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The Journal of Immunology, 2007, 179, 2126 -2133
Copyright © 2007 by The American Association of Immunologists, Inc.
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In Vivo Antigen Stability Affects DNA Vaccine Immunogenicity

Adriaan D. Bins1, Monika C. Wolkers1,2, Marly D. van den Boom, John B. A. G. Haanen and Ton N. M. Schumacher3

Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The factors that determine the immunogenicity of Ags encoded by viral vaccines or DNA vaccines in vivo are largely unknown. Depending on whether T cell induction occurs via direct presentation of vaccine-encoded epitopes or via one of the different proposed pathways for Ag cross-presentation, the effect of intracellular Ag stability on immunogenicity may possibly vary. However, the influence of Ag stability on CD8+ T cell induction has not been addressed in clinically relevant vaccine models, nor has the accumulation of vaccine-encoded Ags been monitored in vivo. In this study, we describe the relationship between in vivo Ag stability and immunogenicity of DNA vaccine-encoded Ags. We show that in vivo accumulation of DNA vaccine-encoded Ags is required for the efficient induction of CD8+ T cell responses. These data suggest that many of the currently used transgene designs in DNA vaccination trials may be suboptimal, and that one should either use pathogen-derived or tumor-associated Ags that are intrinsically stable, or should increase the stability of vaccine-encoded Ags by genetic engineering.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Vaccines that aim to induce cytotoxic T cell responses should achieve two goals, as follows: first, vaccination should lead to the presentation of sufficient densities of the relevant peptide/MHC complexes, and second, such presentation should occur by mature professional APC. Although the signals that lead to maturation of professional APC have been studied in considerable detail (1), our understanding of the factors that lead to efficient MHC class I-restricted presentation of vaccine-encoded Ags to naive T cells is incomplete. Currently, there is solid evidence that peptides can be presented by MHC class I molecules by two distinct pathways (2), as follows: the classical endogenous Ag presentation pathway that processes Ags that are generated within the APC itself (3, 4, 5, 6), and the cross-presentation pathway, in which professional APC present T cell Ags that have been acquired from other cell types (3). Importantly, for a number of viral vectors that are used in vaccination strategies such as vaccinia and adenovirus groups A and C-F, T cell induction depends to a large extent on cross-presentation of the vaccine-encoded Ags (7, 8). Likewise, following intradermal (i.d.) (9) or i.m. DNA vaccination (10, 11), cross-presentation of the DNA vaccine-encoded Ags is a major mechanism for T cell induction.

Several studies have provided evidence that cross-presentation of MHC class I-restricted epitopes involves the transfer of intact Ags from the Ag-donating cell to the APC (12, 13, 14, 15). Conversely, other groups have provided evidence for a role of proteasomal products and intermediates, possibly in the form of heat shock protein-bound peptides, as a main Ag source for cross-presentation (16, 17, 18). Finally, a recent study has indicated that gap junctions can allow transfer of peptide fragments between cells, a pathway that could potentially also be used by professional APC to acquire Ags (19). Based on the nature of the Ag that is transferred, it could be argued that either the level of peptide epitopes or the level of full-length protein Ag would be a relevant factor in vaccine design. This issue is of some relevance for ongoing DNA vaccination trials, because the stability of the vaccine-encoded Ag is generally not taken into account in vaccine design, thereby potentially influencing vaccination efficiency.

To unravel the parameters that determine efficient priming of cytotoxic T cell responses to DNA vaccine-encoded Ags, we have developed a vaccination model that allows us to directly follow vaccine-encoded Ag stability in vivo. We have used this model to determine the effect of Ag stability on CD8+ T cell induction. The data obtained in this model suggest that Ag stability should form an important consideration in the design of DNA vaccines and possibly of other vaccines that rely on Ag cross-presentation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 mice, MHC class II–/– (20), TCRbeta–/– mice (21), and B10F5 mice (22) were obtained from the experimental animal department of The Netherlands Cancer Institute. All animal procedures were performed after approval of the Experimental Animal Committee and in accordance with institutional and national guidelines.

