Accepted Article Preview: Published ahead of advance online publication Modulating APOBEC expression enhances DNA vaccine immunogenicity Rafael Ribeiro Almeida, Rui Andre´ Saraiva Raposo, Fernanda Caroline Coirada, Jamile Ramos da Silva, Luı´ s Carlos de Souza Ferreira, Jorge Kalil, Douglas F Nixon, Edecio Cunha-Neto
Cite this article as: Rafael Ribeiro Almeida, Rui Andre´ Saraiva Raposo, Fernanda Caroline Coirada, Jamile Ramos da Silva, Luı´ s Carlos de Souza Ferreira, Jorge Kalil, Douglas F Nixon, Edecio Cunha-Neto, Modulating APOBEC expression enhances DNA vaccine immunogenicity, Immunology and Cell Biology accepted article preview 8 May 2015; doi: 10.1038/icb.2015.53. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Received 12 March 2015; revised 2 May 2015; accepted 3 May 2015; Accepted article preview online 8 May 2015
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2015 Macmillan Publishers Limited. All rights reserved.
Title Page Title: Modulating APOBEC expression enhances DNA vaccine immunogenicity Authors: Rafael Ribeiro Almeida1,3,*, Rui André Saraiva Raposo5, Fernanda Caroline Coirada1, Jamile Ramos da Silva4, Luís Carlos de Souza Ferreira4, Jorge Kalil1,2,3, Douglas F. Nixon5,# and Edecio Cunha-Neto1,2,3,#
1
Laboratory of Clinical Immunology and Allergy-LIM60, Division of Clinical
Immunology and Allergy, Department of Medicine, University of São Paulo School of Medicine, São Paulo, Brazil 2
Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo, Brazil
3
Institute for Investigation in Immunology-INCT, São Paulo, Brazil
4
Department of Microbiology, Institute of Biomedical Sciences, University of São
Paulo, São Paulo, Brazil 5
Department of Microbiology, Immunology & Tropical Medicine, The George
Washington University, Washington, DC, USA #
Co-senior authors
*Corresponding author Laboratory of Clinical Immunology and Allergy-LIM60, Division of Clinical Immunology and Allergy, Department of Medicine, University of São Paulo School of Medicine, São Paulo, Brazil - Phone: +55 11 30618315; Fax: +55 11 30618392; Email: [email protected]
1
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2015 Macmillan Publishers Limited. All rights reserved.
Abstract DNA vaccines have failed to induce satisfactory immune responses in humans. Several mechanisms of double-stranded DNA sensing have been described, and modulate DNA vaccine immunogenicity at many levels. We hypothesized that the immunogenicity of DNA vaccines in humans is suppressed by APOBEC-mediated plasmid degradation. We showed that plasmid sensing via STING and TBK-1 leads to IFN-β induction, which results in APOBEC3A mRNA upregulation through a mechanism involving PKC signaling. We also showed that murine APOBEC2 expression in HEK293T cells led to 10-fold reduction in intracellular plasmid levels and plasmid-encoded mRNA, and 2.6fold reduction in GFP-expressing cells. A bicistronic DNA vaccine expressing an immunogen and an APOBEC2-specific shRNA efficiently silenced APOBEC2 both in vitro and in vivo, increasing the frequency of induced IFN-γ-secreting T cells. Our study brings new insights into the intracellular machinery involved in double-stranded DNA sensing and how to modulate it to improve DNA vaccine immunogenicity in humans.
