JOURNAL OF VIROLOGY, Nov. 2010, p. 12093–12098 0022-538X/10/$12.00 doi:10.1128/JVI.00406-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
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Stimulation of Human Dendritic Cells by Wild-Type and M Protein Mutant Vesicular Stomatitis Viruses Engineered To Express Bacterial Flagellin䌤 Maryam Ahmed,1* Shelby Puckett,1 Subhashini Arimilli,2 Cassandra L. Braxton,1 Steven B. Mizel,2 and Douglas S. Lyles1 Departments of Biochemistry1 and Microbiology and Immunology,2 Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157 Received 24 February 2010/Accepted 19 August 2010
Vesicular stomatitis viruses (VSVs) containing wild-type (wt) or mutant matrix (M) proteins are being developed as candidate vaccine vectors due to their ability to induce innate and adaptive immunity. Viruses with wt M protein, such as recombinant wild-type (rwt) virus, stimulate maturation of dendritic cells (DC) through Toll-like receptor 7 (TLR7) and its adaptor molecule MyD88. However, M protein mutant viruses, such as rM51R-M virus, stimulate both TLR7-positive and TLR7-negative DC subsets. The goal of this study was to determine whether the ability of rwt and rM51R-M viruses to induce maturation of human DC can be enhanced by engineering these vectors to express bacterial flagellin. Flagellin expressed from the rwt virus genome partially protected human DC from VSV-induced shutoff of host protein synthesis and promoted the production of interleukin 6 (IL-6) and IL-1. In addition, DC infected with rwt virus expressing flagellin were more effective at stimulating gamma interferon (IFN-␥) production from CD8ⴙ allogeneic T cells than DC infected with rwt virus. Although rM51R-M virus effectively stimulated human DC, flagellin expressed from the rM51R-M virus genome enhanced the production of cytokines. Furthermore, mice immunized with both rwt and rM51R-M viruses expressing flagellin had enhanced anti-VSV antibody responses in vivo. Therefore, rwt and rM51R-M viruses expressing flagellin may be promising vectors for the delivery of foreign antigen due to their potential to stimulate DC function. Live attenuated vesicular stomatitis virus (VSV)-based vectors expressing foreign antigens are currently being developed as vaccines due to their abilities to induce potent immune responses and protect against lethal challenge with a variety of different pathogens (15, 22, 31–33). Such vectors are attenuated for virus replication by deletions or mutations in the viral glycoprotein (G protein) (12, 17) and/or rearrangements in the VSV gene order (12, 30). An alternative strategy to decrease VSV pathogenicity is to use matrix (M) protein mutant vectors, such as rM51R-M virus, which are defective in their ability to suppress host innate immunity (5). We have previously shown that rM51R-M virus does not cause disease in mice and induces an antibody response similar to that in mice infected with the wild-type (wt) virus (4). Furthermore, the effectiveness of M protein mutant viruses as vaccine vectors was recently demonstrated using a vaccinia virus challenge model (10). The ability of VSV vectors to function as effective inducers of immune responses is likely due to their ability to induce maturation of dendritic cells (DC) (6, 11, 24). DC are cells of the innate immune system that are key players in the activation of T cells and B cells (reviewed in reference 18). Recently, it has been shown that the potency of viral vectors for inducing maturation of DC can be enhanced by engineering these vectors to express bacterial flagellin (8). Flagellin acts as a potent
* Corresponding author. Mailing address: Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Phone: (336) 716-1589. Fax: (336) 716-7176. E-mail:
[email protected]. 䌤 Published ahead of print on 15 September 2010.