DNA vaccines

DNA vaccines were generated by the introduction of fusion genes into pVAX or pcDNA3.1 (Invitrogen Life Technologies). The fusion genes sNP-GFP-E7 and sE7-GFP-nucleoprotein (NP)4 have been described elsewhere (15) and were cloned into the BamHI and NotI sites of pcDNA3.1. The fusion gene Ub-M-lucNP was generated by addition of the ubiquitin (Ub) gene to the 5' end of a lucNP-encoding vaccine (23). Briefly, two intermediate products were generated with the primers Ub-top, 5'-GGGGGATCCTAAGCCACCATGCAGATC-3', plus Ub-M-bottom, 5'-CTTTATGTTTTTGGCGTCTTCCATACCCCCCCTCAAGCGC-3', and luc-M-top, 5'-GCGCTTGAGGGGGGGTATGGAAGACGCCAAAAACATAAAG-3', plus luc-NP-bottom, 5'-CCCTTTGCGGCCGCTTACATGGCGTCCATGTTCTCGTTGCTGGCGATCTGCACGCCCACGGCGATCTTTCCGCCCTTC-3'. A second round of amplification was performed with Ub-top and Luc-NP bottom primers. To generate Ub-R-lucNP, Ub-top was used together with Ub-R-bottom, 5'-CTTTATGTTTTTGGCGTCTTCGCGACCCCCCCTCAAGCGC-3', and luc-R-top, 5'-GCGCTTGAGGGGGGGTCGCGAAGACGCCAAAAACATAAAG-3' with lucNP-bottom. The variants, Ub-F-lucNP, Ub-G-lucNP, and Ub-S-lucNP, were obtained by changing the first amino acid of the luciferase moiety by exchanging the Ub-X part with Ub-top and Ub-F-bottom, 5'-AAACCCCCCCTCAAGCG-3'; Ub-S-bottom, 5'-CTACCCCCCCTCAAGCG-3'; and Ub-G bottom, 5'-CCACCCCCCCTCAAGCG-3'. The PCR products Ub-F, Ub-G, and Ub-S were digested with BamHI, and ligated into pVAX-Ub-R-lucNP that had been digested with BamHI and NruI.

K14-driven Ub-M-lucNP and Ub-R-lucNP plasmids were constructed by replacing the CMV-IE promoter of the pVAX backbone with the K14 promoter (provided by J. Cho, Pohang University of Science and Technology, Pohang, Korea). UlcNP was generated by relocating the first 150 nt of the lucNP open reading frame into the SSpB1 restriction site at position 492. The fragment was generated with ulcNP1–50 top, 5'-ATATTGTACAAAGACGCCAAAAACATAAAGAAAGG-3' and ulcNP1–50 bottom, 5'-ATATATGTACACGTCCACCTCGATATGTGCATC-3'. A Kozak sequence and start codon were inserted at the 5' end of UlcNP by PCR with ulcNP top, 5'-ATATATGTACACGTCCACCTCGATATGTGCATC-3' and ulcNP bottom, 5'-ATATTGTACAAAGACGCCAAAAACATAAAG AAAGG-3'.

All sequences were confirmed by sequence analysis. DNA was grown in DH5{alpha} and isolated with endotoxin-free DNA purification kit (Qiagen). DNA vaccines for i.d. application were dissolved in water; DNA vaccines for i.m. administration were dissolved in PBS (Invitrogen Life Technologies).

DNA vaccination and intravital imaging

The i.d. vaccination by DNA tattoo and intravital analysis of protein expression was performed, as described previously (23). Briefly, mice were anesthetized, and a volume of 10 µl of a 2 µg/µl DNA solution in water was applied to the shaved skin of the hind leg. The DNA vaccine was applied to the skin by a 20-s tattoo with a sterile disposable 11-needle bar oscillating at a frequency of 100 Hz. For intravital imaging, mice were anesthetized with isofluorane (Abbott Laboratories). An aqueous solution of the substrate luciferin (150 mg/kg; Xenogen) was injected i.p., and 18 min later the luminescence produced by active luciferase was acquired during 30 s in an IVIS system200 charge-coupled device camera (Xenogen). Signal intensity was quantified as the sum of all detected light within the region of interest.