2
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Introduction DNA vaccines are safe, stable and easily produced. Cloning sequences of interest into plasmid vectors provides a simple, fast and cheaper alternative when compared to processes involving microorganism culture and inactivation, or the production of recombinant proteins 1. In vivo plasmid-encoded antigen expression allows the protein to be naturally modified, which may be essential for inducing an effective immune response. Plasmid immunization leads to direct transfection of antigen presenting cells (APCs) and tissue-resident cells, providing local expression of target antigens and subsequent induction of cellular and humoral immunity 1-3. Veterinary DNA vaccines have been used for counteracting viral infections and cancer in different species
4-6
. While successful vaccination leading to vaccine efficacy
in animals has been achieved, human prophylactic and therapeutic clinical trials of DNA vaccines have largely failed 7, despite research efforts to improve adjuvants and delivery systems for DNA- and RNA-based vaccines
8-10
. Several mechanisms of
double-stranded DNA (dsDNA) sensing have been described
11
and are likely to
modulate DNA vaccine immunogenicity at many levels. Among them, Toll-like receptor (TLR)-independent type I interferon (IFN) production has been shown to strongly contribute to DNA vaccine-induced immune responses 10. Intracellular DNA sensing is mediated by a variety of molecules, such as ZDNA-binding protein 1 (ZBP1), Leucin-rich repeat flightless-interacting protein 1 (LRRFIP1), DDX41, IFNγ-inducible protein 16 (IFI16) and the cyclic-GMP-AMP (cGAMP) synthase (cGAS), leading to type I IFN production through a mechanism involving stimulator of IFN genes (STING), interferon-regulatory factor 3 (IRF3) and TANK-binding kinase 1 (TBK-1)
12-16
. STING-deficient mice were shown to have
reduced DNA vaccine-induced cytotoxic T cell responses
17
, suggesting that STING is 3
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2015 Macmillan Publishers Limited. All rights reserved.
crucial for type I IFN-mediated DNA vaccine immunogenicity. As observed for STING, TBK-1-dependent signaling was shown to be essential for type I IFN-mediated adjuvant effect of DNA vaccines, and required for the induction of antigen-specific B and T cells 18
. Type I IFNs induce a variety of innate immunity-related genes
19
, including
members of the apolipoprotein B (APOB) mRNA-editing, catalytic polypeptide (APOBEC) family, which was recently shown to be dependent on protein kinase C (PKC) and signal transducer and activator of transcription 1 (Stat-1)
20-23
. Human
APOBEC3s (A3s) have been implicated in the inhibition of retroviral infections and other retroelements
24-27
. Different A3s were shown to mediate the clearance of foreign
DNA from human cells
28
. APOBEC3A is the most responsive isoform to type I IFN
stimulation, and it is induced by, and exerts the strongest restriction, on transfected plasmids
28
. More recently, it has been shown that A3A genetically edits co-
electroporated luciferase plasmid DNA in mouse skeletal muscle, leading to DNA degradation 29, which suggests that this protein may have an important role in restricting DNA vaccine immunogenicity. However, the mechanisms underlying plasmid-mediated APOBEC3A induction are still unknown. While APOBEC3 has been extensively studied, little is known about APOBEC2 protein. It has been described as heart and skeletal muscle-related involved in muscle development and retina regeneration
31-33
30
and shown to be
. Recent data have also
demonstrated that TNF-α-induced APOBEC2 expression in hepatocytes is implicated in editing activity of certain transcripts
34
. However, the impact of APOBEC2 on
intracellular plasmid clearance and DNA vaccine immunogenicity remains to be determined.
4
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Although it has been shown that STING/TBK-1 pathways are crucial for DNA vaccine immunogenicity, we hypothesized that plasmid-mediated type I IFN production through these pathways is responsible for APOBEC3A induction, and that APOBEC proteins might have an important role in suppressing immunogenicity of DNA vaccines by mediating plasmid degradation. Therefore, we believe that a controlled regulation of plasmid-mediated APOBEC induction would circumvent a possible negative effect of type I IFN on DNA vaccine immunogenicity through the induction of APOBEC proteins. In this study, we first evaluated the role of type I IFN/PKC/STING/TBK-1 pathway in plasmid-mediated APOBEC3A induction in a controlled in vitro setting using human monocytes. Due to the fact that rodents do not express APOBEC3A and that APOBEC2 is expressed in skeletal muscle, where most DNA vaccine are injected, we chose to study the ability of murine APOBEC2 in mediating the clearance of intracellular plasmid DNA and its effects on plasmid-encoded mRNA/protein expression. Finally, we designed and assessed the immunogenicity of a bicistronic DNA vaccine encoding an APOBEC2-specific shRNA and an immunogen (HIVBr18) previously developed by our group. The HIVBr18 immunogen comprises 18 conserved, multiple HLA-DR-binding HIV-1 subtype B peptides and has been shown to induce both CD4+ and CD8+ T-cell responses, but no B-cell responses in HLA-transgenic and BALB/c mice 35, 36.