adjuvant by stimulating DC and other immune cells (20, 28). Flagellin is detected extracellularly by Toll-like receptor 5 (TLR5). In addition to being detected by TLR5, flagellin is also detected intracellularly by the Nod-like receptor (NRL) family member, ICE protease-activating factor (IPAF; also known as CARD12 or NLRC4) (9). The goal of the experiments presented here was to determine whether expression of flagellin would enhance the ability of VSV to induce maturation of human DC. We also considered that the effects of flagellin expression may differ depending on whether the VSV vector had a wt or mutant M protein, since these two types of vectors induce DC maturation by dramatically different pathways. Vectors with wt M protein stimulate DC through TLR7 and its adaptor molecule MyD88 (6, 25). However, TLR7 is present on a limited subset of DC (16). In DC that lack TLR7, the virus with wt M protein rapidly inhibits cellular gene expression, which prevents DC maturation and leads to cell death (2). In contrast to the virus with wt M protein, the rM51R-M virus is defective in the shutoff of host gene expression and induces DC maturation in a wide variety of DC subsets through both TLR-dependent and TLR-independent mechanisms (2, 6). Figure 1A depicts the genomes of the flagellin-expressing viruses used in this study. The gene for Salmonella enterica flagellin (26) was inserted in the backbones of recombinant wild-type (rwt) and rM51R-M viruses in an additional transcription unit between the M and G genes (as described in reference 37) to generate rwt-flagellin and rM51R-flagellin viruses. rwt virus is a recombinant virus obtained from an infectious cDNA clone containing a wt M protein, whereas
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FIG. 1. Protein expression from recombinant rwt-flagellin and rM51R-flagellin viruses. (A) The gene for bacterial flagellin was inserted in a separate transcriptional unit between the M and G genes of rwt and rM51R-M viruses to generate rwt-flagellin and rM51R-flagellin viruses. (B) Monocyte-derived DC were infected with rwt-flagellin and rM51R-flagellin viruses at MOIs of 1, 3, and 10 PFU/cell for 12 and 24 h or were mock infected (mock). Cells were lysed, and the intracellular levels of flagellin were determined by Western blot analysis. The levels of actin were assayed as a loading control. (C) DC were infected with rwt-flagellin, rM51R-flagellin, rwt, and rM51R-M viruses at an MOI of 5 PFU/cell or were mock infected as a control. At different times postinfection, cells were labeled with a 15-min pulse of [35S]methionine (100 Ci/ml) and harvested. Lysates were subjected to SDS-PAGE, and labeled proteins were quantitated by phosphorimaging. A representative image from analysis of virus-infected DC at 12 h postinfection is shown. (D) Host protein synthesis was determined from images similar to that shown in panel C for regions of the gel devoid of viral proteins. The results are shown as percentages of the mock-infected control value and are the means ⫾ the standard errors of three independent experiments.
rM51R-M virus is isogenic to rwt virus except for a mutation that substitutes an arginine for methionine at position 51 of the 229-amino-acid M protein. This mutation renders the virus defective in its ability to inhibit host gene expression but does not compromise the expression of viral genes or the production of infectious progeny (5, 23). The flagellin gene lacked a eukaryotic signal sequence, and thus flagellin was predicted to be primarily intracellular. rwt-flagellin and rM51R-flagellin viruses exhibit growth kinetics similar to those of the parental viruses and produce comparable, and high, levels of intracellular flagellin upon infection of permissive cell lines (data not shown). To determine the ability of the flagellin-expressing viruses to infect human DC, PBMC-derived DC were generated after culturing CD14⫹ cells with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro (34). Cells were infected with rwt-flagellin and rM51R-flagellin viruses at multiplicities of 1, 3, and 10 PFU/cell or were mock infected. At 12 and 24 h postinfection, cells were harvested and lysates were assayed for the presence of flagellin by Western blot analysis (Fig. 1B). rwt-flagellin virus expressed high levels of flagellin in DC when infected at each of the multiplicities of infection (MOIs). In contrast, rM51R-flagellin expressed detectable levels of flagellin only when infected at an MOI of 10 PFU/cell for 12 h. These data indicate that although human
DC support high levels of expression by rwt-flagellin virus, they do not support the efficient expression of viral genes by rM51R-flagellin virus. To determine the rates of viral and host protein synthesis in cells infected with rwt-flagellin and rM51R-flagellin viruses relative to the parental rwt and rM51R-M viruses, cells were infected at a multiplicity of 5 PFU/cell and pulse labeled with [35S]methionine for 15 min at different times postinfection. Proteins were solubilized, and equivalent amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and phosphorescence imaging. A representative image at 12 h postinfection is shown in Fig. 1C. It is apparent from this figure that lower levels of viral proteins were synthesized in DC infected with rwt-flagellin virus than in those infected with rwt virus. In addition, the synthesis of viral proteins in cells infected with rM51R-M and rM51R-flagellin viruses could not be detected above the background of host protein synthesis. The lack of high levels of viral protein synthesis in cells infected with rM51R-flagellin virus is consistent with the low levels of flagellin expression from this virus (Fig. 1B) and our previous data indicating that rM51R-M virus stimulates antiviral responses in murine DC (2), although the level of viral gene expression in human DC shown here is