Detection of NP366- and E749-specific T cells in peripheral blood

PBL were stained with PE-conjugated anti-CD8beta (BD Pharmingen) plus allophycocyanin-conjugated H-2Db/NP366 tetramers or allophycocyanin-conjugated H-2Db/E749 tetramers, at room temperature for 15 min in PBA (PBS, 0.5% BSA, and 0.02% sodium azide). Cells were washed three times in PBA and analyzed by flow cytometry. Live cells were selected based on 7-aminoactinomycin D exclusion.

Analysis of in vitro protein stability by proteasome inhibition

COS cells seeded in 10-mm dishes with a confluency of 80% were transfected with 15 µg of pVAX encoding Ub-M-lucNP or Ub-R-lucNP, together with DEAE dextran, for 30 min at 37°C, and 80 µM chloroquine was added for 2.5 h. DNA-chloroquine-containing medium was aspirated, and cells were treated for 2.5 min with 10% DMSO in DMEM (Invitrogen Life Technologies) containing 10% FBS. The DMSO/medium mixture was aspirated and cells were cultured for 24 h at 37°C. To ensure equal transfection efficiencies for samples, cells were harvested and seeded into six-well plates in triplicates. Forty-eight hours posttransfection, culture medium was aspirated and fresh medium with or without 10 µM MG132 (z-Leu-Leu-Leu-al; Sigma-Aldrich) was added. After 4 h at 37°C, cells were trypsinized, harvested, washed twice with PBS, and resuspended in 20 µl of lysis buffer (Promega). Following a 20-min incubation at room temperature, 5 µl of cell lysate was harvested, 20 µl of substrate buffer (Promega) was added, and luciferase activity was measured.

Analysis of in vitro protein stability by pulse-chase analysis

COS cells at 80% confluency were transfected with 10 µg of DNA encoding Ub-M-lucNP, Ub-R-lucNP, lucNP, or ulcNP using FuGene6, according to the manufacturer’s protocol. The cells were starved in Met/Cys-free DMEM (Invitrogen Life Technologies) for 60 min before a 1-h pulse labeling with 35S-labeled Met/Cys in DMEM. Labeled cells were washed and subsequently cultured in Met/Cys-containing DMEM for the indicated time points. After cell lysis, luciferase variants were immunoprecipitated and analyzed by SDS-PAGE. Protein quantification was performed by phosphor imager analysis (FLA3000; Fujifilm). The images were quantitated using TINA 2.09 analysis software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Epitope location affects DNA vaccine immunogenicity

We have previously shown that tumor-associated T cell epitopes that are derived from signal peptides are cross-presented inefficiently (15). To determine whether for vaccine-encoded Ags a similar bias occurs, we made use of a recently developed multineedle i.d. DNA vaccination strategy (23), hereafter referred to as DNA tattoo. As is the case for gene gun DNA vaccination (9), induction of T cell responses by DNA tattooing occurs at least partially through cross-presentation (see below). To assess the effect of epitope location on vaccine immunogenicity, we generated a pcDNA3.1-based vector that encodes a secreted GFP-based fusion protein, sNP-GFP-E7. This model Ag contains two CD8+ T cell epitopes, as follows: the influenza A nucleoprotein-derived NP366 epitope located within the hydrophilic segment of the signal peptide, and the human papilloma virus-derived E749 epitope located within the COOH-terminal part of the GFP protein (Fig. 1a). The rationale for this design is that it allows the simultaneous measurement of T cell responses against two T cell epitopes, of which one is incorporated in the stable mature GFP protein and the other within the signal peptide that is thought to be short-lived (24, 25). To be able to correct for possible intrinsic differences in immunogenicity between the two T cell epitopes, a second fusion gene (sE7-GFP-NP) containing the same two model Ags, but in reverse order, was used in parallel. Mice were vaccinated with the sNP-GFP-E7 or the sE7-GFP-NP DNA vaccines, and T cell responses were monitored directly ex vivo by MHC tetramer staining (Fig. 1b). Vaccination with these DNA vaccines resulted in considerable frequencies of Ag-specific T cells specific for the Ag expressed within the mature protein, be it either the NP366 epitope or the E749 epitope (Fig. 1c). In striking contrast, when the NP366 epitope or the E749 epitope was encoded within the signal peptide, DNA vaccination failed to induce Ag-specific CD8+ T cell responses above background (Fig. 1c). These data establish that, analogous to tumor cell-associated Ags (15), the immunogenicity of DNA vaccine-encoded Ags is biased toward epitopes in mature proteins, and against epitopes in signal peptides.