5
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Results Type I IFN signaling participates in plasmid-mediated APOBEC3A induction Plasmid transfection induces high levels of both type I IFN and APOBEC3A expression
11, 28
. In order to determine the role of type I IFN in plasmid-mediated
APOBEC3A induction, we first sought to evaluate whether the pro-monocytic cell line THP-1 cells would fit as a feasible model of study. We assessed APOBEC3A and type I IFN expression in THP-1 cells transfected with a plasmid named pVAX-Gag (pVAX1 vector encoding the whole HIV-1 Gag protein). We observed 80-fold, 22.5-fold and 5.75-fold increase in APOBEC3A expression 6h, 12h and 24h after transfection, respectively, when compared to mock-transfected THP-1 cells (Fig. 1a). We also observed 80-fold increase in IFN-β expression 6h after transfection, but no IFN-α expression (Fig. 1a). We then investigated whether type I IFN signaling could play a role in plasmid-mediated APOBEC3A induction. We transfected THP-1 cells with pVAX-Gag and treated the cells with anti-IFN-α and/or anti-IFN-β neutralizing antibodies. Treatment with anti-IFN-β neutralizing antibodies resulted in a significant reduction in APOBEC3A induction by plasmid transfection, while treatment with an isotype control had no effect (p15 SFU/106 splenocytes, which was calculated as the mean response + 3 standard deviations (SD) of splenocytes from non-immunized mice.
Statistical Analysis Statistical significance (p-values) was calculated by using Student’s T test or One-way ANOVA followed by Tukey’s Multiple Comparison Test. Statistical analysis and graphical representation of data was performed using GraphPad Prism version 5.0 software.
20
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Acknowledgments This research was supported by the Brazilian National Research Council (CNPq), grant 420166/2005-0, the São Paulo State Research Funding Agency (FAPESP), grants 2004/15856-9, 2006/50096-0 and 2008/57881-0, and from the NIH, grant AI093179. Rafael Ribeiro Almeida is a recipient of São Paulo State Research Funding Agency (FAPESP) fellowship.
Author contributions Conceived and designed the experiments: RRA, RASR, DFN and EC-N. Performed the experiments: RRA, FCC and JRS. Analyzed the data: RRA, FCC, JRS, RASR, LCSF, JK, DFN and EC-N. Wrote the manuscript: RRA. Revised and approved the final version of the manuscript: FCC, JRS, RASR, LCSF, JK, DFN and EC-N.
Competing financial interests The authors declare no competing financial interests.
21
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Figure legends Figure 1. Type I IFN signaling participates in plasmid-mediated APOBEC3A induction. A) THP-1 cells were transfected with 0.5 µg of pVAX-Gag and APOBEC3A expression was assessed 6h, 12h and 24h after transfection. The expression of IFN-α and IFN-β was assessed 6h after transfection. B) THP-1 cells were transfected with 0.5 µg of pVAX-Gag and immediately treated with 5 µg/mL, 10 µg/mL or 20 µg/mL of anti-IFN-β neutralizing antibody or 20 µg/mL of an isotype control. Total RNA was extracted 8h after transfection for real-time qPCR. C) THP-1 cells were treated with 100 nM or 1 µM of Gö6976 for 16h, transfected with pVAX-Gag and incubated in R10 with the respective concentration of Gö6976 for 8h. Total RNA was extracted for real-time qPCR. mRNA levels are relative to those measured in cells transfected with transfection buffer. Data are shown as mean + SD of 3 independent experiments. One-way ANOVA followed by Tukey’s Multiple Comparison Test were used to determine statistical significance.
Figure 2. STING and TBK-1 knockdown leads to lower IFN-β and APOBEC3A induction by pVAX-Gag transfection. THP-1 cells were transfected with 250 nM of STING and/or TBK-1 Smartpool siRNAs or a control siRNA. STING (a) and TBK-1 (b) mRNA expression were determined by real-time qPCR 3 days after transfection. The cells were then transfected with pVAX-Gag and total RNA was extracted 8h after transfection for analysis of IFN-β (c) and APOBEC3A (d) mRNA expression. mRNA levels from control siRNA-transfected cells were considered as 100% and used to calculate STING and TBK-1 mRNA knockdown in A and B, respectively. mRNA levels in C and D were relative to those measured in cells first transfected with control
27
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siRNA and then with transfection buffer. Data are shown as mean + SD of 2 independent experiments.