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FIG. 2. Maturation of DC by rwt-flagellin and rM51R-flagellin viruses. DC were infected with flagellin-expressing viruses or their parental strains at multiplicities of 5 PFU/cell or were treated with LPS (200 ng/ml) or recombinant flagellin (5 ng/ml). (A) Culture supernatants from infected or treated cells were collected at 24 and 48 h, and the presence of IL-1 was measured by ELISA. Data are expressed in pg cytokine secreted/ml into the supernatant and are representative of two individual experiments. (B) IL-6 levels in culture supernatants from infected cells. Data are expressed in ng cytokine secreted/ml into the supernatant and are the means ⫾ the standard errors of three experiments. (C) The cell surface expression of the costimulatory molecules, CD80 and CD86, was measured by flow cytometry. The geometric mean fluorescence of each sample was determined and used to quantitate the increase in CD80 and CD86 expression over that in untreated cells. Data are the means ⫾ the standard errors of three to
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much lower than that shown in comparable experiments in murine DC (2, 6). Figure 1C also demonstrates that rwt virus effectively inhibited host translation compared to mock-infected cells, as seen in the regions of the gel that are devoid of viral proteins. In contrast, rwt-flagellin virus was less effective at shutting off host protein synthesis than the parental rwt virus. rM51R-M and rM51R-flagellin viruses were defective at inhibiting synthesis of host proteins in human DC, similar to previous results with murine DC (2). Host protein synthesis in virus-infected DC was quantitated by phosphorimaging, and results are shown in Fig. 1D as the percentage of mock-infected cells. The rates of protein synthesis in rM51R-M and rM51R-flagellin virus-infected cells remained similar to those in mock-infected cells, whereas rwt virus and rwt-flagellin viruses effectively inhibited host protein synthesis by 24 h postinfection. However, there was a significant delay in the shutoff of host protein synthesis by rwt-flagellin virus compared to that by rwt virus at 6 and 12 h postinfection. One possible mechanism by which flagellin expressed from rwt virus may delay the shutoff of host protein synthesis is through stimulation of extracellular TLR5 after release from virus-infected apoptotic cells. To address this possibility, DC were infected with rwt virus and treated with extracellular recombinant flagellin (Fig. 1D, rwt ⫹ flagellin). Results show that stimulation of TLR5 by extracellular flagellin did not protect cells from virus-induced inhibition of host gene expression. In fact, there was an enhancement in the shutoff of host protein synthesis in these cells compared to cells infected with the rwt-flagellin virus. Previous studies have suggested that the inhibition of translation of host mRNAs in VSV-infected cells is due to the activation of a stress response (13). It is possible that pretreatment with flagellin enhances pathways leading to cellular stress and thus promotes the inhibition of host translation upon infection with rwt virus, perhaps by enhancing the dephosphorylation of eIF4E. An alternative explanation for the delay in the shutoff of host protein synthesis by rwt-flagellin virus versus that by rwt virus is that intracellularly expressed flagellin may stimulate cytosolic NLRs to promote a cellular response that delays the shutoff of cellular protein synthesis (9). It has been proposed that recognition of cytosolic flagellin by IPAF induces the formation of the inflammasome complex (1). Assembly of this complex leads to the cleavage and activation of caspase 1 and the subsequent cleavage and maturation of the inflammatory cytokines IL-1 and IL-18. To test whether flagellin expressed from VSV stimulates intracellular sensors, we used an enzymelinked immunosorbent assay (ELISA) to determine the production of IL-1 in the supernatants of human DC at 24 and
four experiments. (D) Stimulation of T-cell function by infected DC. DC were mock infected, treated with 200 ng/ml LPS, or infected with each of the viruses at an MOI of 1 PFU/cell. At 24 h postinfection, DC were cultured for 3 days with allogeneic responder T cells. Cells were stained with antibodies for CD8 and IFN-␥ and analyzed by flow cytometry. Data are shown as the total number of IFN-␥-positive CD8⫹ T cells in each culture. Results are means ⫾ standard deviations from four to six experiments. Student’s t test was used to determine significance between samples.
