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. 2012 Sep 25;21(1):251–259. doi: 10.1038/mt.2012.202

Type I IFN Counteracts the Induction of Antigen-Specific Immune Responses by Lipid-Based Delivery of mRNA Vaccines

Charlotte Pollard 1,2,*, Joanna Rejman 3, Winni De Haes 1, Bernard Verrier 4, Ellen Van Gulck 1, Thomas Naessens 2, Stefaan De Smedt 3, Pieter Bogaert 5, Johan Grooten 2, Guido Vanham 1,6, Stefaan De Koker 2
PMCID: PMC3538310  PMID: 23011030

Abstract

The use of DNA and viral vector-based vaccines for the induction of cellular immune responses is increasingly gaining interest. However, concerns have been raised regarding the safety of these immunization strategies. Due to the lack of their genome integration, mRNA-based vaccines have emerged as a promising alternative. In this study, we evaluated the potency of antigen-encoding mRNA complexed with the cationic lipid 1,2-dioleoyl-3trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE ) as a novel vaccination approach. We demonstrate that subcutaneous immunization of mice with mRNA encoding the HIV-1 antigen Gag complexed with DOTAP/DOPE elicits antigen-specific, functional T cell responses resulting in specific killing of Gag peptide-pulsed cells and the induction of humoral responses. In addition, we show that DOTAP/DOPE complexed antigen-encoding mRNA displays immune-activating properties characterized by secretion of type I interferon (IFN) and the recruitment of proinflammatory monocytes to the draining lymph nodes. Finally, we demonstrate that type I IFN inhibit the expression of DOTAP/DOPE complexed antigen-encoding mRNA and the subsequent induction of antigen-specific immune responses. These results are of high relevance as they will stimulate the design and development of improved mRNA-based vaccination approaches.

Introduction

Over the past decades, nucleic acid-based vaccines have emerged as a promising approach for the effective induction of antigen-specific cellular immune responses. As concerns have been raised regarding the safety of DNA and vector-based vaccines, the use of antigen-encoding mRNA for vaccination approaches is increasingly gaining interest.

mRNA-based vaccines present several advantages as compared with antigen delivery by viral vectors or plasmid DNA. First, mRNA cannot integrate in the host genome, therefore rendering its application much safer.1 Second, immunization with mRNA results in transient expression of the encoded protein, thus enabling a more controlled antigen exposure and minimizing the risk of tolerance induction. Third, additional sequences such as plasmid backbone or viral promotors are lacking. Finally, RNA does not need to cross the nuclear barrier for protein expression and therefore offers the possibility to transfect slow or nondividing cells.2

Although intranodal delivery of naked mRNA has been shown to elicit potent CD4+ and CD8+ T cell responses in mouse models,3,4,5 extranodal administration of naked mRNA appears to be more challenging because of its susceptibility to extracellular nucleases. Although some studies have reported the induction of immune responses in mice in response to non-nodal parenteral administration of naked mRNA,6,7,8 these findings have been contradicted by others.3,9,10

Nonviral carriers such as cationic polymers and cationic lipids have been used successfully to stabilize antigen-encoding DNA, thus increasing transfection efficiency and enhancing immune responses in vivo.11 In addition, such particulate delivery systems target antigen-specific cells (APCs) therefore resulting in enhanced antigen presentation and subsequent induction of immune responses.12 Nonetheless, few studies have explored this approach to increase delivery and effectiveness of mRNA vaccines in vivo. As a result, knowledge concerning potency of nonviral delivery of mRNA vaccines is scarce and data describing its immunological properties are largely lacking.9,10,13,14

In this study, we have demonstrated the feasibility of using antigen-encoding mRNA complexed with the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE) as a novel immunization strategy capable of evoking functional T cell responses. Furthermore, by studying the interaction between mRNA/carrier and dendritic cells, we have gained surprising insights regarding the role of type I interferons (IFNs) in modulating the mRNA-based immune response. These findings are of high relevance for the further design and development of RNA based vaccines, and will pave the way toward improved mRNA vaccination approaches.

Results

mRNA complexed with the cationic lipid DOTAP/DOPE induces functional, antigen-specific T-cell responses

In this study, two types of nonviral carriers were evaluated: the polymers poly(β-amino ester) and in vivo jetPEI, and the cationic lipids Lipofectamine 2000 and DOTAP/DOPE. Cationic polymers consist of repeating units of chemical or natural monomers and condense with nucleic acids to form rigid, colloidal nanocomplexes. In contrast, cationic lipids assemble into dynamic spherical vesicles composed of phospholipid bilayers surrounding an aqueous compartment. To determine whether complexation of mRNA by any of these carriers could augment antigen-specific immune responses, mice were immunized with 20 µg of gag mRNA complexed with polymers or cationic lipids. Splenocytes and lymph node cells were isolated 3 weeks after immunization and restimulated with Gag peptides in an IFN-γ enzyme-linked immunosorbent spot (ELISPOT). Gag-specific IFN-γ secreting T cells were observed in spleens and lymph nodes of mice immunized with gag mRNA complexed with the cationic lipids Lipofectamine 2000 and DOTAP/DOPE, but not in mice immunized with naked mRNA or polymer-complexed mRNA (Figure 1a). In addition, DOTAP/DOPE complexed mRNA was capable of evoking IFN-γ secreting T cells to a similar extent as DOTAP/DOPE complexed DNA, suggesting mRNA-based vaccines might equal the potency of DNA based vaccines when stabilized properly (Supplementary Figure S1 online). For the subsequent detailed analysis of the T cell stimulatory capacity of antigen-encoding mRNA, DOTAP/DOPE was used to complex gag mRNA as this carrier induced superior T-cell responses.