Figure 1
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  		FIGURE 1. Ag location affects the immunogenicity of DNA vaccines. C57BL/6 mice were immunized on days 0, 3, and 6 by means of i.d. DNA tattoo with 20 µg of pcDNA3.1-sNP-GFP-E7 or pcDNA3.1-sE7-GFP-NP. At day 12 postvaccination, peripheral blood was drawn and Ag-specific CD8+ T cell responses were measured ex vivo by combined anti-CD8 and Db-NP366 tetramer (top) or Db-E749 tetramer (bottom) staining. a, Schematic representation of sNP-GFP-E7 and sE7-GFP-NP vaccine insert designs. b, Flow cytometric analysis of peripheral blood cells of representative mice for the two groups. c, Symbols represent Ag-specific CD8+ T cell responses of individual mice; bars indicate averages of five mice. Result is representative of two independent experiments.

 
Ag stability determines the immunogenicity of DNA vaccination

It has been proposed that signal peptide-derived epitopes may inefficiently enter the cross-presentation pathway as a consequence of their limited life span (15, 24). However, no measurements exist of the in vivo t1/2 of signal peptides, and it is clearly possible that other characteristics of signal peptides adversely affect T cell priming via cross-presentation. Consequently, whereas the data show that signal peptide-encoded epitopes are strongly disfavored, the broader implications of this finding are unclear. To be able to directly assess whether the t1/2 of a vaccine-encoded Ag could influence its immunogenicity, we generated DNA vaccines encoding an Ag that could be destabilized to a variable degree by a minor modification in its sequence. This Ag consists of three basic components (Fig. 2a). At the COOH terminus of the fusion protein, the NP366 T cell epitope is present to be able to monitor immunogenicity of the Ag. This epitope is preceded by the firefly luciferase that is used to follow in vivo expression of the Ag by intravital imaging. In addition, the luciferase gene product carries a variable residue at its N terminus. Finally, a Ub moiety is encoded N terminus to the luciferase gene product, to control Ag t1/2. This N-terminal Ub moiety is cotranslationally removed by Ub-specific proteases, thereby exposing the variable N-terminal residue of the luciferase gene product. As previously established by Varshavsky and colleagues (26), the nature of this amino acid can determine protein t1/2 in a hierarchical manner known as the N-end rule. Thus, the Ub-X-lucNP fusion proteins described in this study are designed to display a different t1/2, depending on the nature of the X-lucNP N-terminal residue.