Figure 3. APOBEC2 mediates the clearance of plasmid and plasmid-encoded mRNA. HEK293T cells were co-transfected with pmaxGFP and empty pcDNA3.1 (black bars) or pcDNA3.1-APOBEC2 (grey bars). Total DNA and RNA were separately extracted 1 day post transfection. The relative levels of plasmid (a) and plasmidencoded mRNA (b) were determined by real-time qPCR. Data from co-tranfection of pmaxGFP and empty pcDNA3.1 were normalized to one. Bars represent mean + SD of 3 independent experiments. Student’s T test was used to determine statistical significance.
Figure 4. APOBEC2 mediates the reduction of plasmid-encoded protein expression. HEK293T cells were co-transfected with pmaxGFP and empty pcDNA3.1, pcDNA3.1-APOBEC2 or HIVBr18. GFP expression was determined by flow cytometry 1 day (a) and 3 days (b) post transfection. Alternatively, HEK293T cells were transfected with pcDNA3.1, pmaxGFP + pcDNA3.1 or pmaxGFP + pcDNA3.1APOBEC2 and GFP expression was determined by fluorescence microcospy (c). HEK293T cells were also co-transfected with pmaxGFP and empty pcDNA3.1 or decreasing doses of pcDNA3.1-APOBEC2 (d). GFP expression was determined by flow cytometry 1 day post transfection. The amount of DNA used to transfect cells was maintained constant by adding empty pcDNA3.1 to the decreasing doses of pcDNA3.1APOBEC2. Bars represent mean + SD of 4 independent experiments. One-way ANOVA followed by Tukey’s Multiple Comparison Test were used to determine statistical significance. 28
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Figure 5. Schematic diagram of bicistronic DNA vaccines construction. The HIVBr18 gene was subcloned into pcDNA3.1 vector under CMV promoter control using Hind III and Xho I restriction enzymes. To construct the bicistronic DNA vaccines shAPO2Br18 and scBr18, expression cassettes (U6 promoter, poly (A) tail and a DNA sequencing expressing either APOBEC2 shRNA or a scrambled shRNA) were subcloned into pcDNA3.1-HIVBr18 at the MFE I restriction enzyme site.
Figure 6. APOBEC2 knockdown leads to increased DNA vaccine immunogenicity. HEK293T cells were co-transfected with pcDNA3.1-APOBEC2 and scBr18 or shAPO2Br18 and total RNA was extracted 1 day post transfection. Relative APOBEC2 mRNA expression was determined by real-time qPCR (a). Data from co-tranfection of pcDNA3.1-APOBEC2 and scBr18 were normalized to one. BALB/c mice were intramuscularly immunized with 1 dose of shAPO2Br18 or scBr18 (50 µg/animal). Total protein was extracted from injected muscle 4 days after immunization. APOBEC2 expression was determined by Western blotting. The APOBEC2 protein levels were determined by densitometry (b) and GAPDH levels were used for normalization. APOBEC2 and GAPDH levels are shown for individual mice (3 animals per immunization group), which are represented by numbers (c). Alternatively, BALB/c mice (n = 5) were intramuscularly immunized with 2 doses (50 µg/dose) of scBr18 or shAPO2Br18 every 20 days. Twenty days after the last dose mice were euthanized and pooled splenocytes were cultured in the presence of HIVBr18-encoded peptides. The frequency of IFN-γ secreting T cells was determined by ELISPOT (d). Bars represent mean + SD of 3 independent experiments for qPCR, mean + SD of 4 independent experiments for ELISPOT and mean + SD of 3 individual mice per immunization group for densitometry. Student’s T test was used to determine statistical significance. 29
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Cite this article as: Rafael Ribeiro Almeida, Rui Andre´ Saraiva Raposo, Fernanda Caroline Coirada, Jamile Ramos da Silva, Luı´ s Carlos de Souza Ferreira, Jorge Kalil, Douglas F Nixon, Edecio Cunha-Neto, Modulating APOBEC expression enhances DNA vaccine immunogenicity, Immunology and Cell Biology accepted article preview 8 May 2015; doi: 10.1038/icb.2015.53. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Received 12 March 2015; revised 2 May 2015; accepted 3 May 2015; Accepted article preview online 8 May 2015
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2015 Macmillan Publishers Limited. All rights reserved.