Figure 1.

Figure 1

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DOTAP/DOPE complexed antigen-encoding mRNA induces potent antigen-specific immune responses. (a) Mice were immunized s.c. with PBS, 20 µg of Gag-encoding mRNA, either as such (“naked”) or complexed with the indicated carriers. At week 4, mice were boosted with the same formulation. Spleens and lymph nodes were isolated at week 8, and the number of Gag-specific interferon-γ secreting cells was determined by enzyme-linked immunosorbent spot. Means ± SEM are shown of a total of 5 mice per group. PBAE, poly(β-amino ester). (b) Frequencies of CD69+ CD4+ and CD8+ T cells in draining lymph nodes 1 day after footpad injection of 16 µl DOTAP/DOPE (DOTAP), 4ug of naked Gag-encoding mRNA (naked gag) or 4 µg DOTAP/DOPE-complexed Gag-encoding mRNA (DOTAP gag). Means ± SD are shown of a total of 6 mice per group. (c) In vivo killing assay was performed by adoptive transfer of carboxyfluorescein diacetate succinimdyl ester-labeled peptide-loaded splenocytes to mice immunized with 20 µg of naked gag or DOTAP gag. The ratio of Gag-pulsed versus unpulsed cells in 4 mice is shown. ***P < 0.001. (d) Gating strategy for B cells and germinal center (GC) B cells. (e) The frequencies of B cells and GC B cells were determined by flow cytometry 7 days post footpad injection of 16 µl DOTAP, 4 µg naked gag or DOTAP gag. B cells were defined as CD45+ CD19+ CD3− cells, and GC B cells as GL-7+ CD95+ B cells. Means ± SD are shown of a total of 6 mice per group.

Because the activation of naive T cells constitutes the first step in adaptive immunity, we evaluated the expression of the early activation marker CD69 on T cells in the draining lymph nodes of mice injected in the footpad with 4 µg of DOTAP/DOPE-complexed gag mRNA (DOTAP gag). The popliteal lymph nodes were isolated 24 hours later, and the number of CD69+ cells within gated CD4+ and CD8+ T cells was determined by flow cytometry. Our results show that there was a significant increase in the number of CD69+ T cells in the lymph nodes of mice receiving DOTAP gag as compared with DOTAP/DOPE-injected control mice (DOTAP) or mice receiving naked gag (Figure 1b). Induction of strong cytotoxic T cell responses is considered one of the prerequisites for the development of successful therapeutic vaccines against cancer and HIV. To address whether DOTAP/DOPE complexed mRNA-based immunization was capable of inducing functional “killer” responses, we performed an in vivo cytotoxicity assay. Immunization with DOTAP gag resulted in a significant increase of Gag-specific killing as compared with immunization with naked gag or DOTAP/DOPE alone (Figure 1c).

The potency of DOTAP gag-induced CD4 T-helper response to stimulate B cell immunity was evaluated by analyzing the induction of germinal center (GC) B cells. During the development of T cell-dependent antibody responses, GCs are formed by proliferating B cells and provide the main source of memory B cells and long-living plasma cells.15 The number of GC cells (CD3− CD19+ GL-7+ CD95+) within the lymph node B cell population (CD3− CD19+) was quantified 7 days after the administration of DOTAP gag by flow cytometry. We observed a significant increase in GC cells as compared to mice receiving naked gag or DOTAP control (Figure 1d,e).

In conclusion, these findings demonstrate that immunization with DOTAP/DOPE-complexed antigen-encoding mRNA induces antigen-specific, functional T cells resulting in potent cytotoxic T lymphocytes and GC formation.

Prime-boost strategy enhances antigen-specific immune responses in both lymphoid and nonlymphoid tissues

The use of prime-boost strategies, in which prime and boost immunization consist of different vaccine modalities, has been shown to efficiently increase the potency of both DNA and vector-based vaccines. To evaluate whether this principle also applies to mRNA immunization, a protein boost with the HIV-1 capsid protein p24 (processed from the Gag polyprotein) was administered subcutaneously to DOTAP gag primed mice. Boosting with the p24 protein resulted in an over sevenfold increase of antigen-specific IFN-γ secreting cells in both spleens and lymph nodes of DOTAP gag immunized mice (Figure 2a). In addition, induction of antigen-specific interleukin (IL)-2 production was observed in mice receiving DOTAP gag + p24, but not in mice receiving DOTAP gag alone. Surprisingly, Gag-specific IL-2 secreting T cells were also detected in lymph nodes of mice receiving only p24 protein, without DOTAP gag priming.

