Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity
© Tenbusch et al; licensee BioMed Central Ltd. 2008
Received: 07 August 2007
Accepted: 11 April 2008
Published: 11 April 2008
Granulocyte-macrophage colony-stimulating factor (GM-CSF) has shown promising results as a cytokine adjuvant for antiviral vaccines and in various models of tumor gene therapy. To explore whether the targeting of antigens to GM-CSF receptors on antigen-presenting cells enhances antigen-specific CD8 T-cell responses, fusion proteins of GM-CSF and ovalbumin (OVA) were expressed by DNA and adenoviral vector vaccines. In addition, bicistronic vectors allowing independent expression of the antigen and the cytokine were tested in parallel.
In vitro, the GM-CSF ovalbumin fusion protein (GM-OVA) led to the better stimulation of OVA-specific CD8+ T cells by antigen-presenting cells than OVA and GM-CSF given as two separate proteins. However, prime-boost immunizations of mice with DNA and adenoviral vector vaccines encoding GM-OVA suppressed CD8+ T-cell responses to OVA. OVA-specific IgG2a antibody levels were also reduced, while the IgG1 antibody response was enhanced. Suppression of CD8+ T cell responses by GM-OVA vaccines was associated with the induction of neutralizing antibodies to GM-CSF. In contrast, the coexpression of GM-CSF and antigens in DNA prime adenoviral boost immunizations led to a striking expansion of polyfunctional OVA-specific CD8+ T cells without the induction of autoantibodies.
The induction of autoantibodies suggests a general note of caution regarding the use of highly immunogenic viral vector vaccines encoding fusion proteins between antigens and host proteins. In contrast, the expansion of polyfunctional OVA-specific CD8+ T cells after immunizations with bicistronic vectors further support a potential application of GM-CSF as an adjuvant for heterologous prime-boost regimens with genetic vaccines. Since DNA prime adenoviral vector boost regimenes are presently considered as one of the most efficient ways to induce CD8+ T cell responses in mice, non-human primates and humans, further enhancement of this response by GM-CSF is a striking observation.
The induction of strong CTL responses by prophylactic and therapeutic vaccines is considered necessary for the control of chronic viral infections and cancer [1–3]. Genetic vaccines seem to be promising tools, since the expression of antigen by the vaccinee leads to improved MHC-I restricted cellular immune responses. DNA vaccines have been shown to elicit CTL, T helper and antibody responses in a variety of animal models [4–8]. However, DNA vaccines alone stimulated only weak T-cell responses in monkeys  and humans . To enhance antigen expression levels, various viral vector vaccines have been explored. For example, antigens expressing viral vectors based on poxviruses or adenoviruses were shown to be potent inducers of antigen-specific immune responses in SIV/HIV vaccine studies [9, 11]. In addition to increased expression levels, the triggering of innate immune responses by the viral vector particles also seems to contribute to the immunogenicity of viral vector vaccines. However, in contrast to DNA vaccines, repeated immunizations with the same viral vector vaccine appear to be limited by immune responses to the viral vector particles [12, 13]. Thus, DNA prime viral vector boost regimens are considered to be one of the most promising strategies to induce long-lasting CTL responses in humans [9, 14, 15].
GM-CSF expression plasmids were co-injected with plasmids encoding vaccine antigens to examine the adjuvant activity in mouse models for HIV-1 [17–19], Hepatitis C virus [20, 21] and HSV-2  infection. Coexpression of GM-CSF enhanced antigen-specific T-cell proliferation and humoral immune responses, but had little effect on CTL responses. Over-expression of GM-CSF at the injection site led to the increased recruitment of macrophages and dendritic cells (DCs) [23, 24] and influenced the activation status of antigen-presenting cells (APCs) . The temporal and spatial co-expression of antigens and GM-CSF seems to be critical for optimal T-cell priming . In addition, the fusion proteins of antigens and GM-CSF  and DNA vaccines encoding such fusion proteins [28, 29] were shown to improve antigen-specific antibody responses and cancer immunotherapy. The covalent linkage of the antigen and GM-CSF might allow the targeting of APCs expressing GM-CSF receptors, such as DCs. This could improve antigen uptake and presentation and thus also enhance CD8 T cell responses, similar to targeting strategies based on the macrophage mannose receptor or the DEC205 receptor [30, 31]. Therefore, we compared the antigen-specific CD8 T-cell responses induced by DNA vaccines encoding GM-CSF ovalbumin fusion proteins (GM-OVA) with those raised by DNA vaccines coexpressing GM-CSF and ovalbumin (OVA) as two unlinked proteins. Since the antigen expression levels of, and the innate response to, DNA and viral vector vaccines differ considerably, the effect of GM-CSF was determined in DNA immunizations and DNA prime adenoviral vector boost regimens. Surprisingly, immunization with genetic vaccines encoding the GM-CSF ovalbumin fusion protein suppressed CD8+ T cell responses, while coexpression of GM-CSF was found to be a potent stimulator of antigen-specific CD8+ T cell responses. Induction of autoantibodies neutralizing GM-CSF by genetic vaccines encoding the fusion-protein, but not those coexpressing GM-CSF and OVA, might explain the varying effects observed.