Title Page Title: Modulating APOBEC expression enhances DNA vaccine immunogenicity Authors: Rafael Ribeiro Almeida1,3,*, Rui André Saraiva Raposo5, Fernanda Caroline Coirada1, Jamile Ramos da Silva4, Luís Carlos de Souza Ferreira4, Jorge Kalil1,2,3, Douglas F. Nixon5,# and Edecio Cunha-Neto1,2,3,#
1
Laboratory of Clinical Immunology and Allergy-LIM60, Division of Clinical
Immunology and Allergy, Department of Medicine, University of São Paulo School of Medicine, São Paulo, Brazil 2
Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo, Brazil
3
Institute for Investigation in Immunology-INCT, São Paulo, Brazil
4
Department of Microbiology, Institute of Biomedical Sciences, University of São
Paulo, São Paulo, Brazil 5
Department of Microbiology, Immunology & Tropical Medicine, The George
Washington University, Washington, DC, USA #
Co-senior authors
*Corresponding author Laboratory of Clinical Immunology and Allergy-LIM60, Division of Clinical Immunology and Allergy, Department of Medicine, University of São Paulo School of Medicine, São Paulo, Brazil - Phone: +55 11 30618315; Fax: +55 11 30618392; Email: [email protected]
1
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2015 Macmillan Publishers Limited. All rights reserved.
Abstract DNA vaccines have failed to induce satisfactory immune responses in humans. Several mechanisms of double-stranded DNA sensing have been described, and modulate DNA vaccine immunogenicity at many levels. We hypothesized that the immunogenicity of DNA vaccines in humans is suppressed by APOBEC-mediated plasmid degradation. We showed that plasmid sensing via STING and TBK-1 leads to IFN-β induction, which results in APOBEC3A mRNA upregulation through a mechanism involving PKC signaling. We also showed that murine APOBEC2 expression in HEK293T cells led to 10-fold reduction in intracellular plasmid levels and plasmid-encoded mRNA, and 2.6fold reduction in GFP-expressing cells. A bicistronic DNA vaccine expressing an immunogen and an APOBEC2-specific shRNA efficiently silenced APOBEC2 both in vitro and in vivo, increasing the frequency of induced IFN-γ-secreting T cells. Our study brings new insights into the intracellular machinery involved in double-stranded DNA sensing and how to modulate it to improve DNA vaccine immunogenicity in humans.
2
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2015 Macmillan Publishers Limited. All rights reserved.
Introduction DNA vaccines are safe, stable and easily produced. Cloning sequences of interest into plasmid vectors provides a simple, fast and cheaper alternative when compared to processes involving microorganism culture and inactivation, or the production of recombinant proteins 1. In vivo plasmid-encoded antigen expression allows the protein to be naturally modified, which may be essential for inducing an effective immune response. Plasmid immunization leads to direct transfection of antigen presenting cells (APCs) and tissue-resident cells, providing local expression of target antigens and subsequent induction of cellular and humoral immunity 1-3. Veterinary DNA vaccines have been used for counteracting viral infections and cancer in different species
4-6
. While successful vaccination leading to vaccine efficacy
in animals has been achieved, human prophylactic and therapeutic clinical trials of DNA vaccines have largely failed 7, despite research efforts to improve adjuvants and delivery systems for DNA- and RNA-based vaccines
8-10
. Several mechanisms of
double-stranded DNA (dsDNA) sensing have been described
11
and are likely to
modulate DNA vaccine immunogenicity at many levels. Among them, Toll-like receptor (TLR)-independent type I interferon (IFN) production has been shown to strongly contribute to DNA vaccine-induced immune responses 10. Intracellular DNA sensing is mediated by a variety of molecules, such as ZDNA-binding protein 1 (ZBP1), Leucin-rich repeat flightless-interacting protein 1 (LRRFIP1), DDX41, IFNγ-inducible protein 16 (IFI16) and the cyclic-GMP-AMP (cGAMP) synthase (cGAS), leading to type I IFN production through a mechanism involving stimulator of IFN genes (STING), interferon-regulatory factor 3 (IRF3) and TANK-binding kinase 1 (TBK-1)
12-16
. STING-deficient mice were shown to have
reduced DNA vaccine-induced cytotoxic T cell responses
17
, suggesting that STING is 3
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2015 Macmillan Publishers Limited. All rights reserved.