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48 h postinfection (Fig. 2A). Although we were unable to detect the presence of IL-1 in rwt virus-infected cells at these time points, rwt-flagellin virus stimulated the production of high levels of IL-1 in these cells. rM51R-flagellin virus also produced significantly higher levels of IL-1 in infected cells than rM51R-M virus, but levels were lower than those produced by rwt-flagellin virus. Similar results were observed when we assayed for the production of IL-18 (data not shown). These data indicate that flagellin expressed from rwt and rM51R-M viruses stimulates intracellular sensors to promote the production of inflammatory cytokines. To further test the ability of flagellin-expressing viruses to activate DC, we used ELISA to determine the production of the proinflammatory cytokine, IL-6, at 24 h postinfection. In contrast to results obtained with rwt virus, rwt-flagellin virus stimulated the production of IL-6 from DC (Fig. 2B). Infection of cells with rM51R-M virus also stimulated the production of IL-6. However, cells infected with rM51R-flagellin produced even higher levels of IL-6 than those infected with rM51R-M virus, indicating that the expression of flagellin from both wt and M protein mutant viruses promotes the production of the inflammatory cytokine IL-6. We can further conclude that the ability of these viruses to stimulate IL-6 production by human DC is inversely correlated with their ability to inhibit host gene expression (Fig. 1). Treatment of rwt virus-infected cells with flagellin at the time of infection also stimulated the production of IL-6, which we attribute to the early synthesis of IL-6 prior to the shutoff of host protein synthesis. A hallmark of DC maturation is the increase in the cell surface expression of costimulatory molecules (18, 19). To determine the ability of flagellin-expressing VSV to induce the expression of costimulatory molecules, cells were infected with each of the viruses at a multiplicity of 5 PFU/cell and the expression of the costimulatory molecules, CD80 and CD86, was determined at 24 h postinfection by flow cytometry (Fig. 2C). Data are expressed as the fold increase in costimulatory molecule expression over that observed in mock-infected cells. Similar to results in murine DC (2), rwt virus failed to stimulate the production of costimulatory molecules on human DC, most likely due to the induction of cell death (data not shown). Similar results were obtained in DC infected with rwt-flagellin virus. In contrast, rM51R-M and rM51R-flagellin viruses potently stimulated the cell surface expression of CD80 and CD86 to levels comparable to those seen in cells treated with lipopolysaccharide (LPS). Furthermore, levels were higher than those obtained from treatment with recombinant flagellin. These data indicate that the expression of flagellin had little, if any, effect on the expression of costimulatory molecules from both rwt and rM51R-M viruses. Given that both flagellin-expressing viruses promoted the production of IL-6 and IL-1 in infected DC (Fig. 2A and B), this result suggests that the pathway leading to the production of cytokines may be more sensitive to flagellin stimulation than the pathway resulting in the expression of the costimulatory molecules. To test the ability of the flagellin-expressing viruses to activate T cells relative to that of the parental strains, DC were mock infected or infected with each of the viruses at an MOI of 1 PFU/cell. At 24 h postinfection, DC were cultured for 3 days with allogeneic responder T cells, and levels of intracellular IFN-␥ were assayed by flow cytometry. The average num-
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ber of IFN-␥-positive CD8⫹ T cells in each culture is shown in Fig. 2D. DC treated with LPS induced a 10-fold increase in the number of CD8⫹ T cells expressing IFN-␥ compared to mockinfected DC. As expected, rwt virus-infected DC did not promote the activation of T cells. However, DC infected with rwt-flagellin virus showed a statistically significant increase in the total number of IFN-␥-positive CD8⫹ T cells compared to DC infected with the parental rwt virus. In fact, levels were significantly greater than those observed in DC infected with rM51R-M and rM51R-flagellin viruses. Infection with rwtflagellin virus also increased the ability of DC to stimulate IFN-␥ production from CD4⫹ T cells (data not shown). These results are interesting considering the observation that rwtflagellin virus failed to induce the production of costimulatory molecules on the surface of infected DC. However, it is possible that the production of cytokines by this virus is a more important determinant of T-cell activation than the presence of high levels of costimulatory molecules, similar