DNA and adenoviral vector vaccines
Influence of the GM-CSF fusion protein on dendritic cell differentiation and antigen presentation
CD8+ T cell responses after immunization with gene-based vaccines encoding the GM-CSF fusion protein
Humoral immune responses after immunization with gene-based vaccines encoding the GM-CSF fusion protein
Stimulation of CD8+ T cell responses by vaccines coexpressing GM-CSF and ovalbumin
CD8 T cell responses induced by vaccines encoding a biologically inactive GM-CSF ovalbumin fusion protein
In contrast to the GM-OVA vaccines, the coexpression of GM-CSF and ovalbumin did not suppress CD8 T cell responses. This indicates that the suppressive effects of GM-OVA vaccines on the CD8 T cell responses are not simply due to the biological activity of GM-CSF. To exclude the possibility that immune responses were affected by altered antigen expression levels, subcellular localization, and/or stability of the fusion protein, mice were also immunized with DNA and adenoviral vector vaccines expressing a fusion protein of rhesus macaque GM-CSF and ovalbumin. Although fully active on primate cells, rhesus monkey GM-CSF was inactive in rodent cells (Fig. 1C). In all parameters investigated, including Tetramer analyses and intracellular staining for IFN-γ, interleukin 2, and/or CD107a, the immune response induced by the rhesus monkey GM-OVA vaccines did not differ significantly from the response induced by the OVA vaccines (Fig. 5 and 6).
Antibody responses induced by gene-based vaccines encoding bioinactive GM-CSF fusion protein or coexpressing GM-CSF and ovalbumin
Induction of neutralizing antibodies to GM-CSF
Influence of neutralizing antibodies to GM-CSF on immune responses and viral replication in a retroviral infection model
A side-by-side comparison of a DNA prime adenoviral vector boost immunization regimen leading either to the expression of a fusion protein of GM-CSF and the model antigen or to the coexpression of GM-CSF and antigens as two separate proteins revealed striking differences in the induction of polyfunctional antigen-specific CD8+ T cell responses and humoral immune responses. Immunization with gene-based vaccines encoding the bioactive GM-CSF antigen fusion proteins suppressed CD8+ T cell responses, while the coexpression of GM-CSF and antigens stimulated these responses. Although the fusion proteins enhanced the stimulation of antigen-specific CD8+ T cells in coculture experiments with antigen-presenting cells, the induction of neutralizing antibodies to GM-CSF probably counteracted any beneficial effect of the fusion protein on the enhancement of antigen uptake or presentation in vivo. the injection of recombinant GM-CSF proteins or GM-CSF fusion proteins has been shown previously to induce antibodies to GM-CSF in mice and humans [29, 35, 36, 40]. Differences in postranslational modifications such as glycosylation patterns between the recombinant proteins and the endogenously expressed GM-CSF have been postulated to be responsible for this loss of tolerance [35, 40]. Since the GM-CSF expression levels after infection with the bicistronic vector was the same or even higher when compared to the GM-OVA encoding vector, we can exclude that simple overexpression of the cytokine is a reason for the break of tolerance. However, the induction of GM-CSF neutralizing antibodies by genetic vaccines expressing GM-CSF antigen fusion proteins in the vaccinees suggests an alternative mechanism for this autoimmune response. B-cells with antigen receptor specificities for GM-CSF might take up the GM-CSF-antigen fusion protein and present antigen-derived epitopes on MHC-II molecules. T-helper cells specific for the antigen part of the GM-CSF fusion protein could then stimulate these B-cells even in the absence of autoreactive GM-CSF-specific T-helper cells. This would be consistent with a previous hypothesis for the appearance of autoreactive antibodies  and with results from immunization studies with other fusion proteins [42, 43].