crucial for type I IFN-mediated DNA vaccine immunogenicity. As observed for STING, TBK-1-dependent signaling was shown to be essential for type I IFN-mediated adjuvant effect of DNA vaccines, and required for the induction of antigen-specific B and T cells 18
. Type I IFNs induce a variety of innate immunity-related genes
19
, including
members of the apolipoprotein B (APOB) mRNA-editing, catalytic polypeptide (APOBEC) family, which was recently shown to be dependent on protein kinase C (PKC) and signal transducer and activator of transcription 1 (Stat-1)
20-23
. Human
APOBEC3s (A3s) have been implicated in the inhibition of retroviral infections and other retroelements
24-27
. Different A3s were shown to mediate the clearance of foreign
DNA from human cells
28
. APOBEC3A is the most responsive isoform to type I IFN
stimulation, and it is induced by, and exerts the strongest restriction, on transfected plasmids
28
. More recently, it has been shown that A3A genetically edits co-
electroporated luciferase plasmid DNA in mouse skeletal muscle, leading to DNA degradation 29, which suggests that this protein may have an important role in restricting DNA vaccine immunogenicity. However, the mechanisms underlying plasmid-mediated APOBEC3A induction are still unknown. While APOBEC3 has been extensively studied, little is known about APOBEC2 protein. It has been described as heart and skeletal muscle-related involved in muscle development and retina regeneration
31-33
30
and shown to be
. Recent data have also
demonstrated that TNF-α-induced APOBEC2 expression in hepatocytes is implicated in editing activity of certain transcripts
34
. However, the impact of APOBEC2 on
intracellular plasmid clearance and DNA vaccine immunogenicity remains to be determined.
4
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2015 Macmillan Publishers Limited. All rights reserved.
Although it has been shown that STING/TBK-1 pathways are crucial for DNA vaccine immunogenicity, we hypothesized that plasmid-mediated type I IFN production through these pathways is responsible for APOBEC3A induction, and that APOBEC proteins might have an important role in suppressing immunogenicity of DNA vaccines by mediating plasmid degradation. Therefore, we believe that a controlled regulation of plasmid-mediated APOBEC induction would circumvent a possible negative effect of type I IFN on DNA vaccine immunogenicity through the induction of APOBEC proteins. In this study, we first evaluated the role of type I IFN/PKC/STING/TBK-1 pathway in plasmid-mediated APOBEC3A induction in a controlled in vitro setting using human monocytes. Due to the fact that rodents do not express APOBEC3A and that APOBEC2 is expressed in skeletal muscle, where most DNA vaccine are injected, we chose to study the ability of murine APOBEC2 in mediating the clearance of intracellular plasmid DNA and its effects on plasmid-encoded mRNA/protein expression. Finally, we designed and assessed the immunogenicity of a bicistronic DNA vaccine encoding an APOBEC2-specific shRNA and an immunogen (HIVBr18) previously developed by our group. The HIVBr18 immunogen comprises 18 conserved, multiple HLA-DR-binding HIV-1 subtype B peptides and has been shown to induce both CD4+ and CD8+ T-cell responses, but no B-cell responses in HLA-transgenic and BALB/c mice 35, 36.
5
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2015 Macmillan Publishers Limited. All rights reserved.