to results obtained by Arimilli et al. (8). In light of our finding that flagellin expressed from rwt and rM51R-M viruses stimulated DC in culture, we hypothesized that immunization of mice with rwt-flagellin and rM51R-flagellin viruses would boost VSV-specific immune responses in vivo. BALB/c mice were inoculated intranasally with various doses of rwt, rwt-flagellin, rM51R-M, and rM51Rflagellin viruses, and serum was collected at day 18 or 21 postinfection. VSV-specific antibody titers were determined by ELISA using purified VSV as antigen. The limit of detection for the ELISA was a serum dilution of 1:100. Figure 3 shows titers for individual mice inoculated with each of the viruses, with the number of individual mice with titers above the detection limit shown on the x axis. All of the mice infected with rwt and rwt-flagellin viruses had high titers against the VSV antigen (Fig. 3A). However, there was a statistically significant increase in antibody titers upon infection of mice with 105 and 106 PFU of rwt-flagellin virus relative to the parental rwt virus. As shown previously (4), the anti-VSV antibody levels induced by rM51R-M virus were comparable to those induced by rwt virus when inoculated at a dose of 107 PFU/mouse. However, at lower doses, several mice infected with rM51R-M virus had antibody titers that were at or below the limit of detection. In contrast, mice infected with rM51R-flagellin virus had seroconverted at all doses tested. Antibody titers in mice infected with 105 PFU of rM51R-flagellin virus were significantly elevated compared to those in mice infected with rM51R-M virus. However, at 107 PFU, both viruses induced similar antibody titers against VSV. These data indicate that flagellin expressed from rwt and rM51R-M virus enhances the anti-VSV antibody response in vivo. In the study presented here, we expressed bacterial flagellin from the VSV genome in an attempt to design vaccine vectors that are stronger stimulators of DC function. Flagellin was chosen due to its potent immunostimulatory properties and its successful use as a vaccine adjuvant to promote adaptive immunity against antigens from various pathogens (14, 21, 27, 29). Our results demonstrate that flagellin expressed from the rwt virus genome partially protects human DC from VSVinduced shutoff of host protein synthesis and aids in the production of various cytokines, including IL-1. Together with the observation that the addition of extracellular flagellin did
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rM51R-M vectors induce protective immunity against the poorly immunogenic poxvirus antigens LIR and B5R (10). Additional viruses containing mutations in genes whose products suppress host antiviral responses, such as IFN-inducing mutants of bovine respiratory syncytial virus (bRSV) lacking the NS proteins (35), and simian virus 5 (SV5) with P/V mutations (7), are also being developed as vaccine vectors. Studies shown here indicate that rM51R-M virus stimulates human DC, as demonstrated by the increased expression of costimulatory molecules on the surfaces of cells and the production of IL-6. These results are consistent with our previous findings with murine DC indicating that rM51R-M virus promotes the maturation of both myeloid and plasmacytoid DC subsets (2, 6). Our results also indicate that flagellin expressed from the rM51R-M virus genome increases the production of IL-6 and IL-1 from human DC. Therefore, the expression of flagellin from rM51R-M virus has the potential to enhance DC function. In addition to activating DC, flagellin interacts with TLR5 in a variety of other cell types, including macrophages, endothelial cells, and epithelial cells to potently induce innate immunity (20). Therefore, the immune-stimulatory properties of flagellin, together with the capacity of M protein mutant virus to promote DC maturation, suggest that flagellin has the potential to enhance the effectiveness of vaccine vectors with mutant as well as wt M proteins. We are currently testing whether flagellin, expressed from low doses of VSV, has the ability to enhance the antibody response against weakly immunogenic antigens, such as HIV Env. FIG. 3. Immunization with flagellin-expressing viruses promotes high antigen titers against VSV. BALB/c mice (5 to 7 weeks old) were inoculated intranasally with various doses of rwt, rwt-flagellin (A), rM51R-M, and rM51R-flagellin (B) viruses. At day 18 (rwt and rwtflagellin viruses) or 21 (rM51R-M and rM51R-flagellin viruses) postinfection, serum was collected and VSV-specific antibody titers were determined by ELISA using purified VSV as antigen. The limit of detection was a serum dilution of 1:100. Five mice were used per group. The number of mice with detectable titers is shown in parentheses on the x axis.