The consequences of neutralizing GM-CSF antibodies for the host are not well defined. Reconstitution of white blood cells after bone-marrow transplantation and immune responses to a protein vaccine were not impaired in the presence of GM-CSF autoantibodies . Using the FV infection model, we did not observe any negative effects associated with GM-CSF autoantibodies either. Consistently, no pathological alterations have been reported to be associated with anti-GM-CSF antibodies in cancer patients. However, GM-CSF knock out mice were not able to generate CD8+ T cell responses after peptide immunization and IgG2a antibody production was also reported to be delayed after protein immunization . In the present study, induction of GM-CSF neutralizing antibodies coincided with suppression of CD8+ T cell responses induced by a DNA prime adenoviral vector boost regimen. Lack of suppression of CD8+ T cell responses after a single immunization with the same DNA or adenoviral vector vaccine further supports a causal relationship between GM-CSF antibodies and suppression of CD8+ T cell responses, since in these immunization experiments GM-CSF neutralizing antibodies are absent at the time point of T cell priming. Depending on the precise immunostimulatory pathways employed by a vaccine or pathogen, the requirement for GM-CSF might differ significantly, providing an explanation for the different observations made in the mouse models. Although there is no direct evidence of clinical complications so far, possible long-term consequences of the anti-GM-CSF responses, such as involvement in pulmonary alveolar proteinosis (PAP) [45, 46], are difficult to exclude. Given the loss of tolerance by other fusion proteins [42, 43] and the immunogenicity of genetic vaccines, a more general note of caution regarding the use of genetic vaccines encoding fusion proteins between host proteins and heterologous antigens might even be justified.
In contrast to immunization with genetic vaccines encoding the GM-CSF-antigen fusion proteins, which strongly suppressed IgG2a antibody responses, the coexpression of GM-CSF and antigens in the DNA prime adenoviral vector boost regimen did not modulate IgG1 and IgG2a antibody responses notably. This was unexpected, since repeated immunizations with adenoviral vectors coexpressing GM-CSF and the amyloid-beta protein by the intranasal route resulted in a Th2-type immune response . In addition, the coexpression of GM-CSF and antigens in numerous DNA immunization studies increased antibody responses [16, 18, 20–22]. Since the bicistronic expression cassette in our study expresses lower amounts of ovalbumin than the vaccine encoding ovalbumin only, GM-CSF coexpression seems to compensate for the lower antigen expression levels in the sense that in the presence of GM-CSF a lower amount of antigen is needed to induce comparable humoral immune responses than in its absence. Previous studies also show that increased antigen levels correlate with increased antibody titers, but do not influence the dominant immunoglobulin subtype. Thus, the difference in the IgG1/IgG2a ratio observed after immunization with the genetic vaccine encoding the GM-CSF-Ova fusion protein does not seem to be simply due to GM-CSF receptor signalling, since this also occurs by the coexpression of GM-CSF. However, immunization with vaccines encoding rhesus monkey GM-CSF fused to ovalbumin did not lead to a change in the IgG1/IgG2a ratio either, although no differences were observed between the expression levels of the two GM-CSF fusion proteins. This indicates that the change in the antigen-specific IgG1/IgG2a ratio observed after immunization with the fusion protein depends on the GM-CSF bioactivity of the fusion protein and the covalent linkage of GM-CSF with the antigen.
