemmi H, Hochrein H, et al. (2004) Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303:1526.[Abstract/Free Full Text]
  19. Lund JM, Alexopoulou L, Sato A, et al. (2004) Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc. Natl Acad. Sci. USA 101:5598.[Abstract/Free Full Text]
  20. O'Neill LA. (2006) How Toll-like receptors signal: what we know and what we don't know. Curr. Opin. Immunol. 18:3.[CrossRef][Web of Science][Medline]
  21. Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov R. (2001) Toll-like receptors control activation of adaptive immune responses. Nat. Immunol. 2:947.[CrossRef][Web of Science][Medline]
  22. Redecke V, Hacker H, Datta SK, et al. (2004) Cutting edge: activation of Toll-like receptor 2 induces a Th2 immune response and promotes experimental asthma. J. Immunol. 172:2739.[Abstract/Free Full Text]
  23. Lee SE, Kim SY, Jeong BC, et al. (2006) A bacterial flagellin, Vibrio vulnificus FlaB, has a strong mucosal adjuvant activity to induce protective immunity. Infect. Immun. 74:694.[Abstract/Free Full Text]
  24. Ulrich JT and Myers KR. (1995) Monophosphoryl lipid A as an adjuvant. Past experiences and new directions. Pharm. Biotechnol. 6:495.[Medline]
  25. Gustafson GL and Rhodes MJ. (1992) Bacterial cell wall products as adjuvants: early interferon gamma as a marker for adjuvants that enhance protective immunity. Res. Immunol. 143:483.[CrossRef][Web of Science][Medline]
  26. Pulendran B, Kumar P, Cutler CW, Mohamadzadeh M, Van Dyke T, Banchereau J. (2001) Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo. J. Immunol. 167:5067.[Abstract/Free Full Text]
  27. Krieg AM. (2002) CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol. 20:709.[CrossRef][Web of Science][Medline]
  28. Manetti R, Annunziato F, Tomasevic L, et al. (1995) Polyinosinic acid: polycytidylic acid promotes T helper type 1-specific immune responses by stimulating macrophage production of interferon-alpha and interleukin-12. Eur. J. Immunol. 25:2656.[Web of Science][Medline]
  29. Scheel B, Braedel S, Probst J, et al. (2004) Immunostimulating capacities of stabilized RNA molecules. Eur. J. Immunol. 34:537.[CrossRef][Web of Science][Medline]
  30. Westwood A, Elvin SJ, Healey GD, Williamson ED, Eyles JE. (2006) Immunological responses after immunisation of mice with microparticles containing antigen and single stranded RNA (polyuridylic acid). Vaccine 24:1736.[CrossRef][Web of Science][Medline]
  31. Sparwasser T, Hultner L, Koch ES, Luz A, Lipford GB, Wagner H. (1999) Immunostimulatory CpG-oligodeoxynucleotides cause extramedullary murine hemopoiesis. J. Immunol. 162:2368.[Abstract/Free Full Text]
  32. Hemmi H, Kaisho T, Takeuchi O, et al. (2002) Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 3:196.[CrossRef][Web of Science][Medline]
  33. Busch DH, Pilip IM, Vijh S, Pamer EG. (1998) Coordinate regulation of complex T cell populations responding to bacterial infection. Immunity 8:353.[CrossRef][Web of Science][Medline]
  34. Dudani R, Chapdelaine Y, Faassen Hv H, et al. (2002) Multiple mechanisms compensate to enhance tumor-protective CD8(+) T cell response in the long-term despite poor CD8(+) T cell priming initially: comparison between an acute versus a chronic intracellular bacterium expressing a model antigen. J. Immunol. 168:5737.[Abstract/Free Full Text]
  35. Elkins KL, Rhinehart-Jones TR, Stibitz S, Conover JS, Klinman DM. (1999) Bacterial DNA containing CpG motifs stimulates lymphocyte-dependent protection of mice against lethal infection with intracellular bacteria. J. Immunol. 162:2291.[Abstract/Free Full Text]
  36. Klinman DM, Conover J, Coban C. (1999) Repeated administration of synthetic oligodeoxynucleotides expressing CpG motifs provides long-term protection against bacterial infection. Infect. Immun. 67:5658.[Abstract/Free Full Text]
  37. Heit A, Schmitz F, O'Keeffe M, et al. (2005) Protective CD8 T cell immunity triggered by CpG-protein conjugates competes with the efficacy of live vaccines. J. Immunol. 174:4373.[Abstract/Free Full Text]
  38. Martin RM, Brady JL, Lew AM. (1998) The need for IgG2c specific antiserum when isotyping antibodies from C57BL/6 and NOD mice. J. Immunol. Methods 212:187.[CrossRef][Web of Science][Medline]
  39. Heikenwalder M, Polymenidou M, Junt T, et al. (2004) Lymphoid follicle destruction and immunosuppression after repeated CpG oligodeoxynucleotide administration. Nat. Med. 10:187.[CrossRef][Web of Science][Medline]
  40. Krieg AM. (2004) Antitumor applications of stimulating toll-like receptor 9 with CpG oligodeoxynucleotides. Curr. Oncol. Rep. 6:88.[Medline]
  41. Klinman DM. (2006) Adjuvant activity of CpG oligodeoxynucleotides. Int. Rev. Immunol. 25:135.[CrossRef][Web of Science][Medline]
  42. Pascolo S. (2006) Vaccination with messenger RNA. Methods Mol. Med. 127:23.[Medline]
  43. Link BK, Ballas ZK, Weisdorf D, et al. (2006) Oligodeoxynucleotide CpG 7909 delivered as intravenous infusion demonstrates immunologic modulation in patients with previously treated non-hodgkin lymphoma. J. Immunother. 29:558.
  44. Bourquin C, Schreiber S, Beck S, Hartmann G, Endres S. (2006) Immunotherapy with dendritic cells and CpG oligonucleotides can be combined with chemotherapy without loss of efficacy in a mouse model of colon cancer. Int. J. Cancer 118:2790.[CrossRef][Web of Science][Medline]
  45. Mason KA, Ariga H, Neal R, et al. (2005) Targeting toll-like receptor 9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy. Clin. Cancer Res. 11:361.[Abstract/Free Full Text]
  46. Scheel B, Teufel R, Probst J, et al. (2005) Toll-like receptor-dependent activation of several human blood cell types by protamine-condensed mRNA. Eur. J. Immunol. 35:1557.[CrossRef][Web of Science][Medline]
  47. Gorden KB, Gorski KS, Gibson SJ, et al. (2005) Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J. Immunol. 174:1259.[Abstract/Free Full Text]
  48. Hornung V, Rothenfusser S, Britsch S, et al. (2002) Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168:4531.[Abstract/Free Full Text]
  49. Jurk M, Heil F, Vollmer J, et al. (2002) Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat. Immunol. 3:499.[CrossRef][Web of Science][Medline]

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T. Sugiyama, K. Hoshino, M. Saito, T. Yano, I. Sasaki, C. Yamazaki, S. Akira, and T. Kaisho
Immunoadjuvant effects of polyadenylic:polyuridylic acids through TLR3 and TLR7
Int. Immunol., January 1, 2008; 20(1): 1 - 9.
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