Results Type I IFN signaling participates in plasmid-mediated APOBEC3A induction Plasmid transfection induces high levels of both type I IFN and APOBEC3A expression
11, 28
. In order to determine the role of type I IFN in plasmid-mediated
APOBEC3A induction, we first sought to evaluate whether the pro-monocytic cell line THP-1 cells would fit as a feasible model of study. We assessed APOBEC3A and type I IFN expression in THP-1 cells transfected with a plasmid named pVAX-Gag (pVAX1 vector encoding the whole HIV-1 Gag protein). We observed 80-fold, 22.5-fold and 5.75-fold increase in APOBEC3A expression 6h, 12h and 24h after transfection, respectively, when compared to mock-transfected THP-1 cells (Fig. 1a). We also observed 80-fold increase in IFN-β expression 6h after transfection, but no IFN-α expression (Fig. 1a). We then investigated whether type I IFN signaling could play a role in plasmid-mediated APOBEC3A induction. We transfected THP-1 cells with pVAX-Gag and treated the cells with anti-IFN-α and/or anti-IFN-β neutralizing antibodies. Treatment with anti-IFN-β neutralizing antibodies resulted in a significant reduction in APOBEC3A induction by plasmid transfection, while treatment with an isotype control had no effect (p15 SFU/106 splenocytes, which was calculated as the mean response + 3 standard deviations (SD) of splenocytes from non-immunized mice.
Statistical Analysis Statistical significance (p-values) was calculated by using Student’s T test or One-way ANOVA followed by Tukey’s Multiple Comparison Test. Statistical analysis and graphical representation of data was performed using GraphPad Prism version 5.0 software.
20
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2015 Macmillan Publishers Limited. All rights reserved.
Acknowledgments This research was supported by the Brazilian National Research Council (CNPq), grant 420166/2005-0, the São Paulo State Research Funding Agency (FAPESP), grants 2004/15856-9, 2006/50096-0 and 2008/57881-0, and from the NIH, grant AI093179. Rafael Ribeiro Almeida is a recipient of São Paulo State Research Funding Agency (FAPESP) fellowship.
Author contributions Conceived and designed the experiments: RRA, RASR, DFN and EC-N. Performed the experiments: RRA, FCC and JRS. Analyzed the data: RRA, FCC, JRS, RASR, LCSF, JK, DFN and EC-N. Wrote the manuscript: RRA. Revised and approved the final version of the manuscript: FCC, JRS, RASR, LCSF, JK, DFN and EC-N.
Competing financial interests The authors declare no competing financial interests.
21
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2015 Macmillan Publishers Limited. All rights reserved.
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Figure legends Figure 1. Type I IFN signaling participates in plasmid-mediated APOBEC3A induction. A) THP-1 cells were transfected with 0.5 µg of pVAX-Gag and APOBEC3A expression was assessed 6h, 12h and 24h after transfection. The expression of IFN-α and IFN-β was assessed 6h after transfection. B) THP-1 cells were transfected with 0.5 µg of pVAX-Gag and immediately treated with 5 µg/mL, 10 µg/mL or 20 µg/mL of anti-IFN-β neutralizing antibody or 20 µg/mL of an isotype control. Total RNA was extracted 8h after transfection for real-time qPCR. C) THP-1 cells were treated with 100 nM or 1 µM of Gö6976 for 16h, transfected with pVAX-Gag and incubated in R10 with the respective concentration of Gö6976 for 8h. Total RNA was extracted for real-time qPCR. mRNA levels are relative to those measured in cells transfected with transfection buffer. Data are shown as mean + SD of 3 independent experiments. One-way ANOVA followed by Tukey’s Multiple Comparison Test were used to determine statistical significance.
Figure 2. STING and TBK-1 knockdown leads to lower IFN-β and APOBEC3A induction by pVAX-Gag transfection. THP-1 cells were transfected with 250 nM of STING and/or TBK-1 Smartpool siRNAs or a control siRNA. STING (a) and TBK-1 (b) mRNA expression were determined by real-time qPCR 3 days after transfection. The cells were then transfected with pVAX-Gag and total RNA was extracted 8h after transfection for analysis of IFN-β (c) and APOBEC3A (d) mRNA expression. mRNA levels from control siRNA-transfected cells were considered as 100% and used to calculate STING and TBK-1 mRNA knockdown in A and B, respectively. mRNA levels in C and D were relative to those measured in cells first transfected with control
27
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siRNA and then with transfection buffer. Data are shown as mean + SD of 2 independent experiments.