The immunomodulatory properties of GM-CSF could also differ substantially with the type of antigens, the route of immunization, and/or the type of genetic vaccine used. This also seems to apply to the effect of GM-CSF on CD8+ T cell responses. Coexpression of GM-CSF and antigens by DNA vaccines was shown to stimulate CD8+ T cell responses in some studies [17, 49] but not in others [19, 50]. In the context of a DNA prime adenoviral vector boost regimen, we observed that GM-CSF was a strong stimulator of CD8+ T cell responses. Coexpression of GM-CSF by the DNA and adenoviral vector vaccines enhanced the percentage of CD8+ T cells specific for the immunodominant epitope of the antigens from approximately 11% to more than 25% as determined by tetramer staining and intracellular IFN-γ staining. Although we can not completely rule out that the different antigen expression levels influence the strength of immune response, it seems unlike that lower antigen levels induce stronger CTL responses. Rather, it has been reported that increased antigen expression levels obtained by codon-optimization or enhanced promotor activities correlate with the better induction of IFN-γ producing T-cells or cytotoxic T-cells [48, 51].
Coexpression of GM-CSF and ovalbumin by DNA prime adenoviral vector boost immunizations led to a strong CD8+ T cell response, with more than a quarter of all CD8+ T cells being specific for a single immunodominant peptide. Although the percentage of CD8+ T cell specific for other antigens might well be lower than those obtained in the present study with the ovalbumin model antigens, the enhancement of CD8+ T cells responses by GM-CSF is encouraging. The antigen-specific CD8+ T cells induced in the presence of GM-CSF were not only numerous, but also displayed markers of polyfunctional T cells such as the coexpression of IFN-γ, IL-2, and the CD107a degranulation marker. T cells that produce both IFN-γ and IL-2 were recently proposed to be important in the control of chronic viral infections, like CMV, EBV or in HIV LTNP and might be indicative of long-lived memory T-cells . Although the functional relevance of the CD8+ T cell response induced by DNA and adenoviral vector vaccines coexpressing GM-CSF and antigens needs to be confirmed in relevant tumor and infection models, GM-CSF should be considered as a CD8+ T-cell adjuvant in DNA prime adenoviral vector immunization regimens.
Genetic vaccines encoding fusion proteins between antigens and a host protein led to the rapid induction of autoantibodies. Therefore a more general note of caution regarding the use of highly immunogenic viral vector vaccines encoding such fusion proteins seems to be justified. In contrast, the coexpression of GM-CSF and antigens as two separate proteins in DNA prime adenoviral vector immunization regimens led to the enhanced induction of polyfunctional CD8+ T-cells, further supporting a potential application of GM-CSF as an adjuvant not only for DNA, but also for viral vector vaccines. Given the fact that the DNA prime adenoviral vector boost regimen is presently one of the most efficient ways to induce CD8+ T cell responses in mice, non-human primates and humans, the enhancement of this response by GM-CSF is a striking observation.
DNA and adenoviral vector vaccines
The DNA constructs used for the immunization studies are all based on the expression plasmid pcDNA3.1 (Invitrogen, Karlsruhe, Germany) or pShuttle-CMV . The coding sequence of murine GM-CSF was amplified by RT-PCR from RNA isolated from stimulated mouse splenocytes and cloned into the pCR-2.1-TOPO vector (Invitrogen). All other plasmids were constructed via standard cloning techniques including overlap extension PCR. Transgene expression is driven in all constructs by the immediate early promoter/enhancer region of human cytomegalovirus. The open reading frame of the fusion protein GM-OVA consisting of murine GM-CSF, a [Gly4Ser]3 linker and OVA was cloned into the pcDNA3.1 vector. pOVA, also a pcDNA3.1 derivative, encodes ovalbumin itself. Both proteins have a C-terminal HIS6-tag. The plasmids pGMrh-OVA, pGM-DP-OVA, and pΔGM-OVA are based on the pShuttle-CMV vector. The GMrh-OVA fusion protein is equivalent to the mouse one with the sequence of murine GM-CSF being replaced by the rhesus monkey homologue. In the plasmid pGM-DP-OVA, transgene expression is driven by a bidirectional version of the CMV/TetO2 promoter . In pΔGM-OVA the coding sequence of ovalbumin is preceeded by the 17 amino acid long signal peptide of murine GM-CSF. All DNA preparations for immunizations were carried out with the Endofree Plamid Mega or Giga Kit (Qiagen, Hilden, Germany).