not protect cells from rwt virus-induced inhibition of host gene expression, these data suggest that intracellularly expressed flagellin stimulates cytosolic sensors to induce antiviral effects. In addition to the activation of caspase 1 and production of active IL-1 and IL-18, studies have shown that NLR proteins and their cytosolic protein complexes promote the activation of NFB and mitogen-activated protein kinase (MAPK) pathways (9, 36), which may be responsible for the delay in the shutoff of host protein synthesis. In addition, the NF-B pathway is an important mediator for activation of the IL-6 gene (9). Therefore, our data suggest that expression of flagellin from the rwt virus genome may enhance the activation of innate immune mechanisms to aid in the stimulation of DC. Furthermore, the ability of intracellular flagellin to overcome the suppression of DC function by rwt virus may contribute not only to the effectiveness of rwt-flagellin virus as a vaccine vector but also to its safety in vivo. rM51R-M virus shows great potential as an effective vaccine vector for the delivery of foreign antigens due its ability to stimulate innate and adaptive immunity in vivo, without causing disease (3–5). In fact, we have recently shown that
We thank Griffith Parks for helpful discussions and review of the manuscript. This study was supported by NIH program project grants P01AI060642 and P01-AI082325. REFERENCES 1. Abdelaziz, D. H., K. Amr, and A. O. Amer. 2010. Nlrc4/Ipaf/CLAN/ CARD12: more than a flagellin sensor. Int. J. Biochem. Cell Biol. 24:789– 791. 2. Ahmed, M., K. L. Brzoza, and E. M. Hiltbold. 2006. Matrix protein mutant of vesicular stomatitis virus stimulates maturation of myeloid dendritic cells. J. Virol. 80:2194–2205. 3. Ahmed, M., S. D. Cramer, and D. S. Lyles. 2004. Sensitivity of prostate tumors to wild type and M protein mutant vesicular stomatitis viruses. Virology 330:34–49. 4. Ahmed, M., T. R. Marino, S. Puckett, N. D. Kock, and D. S. Lyles. 2008. Immune response in the absence of neurovirulence in mice infected with M protein mutant vesicular stomatitis virus. J. Virol. 82:9273–9277. 5. Ahmed, M., M. O. McKenzie, S. Puckett, M. Hojnacki, L. Poliquin, and D. S. Lyles. 2003. Ability of M protein of vesicular stomatitis virus to suppress interferon beta gene expression is genetically correlated with the inhibition of host RNA and protein synthesis. J. Virol. 77:4646–4657. 6. Ahmed, M., L. M. Mitchell, S. Puckett, K. L. Brzoza-Lewis, D. S. Lyles, and E. M. Hiltbold. 2009. M protein mutant vesicular stomatitis virus stimulates maturation of TLR7-positive dendritic cells through TLR-dependent and -independent mechanisms. J. Virol. 83:2962–2975. 7. Arimilli, S., M. A. Alexander-Miller, and G. D. Parks. 2006. A simian virus 5 (SV5) P/V mutant is less cytopathic than wild-type SV5 in human dendritic cells and is a more effective activator of dendritic cell maturation and function. J. Virol. 80:3416–3427. 8. Arimilli, S., J. B. Johnson, K. M. Clark, A. H. Graff, M. A. Alexander-Miller, S. B. Mizel, and G. D. Parks. 2008. Engineered expression of the TLR5 ligand flagellin enhances paramyxovirus activation of human dendritic cell function. J. Virol. 82:10975–10985. 9. Benko, S., D. J. Philpott, and S. E. Girardin. 2008. The microbial and danger signals that activate Nod-like receptors. Cytokine 43:368–373. 10. Braxton, C. L., S. Puckett, S. B. Mizel, and D. S. Lyles. 2010. Protection against lethal vaccinia virus challenge using an attenuated matrix protein mutant vesicular stomatitis virus vaccine vector expressing poxvirus antigens. J. Virol. 84:3552–3561. 11. Ciavarra, R. P., A. Stephens, S. Nagy, M. Sekellick, and C. Steel. 2006.
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