Figure 2.

Figure 2

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Prime-boost strategy enhances antigen-specific immune responses. Mice were immunized by 2 s.c. injections of 20 µg of naked or DOTAP gag with a 3-week interval. At week 8, some mice were boosted with 20 µg of the capsid protein p24 either subcutaneously (a and b) or intratracheally (c). T cell and B cell responses were analyzed 10 days after the p24 protein boost. (a) Number of Gag-specific interferon (IFN)-γ and interleukin-2 secreting T cells was determined by enzyme-linked immunosorbent spot (ELISPOT) on isolated spleen and lymph node cells. Means ± SEM are shown of a total of 5 mice per group. (b) Anti-p24 immunoglobulin (Ig)M, IgG1, and IgG2c titers were determined by serum enzyme-linked immunosorbent assay. Means ± SEM are shown for 5 mice, immunized with naked gag and 5 with DOTAP gag, each followed by p24 boost. (c) Number of Gag-specific IFN-γ secreting CD4 and CD8 T cells was determined by ELISPOT on isolated lung cells. Means ± SEM are shown of a total of 5 mice per group.

Next, we determined whether the p24 protein boost could induce p24-specific humoral responses. A serum enzyme-linked immunosorbent assay was performed to assess the levels of p24-specific immunoglobulin (Ig)M, IgG1, and IgG2c antibodies. Of note, immunization with DOTAP gag plus p24 led to relatively high antibody titers, whereas naked gag plus p24 (which was able to induce IL-2) failed to induce p24-specific antibodies (Figure 2b). Serum levels of IgG1 were relatively low in comparison with IgG2c, thereby indicating a preference for Th1-dependent class switching.

Vaccine-elicited effector memory T cells that migrate from the lymphoid site of immune induction to the peripheral site of pathogen entry where they can exert their effector functions without requiring preceding rounds of antigen stimulation and cell division in the lymph nodes, might be of great importance to eliminate infection very early upon pathogen entry. Therefore, the induction of such effector memory T cells might be crucial for the development of an effective vaccine against insidious pathogens such as HIV.16 To address the capacity of parenteral mRNA-based vaccination in evoking the recruitment of effector T cells toward peripheral (mucosal) tissues, CD4 and CD8 T cells were isolated from the lungs one month following the subcutaneous booster immunization and subsequently restimulated with Gag peptides in an IFN-γ ELISPOT assay. Remarkably, even without preceding pulmonary antigen challenge, high levels of Gag-responsive T cells were present in the lung, capable of rapidly secreting IFN-γ upon antigenic stimulation. Although low levels CD4 T cell responses were detectable, T cell responses appeared to be largely limited to the CD8 T cell compartment, a phenomenon easily anticipated given the need for endosomal mRNA escape and subsequent cytosolic expression of the encoded antigens, which are consequently presented via major histocompatibility complex (MHC)-I. Administration of a local pulmonary p24 protein boost dramatically increased the level of the CD4 T cell response (40-fold), and even further amplified the CD8 T cell response, albeit to a less degree. Whether these amplified T cell responses are a result from local antigen presentation and T cell proliferation, or whether they represent newly recruited T cells from the draining lymph nodes remains to be determined. In any case, delivering a protein boost appears to dramatically amplify the CD4 T cell response, a feature that can be explained by the mainly MHC-II mediated route of antigen presentation of soluble antigens. Given the role of CD4 T cells in supporting CD8 T cell function, the administration of a protein boost might significantly enhance the quality and longevity of the mRNA evoked CD8 T cell response.

DOTAP/DOPE complexed mRNA displays intrinsic adjuvant activity

Most traditional vaccines consisting of recombinant protein antigens fail to activate pattern recognition receptors, therefore requiring the addition of adjuvants to evoke effector responses.17 In this respect, the use of mRNA vaccines may confer an extra advantage as in addition to encoding the antigen, DOTAP/DOPE complexed mRNA might also display intrinsic adjuvant capacity, as dendritic cells (DCs) express various pattern recognition receptors including TLR3 and TLR7/8 that recognize respectively double stranded RNA and single stranded RNA.18,19

A first indication that DOTAP/DOPE complexed mRNA entails intrinsic adjuvant properties came from assessing the maturation status of bone marrow-derived DCs transfected with 2.5 µg of DOTAP gag. As depicted in Figure 3a, successfully transfected cells (Gag+ DCs) expressed elevated levels of the co-stimulatory markers CD40, CD80, and CD86 as compared with their nontransfected counterparts (Gag- DCs). Moreover, quantitative real-time reverse transcript PCR (RT-qPCR) analysis of DCs incubated with DOTAP gag showed a modest increase in expression levels of the proinflammatory cytokines IL-6 and IL-1β, and a profound upregulation of the type I IFNs IFN-α and IFN-β (Figure 3b). This massive increase in expression levels of type I IFN was not observed in DCs exposed to naked gag or DOTAP alone.

Figure 3.