Figure 2
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  		FIGURE 2. Structure and in vitro stability of Ub-M-lucNP and Ub-R-lucNP fusion proteins. a, Schematic representation of the design of the Ub-M-lucNP and Ub-R-lucNP fusion gene constructs. The arrowhead indicates the cotranslational cleavage site of Ub by Ub-specific proteases. M and R indicate the methionine and arginine starting amino acids. b, COS cells were transiently transfected with Ub-M-lucNP (left) or Ub-R-lucNP (right), and incubated with the proteasomal inhibitor MG132 (z-Leu-Leu-Leu-al) for 4 h (+), or left untreated (–). After cell lysis, luciferase activity of the lysate was determined. Results are representative of three independent experiments. c, COS cells that were transiently transfected with Ub-M-lucNP or Ub-R-lucNP were starved in Met/Cys-free DMEM for 60 min before a 1-h pulse labeling with 35S-labeled Met/Cys in DMEM. Labeled cells were washed and subsequently cultured in Met/Cys-containing DMEM for the indicated time points. After cell lysis, luciferase variants were immunoprecipitated, and samples were analyzed by SDS-PAGE. Based on the molecular mass of the observed proteins (62 kDa), removal of the Ub moiety is complete at all time points examined (data not shown). d, Graphical representation of an automated quantification of c.

 
To determine whether the intracellular stability of X-Luc-NP fusion proteins is controlled by the N-end rule pathway, we transfected COS cells with plasmids encoding Ub-M-lucNP or Ub-R-lucNP (in which M and R are stabilizing and destabilizing residues, respectively) (26) and determined lucNP expression level by measuring luciferase activity (Fig. 2b). Both X-lucNP variants were enzymatically active, as judged by bioluminescence measurements after addition of the substrate luciferin (Fig. 2b). To determine whether the t1/2 of the Luc-NP fusion proteins is dependent on the nature of the N-terminal amino acid, proteasomal activity was inhibited by addition of MG132 (z-Leu-Leu-Leu-al). A 4-h inhibition of proteasomal activity did not measurably alter bioluminescence of cells transfected with Ub-M-lucNP DNA. In contrast, luciferase activity of cells transfected with Ub-R-lucNP DNA was increased ~4-fold upon inhibition of proteasomal activity, indicating that the resulting R-lucNP protein has a significantly shorter t1/2 than M-LucNP. In line with this, immunoprecipitation of luciferase from pulse-labeled cells transfected with Ub-R-lucNP or Ub-M-lucNP showed a substantial difference in t1/2 between the two proteins (Fig. 2, c and d).

The activity of the E3 ligases that recognize different N-terminal residues may be cell-type dependent (27). Therefore, it was essential to determine the in vivo t1/2 of vaccine-encoded Ub-M-lucNP and Ub-R-lucNP fusion proteins. To this end, mice received a single i.d. DNA vaccination with either Ub-M-lucNP or Ub-R-lucNP DNA, and in vivo protein stability was subsequently determined by measuring luciferase activity at several time points post-DNA application (Fig. 3a). Within the first 12 h postimmunization, luciferase activity increased rapidly and (as expected; see legend to Fig. 3) did not substantially differ between Ub-M-lucNP- and Ub-R-lucNP-vaccinated animals. However, at later time points, the luciferase activity in the Ub-R-lucNP group decreased much more rapidly than in the Ub-M-lucNP group. Specifically, at 72 h postvaccination, luciferase activity in Ub-M-lucNP-vaccinated animals was ~100-fold higher than that in Ub-R-lucNP-vaccinated animals. Based on the rate of decay of luciferase activity from 24 h onward, the in vivo t1/2 of R-lucNP and M-lucNP are 5 and 11 h, respectively. It is noted that these numbers may underestimate the actual difference in Ag t1/2 of the two proteins, because it assumes that LucNP synthesis has ceased after 24 h, whereas low-level de novo production may still occur at this time point.