Figure 3. APOBEC2 mediates the clearance of plasmid and plasmid-encoded mRNA. HEK293T cells were co-transfected with pmaxGFP and empty pcDNA3.1 (black bars) or pcDNA3.1-APOBEC2 (grey bars). Total DNA and RNA were separately extracted 1 day post transfection. The relative levels of plasmid (a) and plasmidencoded mRNA (b) were determined by real-time qPCR. Data from co-tranfection of pmaxGFP and empty pcDNA3.1 were normalized to one. Bars represent mean + SD of 3 independent experiments. Student’s T test was used to determine statistical significance.
Figure 4. APOBEC2 mediates the reduction of plasmid-encoded protein expression. HEK293T cells were co-transfected with pmaxGFP and empty pcDNA3.1, pcDNA3.1-APOBEC2 or HIVBr18. GFP expression was determined by flow cytometry 1 day (a) and 3 days (b) post transfection. Alternatively, HEK293T cells were transfected with pcDNA3.1, pmaxGFP + pcDNA3.1 or pmaxGFP + pcDNA3.1APOBEC2 and GFP expression was determined by fluorescence microcospy (c). HEK293T cells were also co-transfected with pmaxGFP and empty pcDNA3.1 or decreasing doses of pcDNA3.1-APOBEC2 (d). GFP expression was determined by flow cytometry 1 day post transfection. The amount of DNA used to transfect cells was maintained constant by adding empty pcDNA3.1 to the decreasing doses of pcDNA3.1APOBEC2. Bars represent mean + SD of 4 independent experiments. One-way ANOVA followed by Tukey’s Multiple Comparison Test were used to determine statistical significance. 28
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Figure 5. Schematic diagram of bicistronic DNA vaccines construction. The HIVBr18 gene was subcloned into pcDNA3.1 vector under CMV promoter control using Hind III and Xho I restriction enzymes. To construct the bicistronic DNA vaccines shAPO2Br18 and scBr18, expression cassettes (U6 promoter, poly (A) tail and a DNA sequencing expressing either APOBEC2 shRNA or a scrambled shRNA) were subcloned into pcDNA3.1-HIVBr18 at the MFE I restriction enzyme site.
Figure 6. APOBEC2 knockdown leads to increased DNA vaccine immunogenicity. HEK293T cells were co-transfected with pcDNA3.1-APOBEC2 and scBr18 or shAPO2Br18 and total RNA was extracted 1 day post transfection. Relative APOBEC2 mRNA expression was determined by real-time qPCR (a). Data from co-tranfection of pcDNA3.1-APOBEC2 and scBr18 were normalized to one. BALB/c mice were intramuscularly immunized with 1 dose of shAPO2Br18 or scBr18 (50 µg/animal). Total protein was extracted from injected muscle 4 days after immunization. APOBEC2 expression was determined by Western blotting. The APOBEC2 protein levels were determined by densitometry (b) and GAPDH levels were used for normalization. APOBEC2 and GAPDH levels are shown for individual mice (3 animals per immunization group), which are represented by numbers (c). Alternatively, BALB/c mice (n = 5) were intramuscularly immunized with 2 doses (50 µg/dose) of scBr18 or shAPO2Br18 every 20 days. Twenty days after the last dose mice were euthanized and pooled splenocytes were cultured in the presence of HIVBr18-encoded peptides. The frequency of IFN-γ secreting T cells was determined by ELISPOT (d). Bars represent mean + SD of 3 independent experiments for qPCR, mean + SD of 4 independent experiments for ELISPOT and mean + SD of 3 individual mice per immunization group for densitometry. Student’s T test was used to determine statistical significance. 29
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2015 Macmillan Publishers Limited. All rights reser
©
2015 Macmillan Publishers Limited. All rights reserved.
2015 Macmillan Publishers Limited. All rights rese
©
2015 Macmillan Publishers Limited. All rights reserved.
©
2015 Macmillan Publishers Limited. All rights reserved.
©
2015 Macmillan Publishers Limited. All rights reserved.