Figure 3

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DOTAP/DOPE complexed mRNA displays intrinsic adjuvant activity. (a) Expression pattern of maturation markers of bone marrow-derived DCs (BMDCs) transfected with DOTAP gag. BMDCs were transfected with 2.5 µg DOTAP gag and the expression of major histocompatibility complex (MHC)-II, CD80, CD86, and CD40 on Gag+ and Gag-CD11c+ cells was analyzed 24 hours later by flow cytometry. (b) Transcript levels of interleukin (IL)-6, IL-1β, IFN-α and interferon (IFN)-β mRNA were determined by RT-qPCR 24 hours (IL-6 and IL-1β) or 6 hours (IFN-α and IFN- β) after transfection of BMDCs with 2.5 µg DOTAP gag. Results represent mean n-fold induction levels compared to unstimulated control cells ± SD from triplicate PCR reactions. (c and d) Nuclear translocation of IFN regulatory factors (IRF)-3 and IRF-7 was visualized by confocal microscopy at different time points after stimulation of BMDCs with 2.5 µg DOTAP gag. (c) Confocal images taken 5 minutes after incubation with DOTAP or DOTAP gag and (d) ratios of protein present in the nucleus to total protein. Results represent mean ± SD of 100 cells. (e) Transcript levels of IFN-α and IFN-β mRNA were determined by RT-qPCR 6 hours after transfection of WT, MyD88−/− or TRIF−/− DCs with 2.5 µg DOTAP gag. Results represent mean n-fold induction levels compared to unstimulated control cells ± SD from triplicate PCR reactions. TRIF, TIR-domain-containing adapter-inducing interferon-β. (f) Mice were injected i.v. with 20 µg naked gag or DOTAP gag. Serum levels of IFN-α were determined 1, 3, 6, and 24 hours later by enzyme-linked immunosorbent assay. Mean ± SEM of 4 mice per group is shown. (g) Flowcytometric analysis of DC subsets and inflammatory monocytes present in the draining lymph nodes 1 and 3 days after footpad injection of 4µg of DOTAP gag. Conventional DCs were defined as CD11chi MHCIIint, Tissue-derived DCs were CD11cint MHCIIhi and inflammatory monocytes were MHC-II+ Ly6chi CD11bhi. Mean ± SEM 6 mice per group is shown. ***P < 0.001 as compared with the corresponding inflammatory monocyte numbers in DOTAP injected control mice and mice injected with naked gag.

These findings were confirmed by studying the nuclear translocation of the IFN regulatory factors (IRF) IRF-3 and IRF-7 using confocal microcopy. Upon activation, IRF-3/7 dimerizes and translocates to the nucleus to activate transcription of type I IFN genes. As shown in Figure 3c,d, both IRF-3 and IRF-7 rapidly migrated to the nucleus after in vitro stimulation of DC with DOTAP gag.

To evaluate to what extent the strong type I IFN response to DOTAP/DOPE complexed mRNA was dependent on TLR3 and/or TLR7 mediated signaling, we compared expression levels of type IFN in bone marrow-derived DCs isolated from MyD88−/− (defective in TLR7 signaling), TIR-domain-containing adapter-inducing interferon-β (TRIF)−/− (defective in TLR3 signaling) and wild-type (WT) mice. Although MyD88 deficiency had very little impact on type I IFN expression, TRIF deficiency in contrast evoked a relatively strong decrease in IFN-α expression levels and a moderate downregulation of IFN-β expression (Figure 3e).

The capacity of DOTAP/DOPE complexed mRNA to induce type I IFN was also confirmed in vivo by performing enzyme-linked immunosorbent assay assays on serum isolated 1, 3, 6, and 24 hours after i.v. administration of 20 µg DOTAP gag. We observed a transient induction of IFN-α, with serum levels peaking at 3–6 hours (Figure 3f).

Furthermore, footpad injection of DOTAP gag resulted in a rapid, but transient influx of inflammatory monocytes (MHC-II+ Ly6Chi CD11bhi cells) to the draining lymph nodes (Figure 3g). Since the recruitment of inflammatory monocytes is typically observed following microbial infection or adjuvant injection, these data confirm the intrinsic immune-stimulatory properties of DOTAP/DOPE complexed mRNA.20

Expression of antigen-encoding mRNA and subsequent induction of immune responses is inhibited by type I IFN

Although type I IFNs are considered to be important promoters of cellular immunity,21,22,23,24 their induction might interfere with the potency of mRNA-based vaccines by inducing antiviral responses resulting in break-down of exogenous mRNA and inhibition of translation.25 To address these issues, IFNαR−/− mice were immunized with DOTAP gag. Surprisingly, we observed a strong increase in the levels IFN-γ and IL-2 secreting T cells as compared with WT mice, suggesting a rather negative effect of type I IFN (Figure 4a).

Figure 4.