Figure 3
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  		FIGURE 3. Ag stability promotes DNA vaccine immunogenicity. a, In vivo kinetics of luciferase activity upon i.d. DNA vaccination with Ub-M-lucNP or Ub-R-lucNP. Mice were injected once by DNA tattoo with 20 µg of Ub-M-lucNP ({diamondsuit}) or Ub-R-lucNP ({blacktriangleup}). At the indicated time points postvaccination, luciferase expression was determined by intravital imaging. It is noted that the difference in luciferase activity resulting from a differential t1/2 is expected to be maximal at late time points at which de novo Ag production is low or has ceased, and will be suppressed at early time points during which Ag production is still rising. b, Immunogenicity of Ub-M-lucNP and Ub-R-lucNP DNA vaccines. Mice were vaccinated with 20 µg of Ub-M-lucNP ({diamondsuit}) or Ub-R-lucNP ({blacktriangleup}) on days 0, 3, and 6. At the indicated time points, peripheral blood was drawn and Ag-specific CD8+ T cell responses were determined ex vivo by Db-NP366 tetramer staining. Symbols represent CD8+ T cell responses of individual mice; bars indicate group averages. Result is representative of three independent experiments.

 
To examine whether the in vivo difference in intracellular Ag t1/2 affects the immunogenicity of these DNA vaccines, we measured NP-specific CD8+ T cell responses by ex vivo MHC tetramer staining of peripheral blood samples upon vaccination with either Ub-M-lucNP or Ub-R-lucNP. Whereas vaccination with Ub-M-lucNP DNA yielded NP366-specific CD8+ T cell responses with a peak average of 10.4% (Fig. 3b), vaccination with the destabilized variant Ub-R-lucNP resulted in a 4.7-fold lower Ag-specific T cell response (2.2% on average at the peak; p = 0.008). These data suggest that Ag t1/2 is a major determinant of the efficacy of i.d. DNA vaccines in the induction of CD8+ T cell responses.

Ag stability influences cross-presentation of MHC class I-restricted epitopes in the draining lymph nodes (DLN)

The above data demonstrate the effect of in vivo Ag stability on the immunogenicity of i.d. DNA vaccines, but do not reveal whether this effect is due to an effect on cross-presentation or on endogenous presentation by transfected APC. To directly test whether the induction of CD8+ T cell responses via cross-presentation is dependent on Ag stability, mice were vaccinated with DNA vectors encoding the Ub-M-lucNP and Ub-R-lucNP variants under control of the K14 promoter. As opposed to the CMV-IE promoter used in Figs. 1 and 3 (and in the majority of human vaccination trials (28)), the K14 promoter is solely active in epidermal cells (9), thereby eliminating a contribution of directly transfected professional APC to Ag presentation. Mice that were vaccinated with the (K14)-Ub-M-lucNP DNA vaccine mounted peak NP366-specific T cell responses of on average 1.4% (Fig. 4a). In contrast, after vaccination with the (K14)-Ub-R-lucNP DNA vaccine, T cell responses remained close to background levels (0.1% on average, p = 0.014). These data indicate that Ag stability substantially affects the immunogenicity of DNA vaccines in a setting in which T cell induction can only occur through epitope presentation by APC that have obtained Ag from other cells. Of note, the magnitude of T cell responses induced by K14-driven DNA vaccines is close to 10-fold lower as compared with those induced by CMV IE-driven vaccines (Fig. 3b vs Fig. 4a). This difference is likely to be a consequence of the 10- to 50-fold lower level of protein production from K14-driven vaccines (data not shown). In addition, it is possible that endogenous Ag presentation by transfected APC enhances T cell induction (29) in the case of CMV-IE-driven vaccines.


Figure 4
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  		FIGURE 4. Ag stability promotes vaccine immunogenicity through enhanced cross-presentation. a, Mice were vaccinated on days 0, 3, and 6 with 20 µg of pVAX(K14)-Ub-M-lucNP ({diamondsuit}) or pVAX(K14)-Ub-R-lucNP ({blacktriangleup}), and induction of NP366-specific CD8+ T cell responses was measured by Db-NP366 tetramer staining. The percentage of NP366-specific CD8+ T cells at various time points postvaccination is plotted. b, Ag stability affects MHC class I-restricted Ag presentation in the DLN. Mice were vaccinated with 20 µg of Ub-M-lucNP, Ub-R-lucNP, or a control plasmid. At day 7 postvaccination, 2 x 106 NP366-specific CFSE-labeled F5 T cells that were depleted for CD4+ cells by magnetic bead separation with a CD4-specific Ab were injected into TCRbeta–/– mice. Four days later, DLN ({diamondsuit}) and non-DLN (224) were excised and analyzed for dividing F5 T cells, as determined by the percentage of CFSE-dull Db-NP366 tetramer-positive cells. c, MHC-II–/– mice were vaccinated on days 0, 3, and 6 by DNA tattooing with 20 µg of Ub-M-lucNP ({diamondsuit}) or Ub-R-lucNP ({blacktriangleup}). On days 8, 10, and 11, peripheral blood was drawn and Ag-specific CD8+ T cell responses were determined ex vivo by Db-NP366 tetramer staining.