All E1-deleted, replication-defective adenoviruses with the corresponding expression cassettes (Ad-GM-OVA, Ad-GM-DP-OVA, Ad-rhGM-OVA, Ad-ΔGM-OVA) were generated by the AdEasy-system . The pShuttle plasmids and pAdEasy1 were electroporated into BJ5183 bacteria as previously described . Correctly recombined plasmids were transfected into 293 cells. Viral vectors growing out were checked for transgene expression by Western Blot analyses and GM-CSF bioactivity, if applicable. Vector particles were purified by CsCl gradient centrifugation and quantified by optical density measurements. In addition, the TCID50of the vectors were determined on 293 cells. The adenoviral vector preparations were also tested for endotoxin levels with the LAL quantification assay (Cambrex Bio Science, Verviers, Belgium), confirming that the dose used for immunization of mice contained less than 0,1 EU.
Cell culture media and reagents
HEK293 and 293T cells were cultured in DMEM containing 10% FCS and 1% penicillin/streptomycin. RPMI 1640 supplemented with 10% FCS, 2 mM L-Glutamine, 10 mM HEPES, 50 μM β-Mercaptoethanol and 1% antibiotic/antimycotic (all Gibco, Karlsruhe, Germany) was used for the lymphocyte cultures (R10-medium). Bone-marrow cultures were grown in RPMI 1640, supplemented with 10% FCS, 1% penicillin/streptomycin, 4 mM L-Glutamine, 1 mM sodium pyruvate and recombinant mouse IL-4 (1 ng/ml) and GM-CSF (5 ng/ml) (Biomol, Hamburg, Germany).
Expression and bioactivity of GM-OVA
293T cells were transfected using the calcium/phosphate precipitation method. Supernatants were collected 48 h after transfection and tested for protein expression by Western Blot analysis. A combination of rabbit-α-OVA (Chemicon International, LTD, Hampshire, UK) and goat-α-rabbit-HRP (Sigma, Munich, Germany) antibodies was used for detection. To confirm the bioactivity of GM-OVA, it was purified from supernatants of transfected cells by Ni-NTA-affinity chromatography as described by the manufacturer (Qiagen, Hilden, Germany). The purified protein migrated as a single band of the expected size in Coomassie stained polyacrylamide gels. The GM-CSF-dependent FDCP-1 cells were incubated with the purified GM-OVA or supernatants from NIH 3T3 cells stably expressing GM-CSF. The GM-CSF content of the supernatants had been determined by ELISA. After 48 h, GM-CSF-dependent cell growth was monitored by MTT-assay as described elsewhere [55, 56]. Additionally, the ability to generate DCs from bone marrow cultures was tested as previously described . Briefly, bone marrow derived monocytes were cultured in the prescence of IL-4 and recombinant GM-CSF or GM-OVA for 8 days, with or without LPS maturation for 24 h. Their phenotype was characterized by surface staining with the antibodies α-CD11c-APC, α-CD80-FITC, α-CD86-FITC (all BD Bioscience, Heidelberg, Germany) and α-CD83-PE (eBioscience, San Diego, USA) in FACS analyses.
OT-I proliferation assay
Splenocytes of transgenic OT-I mice were incubated with CFSE (3μM; Molecular Probes, Eugene, Oregon, USA) at a density of 8 × 107 cells/ml for 6 min at room temperature with gentle mixing. The labelling reaction was stopped by adding one volume of FCS after which the cells were washed twice with PBS. Thereafter, cells were plated into 96-well plates at a density of 1 × 106 cells/well and incubated with different concentrations of either GM-OVA or OVA (500 to 0,5 ng/ml) for 4 days. After washing the cells twice with PBS containing 0,5% BSA and 1 mM sodium azide (PBS/BSA/Azid), cell proliferation was measured in FACS analysis. Cells, which were incubated with OT-I peptide, were used as positive controls whereas non-stimulated cells served as negative ones.
Animals and immunizations
6–8 week old female C57BL/6N mice were purchased from Janvier (Le Genest-ST-Isle, France) and housed in singly-ventilated cages in accordance with the national law and institutional guidelines.