Figure 4

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Type I IFN inhibitexpression of antigen-encoding mRNA and subsequent induction of immune responses. (a) Wild-type (WT) and interferon (IFN)αR−/− mice were immunized with 20µg DOTAP gag as previously described. Gag-specific IFN-γ and interleukin-2 secreting T cells were determined by enzyme-linked immunosorbent spot on isolated spleens. Mean of 5 mice per group is shown. ***P < 0.001. (b and c) WT, MyD88−/−, TRIF−/−, and IFNαR−/− bone marrow-derived DCs were transfected with 2.5 µg of DOTAP/DOPE-complexed Gag-encoding mRNA. Mean percentage ± SD of Gag+ CD11c+ cells (b) and expression pattern of the maturation markers major histocompatibility complex (MHC)-II, CD80, CD86, and CD40 on Gag+ and Gag- CD11c+ cells (c) was determined 24 hours later by flowcytometry. TRIF, TIR-domain-containing adapter-inducing interferon-β. (d) Flowcytometric analysis of DC subsets and inflammatory monocytes present in the draining lymph nodes of WT and IFNαR−/− mice 1 day post footpad injection of 4µg DOTAP gag. Conventional DCs were defined as CD11chi MHCIIint, tissue-derived DCs were CD11cint MHCIIhi and inflammatory monocytes were MHC-II+ Ly6chi CD11bhi. Mean ± SEM of 6 mice per group is shown.

To investigate whether this effect might indeed be due to type I IFN-mediated inhibition of antigen expression, we compared efficiency of Gag expression following DOTAP gag transfection in WT and IFNαR−/− DCs. As depicted in Figure 4b, the frequency of Gag positive DCs was clearly elevated in IFNαR−/− DCs. Similar results were obtained with egfp mRNA (Supplementary Figure S2 online), suggesting that this phenomenon is independent of the specific mRNA sequence of Gag. MyD88−/− DCs showed no increase in Gag expression, which is in agreement with the abovementioned finding that expression of type I IFN is not affected in MyD88−/− DCs. Although Gag expression did not reach the same level as in IFNαR−/− DCs, TRIF−/− DCs showed a clear increase in Gag expression as compared to WT cells. Again, these data are in accordance with the earlier observed intermediate decrease in type I IFN in TRIF−/− DCs, indicating that DOTAP gag-induced expression of type I IFN is partially dependent on TLR3 signaling.

Although the capacity of type I IFN to link innate to adaptive immunity has been shown to be crucial for the adjuvant activity of TLR3 and TLR7 agonists,26,27 they clearly interfere with the potency of mRNA-based vaccination. Nevertheless, activation of innate immune responses is a critical prerequisite for all vaccines to induce adaptive immune responses. To determine to what extent IFNαR signaling is required for the DOTAP gag-mediated activation of innate immunity, we evaluated the capacity of DOTAP gag to activate IFNαR−/− DCs and to promote the recruitment of inflammatory monocytes following subcutaneous injection in IFNαR−/− mice. Similar to WT cells, we observed an upregulation of the maturation markers CD40 and CD86 (but not CD80) on DOTAP gag-transfected IFNαR−/− bone marrow-derived DCs (Figure 4c). In addition, subcutaneous injection of DOTAP/DOPE complexed mRNA still resulted in the infiltration of inflammatory monocytes in the draining lymph nodes (Figure 4d). When compared with WT mice injected with DOTAP gag, total numbers of recruited inflammatory monocytes were lower in IFNαR−/− mice, a phenomenon which might however be at least partially explained by the basal lower counts of DCs and monocytes in the lymph nodes of IFNαR−/− mice.

Taken together, these results demonstrate that IFNαR signaling is not required for the innate immune activation and recruitment of DCs in DOTAP gag immunized mice.

Discussion

In recent years, antigen-encoding mRNA has emerged as a promising alternative to classical DNA and viral vector-based immunization approaches. mRNA vaccines have sparked interest particularly in the field of tumor vaccination, as these strategies hold the potential to introduce a broad spectrum of patient-specific tumor associated antigens.28,29 Moreover, the use of mRNA vaccines could also be beneficial in the context of therapeutic HIV vaccination as it enables the delivery of multiple HIV genes and their mutations, thus allowing the approximate patient-specific representation of the quasi species of the virus.30

In this work, we have evaluated the potency of an mRNA vaccine complexed with the cationic lipid DOTAP/DOPE in an in vivo preclinical mouse model. In addition, we have investigated the mechanisms contributing to the immunogenicity of DOTAP/DOPE mediated delivery of mRNA. Finally, we have studied the role of type I IFN in the induction of immune responses against the encoded antigen.

In a first set of experiments, we assessed the capacity of different nonviral carriers to induce antigen-specific immune responses against mRNA encoding the HIV-1 protein Gag. Our results identified DOTAP/DOPE as a potent carrier for subcutaneous delivery of mRNA to induce immune responses, a feature which could not be achieved using naked mRNA. DOTAP/DOPE mRNA vaccination appeared to strongly evoke CD8 T cells, which were fully capable of recognizing and killing peptide-pulsed target cells in vivo. Of note, subcutaneous immunization allowed the emigration of effector T cells to nonlymhoid tissues, as was demonstrated by the high numbers of Gag-responsive CD8 T cells in the lungs of DOTAP/DOPE-complexed gag mRNA immunized mice. The presence of high numbers of such effector memory cells at the site of pathogen entry might be of significant importance for the development of effective vaccines against insidious pathogens that rapidly spread or evade immunity. Further studies are needed to address whether such CD8 T cells are also present in the gastrointestinal and/or genital tract, the sites of HIV entry. As could be anticipated by the cytosolic expression of the mRNA encoded antigen, mRNA-based vaccination mainly promotes the induction of CD8 T cell responses. Nevertheless, low levels of antigen-specific CD4 T cells were present in the pulmonary compartment, and their numbers could be dramatically increased by delivering a protein boost, which also strongly amplified the T cell response in the lymphoid compartment and enabled the generation of a Th1-skewed humoral immune response.

During the last decade, it has become increasingly clear that the induction of effector T- and B cell responses largely depends on the initial activation of the innate immune system via a set of pattern recognition receptors. Given the robust induction of T- and B cell responses observed in response to lipid-based mRNA vaccination, we speculated that DOTAP/DOPE complexed mRNA may act as a viral mimic by triggering RNA recognizing pattern recognition receptors in the endosomes or cytosol of DCs.

We showed that bone marrow-derived DCs upregulated CD40 and CD86 in response to DOTAP gag, indicating activation of DCs. Moreover, we observed a moderate increase of IL-6, IL-1β, and a profound induction of type I IFN, a cytokine typically released in response to viral RNA.31 Data obtained from TRIF knockout DCs indicated that DOTAP gag-induced DC activation is partially dependent on TLR3 signaling. However, it is likely that other signaling pathways are also involved, as endosomal escape of lipoplexes may enable activation of cytosolic RNA sensors.

The intrinsic capacity of DOTAP/DOPE complexed antigen-encoding mRNA to activate the innate immune system was also confirmed in vivo by the systemic release of type I IFN and the rapid recruitment of inflammatory monocytes to the draining lymph nodes of DOTAP gag immunized mice.

The induction of type I IFN in response to antigen-encoding mRNA is possibly a double edged sword. Although systemic production of type I IFN has been shown to be crucial to the adjuvant activity of polyI:C and polyUs21,26,27 type I IFN mediate antiviral responses resulting in break-down of exogenous mRNA and inhibition of translation.25 This may be particularly unfavorable when using mRNA to encode antigens for vaccination.

Supporting this hypothesis, we found that transfection of IFNαR−/− DCs resulted in higher numbers of positively-transfected cells as compared with WT cells. Moreover, immunization experiments in IFNαR−/− mice resulted in a tremendous increase in Gag-specific T cell responses as compared with WT mice. In addition, we have demonstrated that IFNαR signaling is not crucial for the innate immune activation and recruitment of DCs in DOTAP gag immunized mice.

These findings are of high significance as they demonstrate that RNA vaccines should be designed to minimize the induction of type I IFN, therefore enhancing the potency of such vaccines. The use of modified RNA nucleotides could be a feasible and potent way to enhance the efficacy of RNA vaccines, as they have been reported to release less type I IFN.32

In conclusion, we have demonstrated that DOTAP/DOPE complexed antigen-encoding mRNA potently induces systemic T cell immunity. In addition, we have identified type I IFN as a negative factor in the induction of immune responses by antigen-encoding mRNA. These findings are of high relevance as they will impact the design and development of novel RNA vaccines.

Methods

Mice. Female C57BL/6 mice were purchased from Janvier (Le Genest Saint Isle, France). IFNαR−/−, MyD88−/−, and TRIF−/− mice were bred at the breeding facility of the Vlaams Instituut voor Biotechnolgoy (VIB). Mice were 6–8 weeks old at the start of the experiment and maintained under specific pathogen-free conditions. All experiments were approved by the local ethical committee for animal experiments.

Production of in vitro transcribed mRNA. The pGEM4Z/hHxB-2-gag/A64 plasmid, encoding a “humanized”, codon-optimized HxB-2 HIV-1 Gag protein, was generated as previously described.33 The pGEM4Z/ enhanced green fluorescent protein (EGFP)/A64 and was kindly provided by Dr David Boczowski from Duke University. The plasmids were propagated in Escherichia coli supercompetent cells (Stratagene, La Jolla, CA) and purified using endotoxin-free QIAGEN-tip 500 columns (Qiagen, Chatsworth, CA). The pGEM4Z/hHxB-2-gag/A64 and pGEM4Z/EGFP/A64 plasmids were linearized with SpeI (MBI Fermentas, St Leon-Rot, Germany) and purified using a PCR purification kit (Qiagen, Venlo, The Netherlands). RNA was transcribed from the linearized plasmids using the T7 mMessageMachine Kit (Ambion, Austin, TX) according to the manufacturer's instructions.