All vaccines were diluted in PBS and injected subcutaneously in both hind foot pads. For single dose experiments, mice were immunized on day 1 with either 50 μg of DNA (pOVA or pGM-OVA) or 5 × 109 adenoviral particles (Ad-ΔGM-OVA resp. Ad-GM-OVA) corresponding to 2 × 108 50% tissue culture infectious doses (TCID50). After one week, serum samples were collected and on day 8 animals were sacrificed to analyze the CTL responses. In the prime-boost experiments, all animals received an additional DNA injection (pOVA or pGM-OVA) 5 weeks prior to the above mentioned protocol. Serum samples were collected 28 days after the first and 7 days after the second immunization, whereas the T-cell assays were carried out 8 days after the second immunization.
OVA specific antibody ELISA
Blood was taken retro-orbitally and serum was collected after centrifugation for 5 min at 5.000 g in a table top centrifuge. Ovalbumin protein (Sigma) was coated on 96-well plates (MaxiSorb, Nunc, Wiesbaden, Germany) at a final concentration of 5 μg/ml. After blocking with 5% milk powder, serum samples were added at appropriate dilutions and incubated for 1 h, followed by intensive washing. Alkaline phosphatase-coupled antibodies against mouse IgG1 or IgG2a antibodies (BD Bioscience) were added and incubated for 1 h. The enzymatic reaction was developed with the pNPP substrate (Sigma) for 30 min. Reaction was stopped by sodium hydroxide solution (1 M) and the optical densities were measured at a wavelength of 405 nm.
Tetramer and intracellular cytokine staining (ICS)
Splenocytes were collected at indicated time points. After red blood cell lysis, 1 × 106 cells were plated in 96-well round-bottom plates (Nunc) for each staining.
For the tetramer staining, cells were washed once and incubated with 2 μl of SIINFEKL/H-2Kb-APC tetramers (Sanquin, Amsterdam, NL) in total volume of 100 μl PBS/BSA/Azid for 40 min at room temperature. After surface staining with α-CD8-FITC, cells were incubated with 7-amino-actinomycin D (7-AAD) for 5 min to exclude dead cells from subsequent FACS analyses. In some experiments, α-CD62L-PE antibodies were included in the tetramer analysis to characterize the CD8+ activation status.
For ICS, samples were stimulated for 6 h in the presence of 2 μM Monensin, which inhibits the cytokine secretion, and 1 μl α-CD107a-FITC, which is a marker for lymphocyte degranulation . Cells were either stimulated by α-CD3 and α-CD28 antibodies (2 μg/ml and 1 μg/ml, respectively) or the OT-I/SIINFEKL peptide (2 μg/ml, Genaxxon, Biberbach, Germany) and compared to non-stimulated cultures. After stimulation, surface staining was carried out with αCD8-PerCP or αCD4-FITC (BD Bioscience). Cells were fixed in 2% paraformaldehyde, followed by permeabilisation with 0,5% Saponin in PBS/BSA/Azid buffer. Cytokines were detected with αIFN-γ-PE and αIL-2-AlexaFluor647.
Friend virus challenge and virus detection
Mice were injected intravenously with 0.5 ml phosphate buffered saline (PBS) containing 3,000 spleen focus-forming units (SFFU) of the Friend virus complex (FV). The B-tropic, polycythemia-inducing FV complex used in all experiments was from uncloned virus stocks obtained from 10% spleen cell homogenates as described previously .
For infectious center assays, single-cell suspensions from infected mouse spleens were cocultivated with Mus dunnis cells at 10-fold dilutions. Cultures were incubated for 5 days, fixed with ethanol, stained with F-MuLV envelope-specific monoclonal antibody 720, and developed with peroxidase-conjugated goat antimouse antibody and aminoethylcarbazol to detect foci. Tetramer analyses were done by flow cytometry as described previously . For the quantification of Friend virus-infected blood cells, single-cell suspensions of nucleated, live cells were analyzed by flow cytometry. To detect Friend virus infection cells were stained as described previously with tissue culture supernatant containing Friend murine leukemia virus glycosylated Gag-specific monoclonal antibody 34 (AB34) .
Results are expressed as the means ± standard errors of the means (SEM). Statistical comparisons were performed by one-way ANOVA test, followed by a Bonferroni post test using the Prism 4.0, GraphPad Software. P < 0,05 was considered as statistically significant.
This work was supported by grants from the German Research Foundation (GK1045/1), the Wilhelm Sander Stiftung (2004.107.1), and the European Commission FP6 program (TIP-VAC, LSHP-CT-2004-012116).
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