Immunization experiments. C57BL/6 mice were immunized twice s.c. at a 3-week interval with 20 µg of Gag-encoding mRNA complexed with 20 µl Lipofectamine 2000 (Invitrogen, Merelbeke, Belgium), 20 µl poly(β-amino ester) (produced by Joanna Rejman, Ghent University), 3.2 µl in vivo jetPEI (PolyPlus Transfection, Illkirch, France) or 40 µl of DOTAP/DOPE (Avanti Polar Lipids, Alabaster, AL) in a total volume of 200 µl of PBS (Lipofectamine and poly(β-amino ester)) or 5% glucose water (DOTAP/DOPE and in vivo jetPEI). Spleens, lymph nodes and serum were isolated 3 weeks post booster-immunization. In specified experiments, mice immunized with DOTAP/DOPE-complexed gag mRNA (DOTAP gag) received a protein boost of 20 µg p24 (produced by Bernard Verrier, Université Claude Bernard de Lyon) either subcutaneously or intratracheally 3 weeks after the last injection of DOTAP gag. For comparison of Gag-encoding mRNA versus plasmid DNA vaccination, mice were injected with 10 µg of DOTAP/DOPE complexed gag mRNA or 10 µg of DOTAP/DOPE complexed plasmid encoding the hHxB-2-gag sequence after a CMV promoter.

Flow cytometry. All flow cytometric experiments were performed on a triple-laser (B-V-R) LSR-II (Becton Dickinson, San Jose, CA) and analyzed with FACSDiva (Becton Dickinson) and FlowJo (Treestar, OR). Cells were stained with α-CD16/CD32 (BD Biosciences, San Diego, CA) to block nonspecific FcR binding, and with Live/Dead Fixable Aqua stain (Invitrogen) to eliminate dead cells from analysis. Antibodies used are α-CD3 Pacific Blue or PE, α-CD4 FITC, α-CD8 PerCP, α-CD69 PE-Cy7, α-CD45 Horizon-V450, α-CD19 APC-Cy7, α-GL-7 FITC, α-CD95 PE-Cy7, α-CD11c APC, α-CD40 biotin/streptavidin-PerCP, α-CD80 FITC, α-CD86 PE-Cy7, α-MHC-II PE-Cy7, α-CD11b APC-Cy7, α-Ly-6C Horizon-V450 (all BD Biosciences), and α-MHC-II eFluor450 (eBioscience, Vienna, Austria).

T cell activation analysis. Mice were injected in the footpad with 4 µg of gag mRNA complexed with 8 µl DOTAP/DOPE. Popliteal lymph nodes were isolated 24 hours later and digested with 150 u/ml collagenase II (Sigma-Aldrich, St Louis, MO) to obtain single cell suspensions. Cells were stained with α-CD3, α-CD4, α-CD8, and α-CD69 and analyzed by flow cytometry.

ELISPOT. Spleens and lymph nodes of immunized mice were passed through 70 µm nylon meshes (BD Biosciences) to obtain single cell suspensions. Red blood cells were lysed using ACK red blood cell lysis buffer (BioWhittaker, Wakersville, MD) and 2.5 × 105 cells were cultured for 24 hours on IL-2 and IFN-γ (Diaclone, Besançon, France) pre-coated 96-well plates in the presence of 2 µg/ml Gag peptide pool (NIH, Germantown, MD). For ELISPOT analysis on lung T cells, lungs of immunized mice were digested with 150 u/ml collagenase II and passed through 70 µm nylon meshes to obtain single cell suspensions. CD4 and CD8 T cells were isolated using CD4 and CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. 1.0 × 105 cells were cultured for 24 hours on IFN-γ pre-coated 96-well plates in the presence of 2 µg/ml Gag peptide pool. 1.0 × 105 splenocytes from naive mice were added to each well as antigen-presenting cells. ELISPOTs were analyzed according to the manufacturer's instructions.

In vivo cytotoxicity assay. Splenocytes from female C57BL/6 mice were pulsed with 30 µmol/l of Gag peptide or left unpulsed before labeling with 5µmol/l carboxyfluorescein diacetate succinimdyl ester or 20 µmol/l chloromethyl benzoyl aminotetramethylrhodamine, respectively (both purchased from Invitrogen). Labeled cells were mixed at a 1:1 ratio, and a total of 1 × 108 cells mixed cells were adoptively transferred into immunized mice. Splenocytes from host mice were analyzed 4 days later by flow cytometry. Percentage antigen-specific cytotoxicity was determined using the following formula: (percentage of unpulsed cells)/(percentage of cells pulsed with Gag peptide) × 100.

GC B cell analysis. Mice were vaccinated in the footpad with 4 µg of gag mRNA complexed with 8 µl DOTAP/DOPE. Single cell suspensions of the draining popliteal lymph nodes were prepared 7 days post footpad injection. Cells were stained with α-CD3, α-CD45, α-CD19, α-GL-7, and α-CD95 and analyzed by flow cytometry.

Enzyme-linked immunosorbent assay for antibody responses. Maxisorp plates (Nunc, Zaventem, Belgium) were coated overnight at 4 °C with 5 µg/ml p24 protein and incubated with serial dilutions of serum. P24-specific antibodies were determined by use of horseradish peroxidase -coupled anti-mouse immunoglobulin M (AbD Serotec, Dusseldorf, Germany), IgG1 (Southern Biotech, Birmingham, AL) and IgG2c (ICL, Portland, OR) antibodies.

Transfection of DCs. DC primary cultures were generated from bone marrow of WT C57BL/6 mice or IFNαR−/− mice. At day 8.5 × 105 cells were transfected with 2.5 µg of Gag or EGFP-encoding mRNA complexed with 15 µl DOTAP/DOPE for 20 hours. DOTAP egfp and DOTAP gag-transfected cells were stained with α-CD11c. Subsequentely, DOTAP gag-transfected cells were fixed and permeabilized using LEUKOPERM kit (AbD Serotec, Düsseldorf, Germany) and stained with anti-Gag KC57-RD-1 (Beckman Coulter, Suarlée, Belgium). Gag and EGFP expression was analyzed by flow cytometry. Expression of maturation markers was analyzed by flow cytometry after staining the cells with, α-MHC-II, α-CD40, α-CD11c, α-CD80, and α-CD86.

Quantitative reverse transcriptase PCR. RNA isolation was performed using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's protocol. cDNA was synthesized using a Superscript II Reverse Transcription Reagent Kit (Invitrogen). Real-time quantitative PCR (qPCR) was performed on a Lightcycler 480 using a qPCR kit for SYBR Green I (both Roche Diagnostics, Mannheim, Germany). Real-time qPCR amplification was performed in triplicate reactions. mRPL13a mRNA was used as reference housekeeping gene for normalization. All primers were purchased from Invitrogen.

Confocal microscopy. 2.5 × 104 bone marrow-derived DCs were incubated with 2.5 µg DOTAP gag in 8-well microscopy chambers. Subsequentely, cells were fixed with 2 % paraformaldehyde and permeabilized with 0.2 % triton-100. Samples were stained with rabbit polyclonal anti-mouse IRF-3 and IRF-7 (Santa Cruz Biotechnology, Heidelberg, Germany). Anti-rabbit IgG Alexa Fluor 633 (Invitrogen) was used as a secondary antibody. Cell nuclei were stained with 4′-6-diamidino-2-phenylindole and Vectashield (Vector Laboratories, Peterborough, UK) was used to mount the samples. Confocal microscopy images were recorded on a Leica SP5 confocal microscope equipped with a 40×/1.25 oil immersion objective. Ratio of protein in the nucleus to total protein was determined by using Volocity software (Perkin Elmer, Watlham, MA) to calculate voxel counts.

Characterization of DC subsets. Mice were vaccinated in the footpad with 4 µg of gag mRNA complexed with 8 µl DOTAP/DOPE. Single cell suspensions of the draining popliteal lymph nodes were prepared 1 or 3 days post footpad injection. Cells were stained with α-MHC-II, α-CD11b, α-CD11c, and α-Ly-6C.

Measurement of in vivo IFN-α secretion. Mice were injected i.v. with 20 µg of naked or DOTAP/DOPE complexed Gag-encoding mRNA and serum was collected at the indicated time points. Production of IFN-α was determined by enzyme-linked immunosorbent assay (PBL IFN Source, Piscataway, NJ) according to the manufacturer's instructions.

Statistical analysis. Statistics were performed using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA). Statistical significance was determined using a two-tailed Student's t-test with a 95% confidence interval when comparing two independent experimental groups, or one-way ANOVA followed by Newman-Keuls post-test when comparing multiple groups.

SUPPLEMENTARY MATERIAL Figure S1. Immunization with DOTAP/DOPE-complexed mRNA or DNA elicits similar amounts of antigen-specific T cells. Figure S2. Type I IFN inhibit expression of antigen-encoding mRNA and subsequent induction of immune responses.

Acknowledgments

We are grateful to the NIH AIDS Research and Reference Reagent Program for providing the peptides, to Benjamin Vandendriessche (Laboratory of Molecular Pathophysiology and Experimental Therapy, Ghent University, Ghent, Belgium) for his help with i.v. injections and to Amanda Goncalves for her support with the confocal microscope. This work was supported by grants from IUAP (Inter-University Attraction Poles, P6/41 of the Belgian Government), FWO (Fund for Scientific Research Flanders, project number G.0226.10), SBO (Strategic Basic Research Program of the Flemish government, project number 80016), Europrise (European Vaccines and Microbicides Enterprise, project number 037611), Ghent University (Methusalem BOF09/01M00709) and SOFI-B (Secondary Research Funding ITM). C.P. acknowledges the SOFI-B for a PhD scholarship. W.D.H is a predoctoral fellow of the agency for innovation by Science and Technology in Flanders (IWT). The authors declare no conflict of interest.

Supplementary Material

Figure S1.

Immunization with DOTAP/DOPE-complexed mRNA or DNA elicits similar amounts of antigen-specific T cells.

Click here for additional data file. (1.6MB, tiff)
Figure S2.

Type I IFN inhibit expression of antigen-encoding mRNA and subsequent induction of immune responses.

Click here for additional data file. (2.1MB, tiff)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Immunization with DOTAP/DOPE-complexed mRNA or DNA elicits similar amounts of antigen-specific T cells.

Click here for additional data file. (1.6MB, tiff)
Figure S2.

Type I IFN inhibit expression of antigen-encoding mRNA and subsequent induction of immune responses.

Click here for additional data file. (2.1MB, tiff)

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