 
Because CD4+ T cell help is required for efficient DNA vaccination (30), we set out to determine whether the higher immunogenicity of stable Ags correlates with enhanced MHC class I-restricted cross-presentation, or can be explained by more effective CD4+ T cell help. To this end, CFSE-labeled NP366-specific F5 TCR transgenic CD8+ T cells were transferred into TCRbeta-deficient mice that lack both CD4+ and CD8+ T cells. Subsequently, the mice received DNA vaccines encoding Ub-M-lucNP or Ub-R-lucNP, and at day 7 postvaccination the DLN were excised and analyzed for division of F5 T cells. In mice that were vaccinated with Ub-M-lucNP, on average 7.0% of the F5 T cells had divided (Fig. 4b), whereas vaccination with Ub-R-lucNP led to considerably less T cell division (2.8% on average, p = 0.03). As a second approach, we determined the induction of NP366-specific T cell responses in MHC class II-deficient mice that lack CD4+ T cells. Consistent with prior data on the requirement for CD4+ T cell help in DNA vaccination (9, 30), overall CD8+ T cell responses in MHC class II-deficient mice are very low. However, Ub-M-lucNP vaccination still induced significantly higher CD8+ T cell responses than vaccination with Ub-R-lucNP (0.7 vs 0.2%, on average at the peak of the response, p = 0.0003; Fig. 4c). These results demonstrate that for i.d. DNA vaccines, the stability of the Ag correlates with the efficiency of cross-presentation of MHC class I-restricted epitopes in the DLN.

To further dissect the effect of Ag stability on T cell induction, we generated three more variants of lucNP that encoded glycine (G), serine (S), or phenylalanine (F) as the N-terminal amino acid that is created upon Ub removal (Ub-G-lucNP, Ub-S-lucNP, and Ub-F-lucNP). These residues were chosen because they were previously found to yield fusion proteins with a range of t1/2 in mouse cells in vitro (31). Specifically, a serine-based fusion protein displayed an identical or marginally higher stability in L cells as compared with the wild-type Ub-M-lucNP in a 1-h chase period, and glycine- and phenylanaline-based fusion proteins have an intermediate and low t1/2 relative to M- and R-based fusion proteins.

First, we assessed the in vivo stability of these variants by intravital bioluminescence imaging. Interestingly, comparison of the in vivo t1/2 revealed that luciferase activity upon Ub-S-lucNP DNA vaccination persisted substantially longer than after Ub-M-lucNP vaccination (Fig. 5a; 15 vs 11 h, based on decay in luciferase activity from 24 h onward), whereas the in vivo t1/2 of Ub-G-lucNP and Ub-F-lucNP fell in between that of Ub-M-lucNP and Ub-R-lucNP (9 and 7 h, respectively). We then tested the immunogenicity of DNA vaccines encoding these three additional stability variants in parallel with DNA vaccines encoding Ub-M-lucNP and Ub-R-lucNP. In accordance with the hypothesis that Ag t1/2 controls vaccine immunogenicity, T cell responses induced by Ub-S-lucNP were highly robust and exceeded that of Ub-M-lucNP. Furthermore, the magnitude of the T cell responses induced by Ub-G-lucNP and Ub-F-lucNP ranked in between Ub-M-lucNP and Ub-R-lucNP, and correlated with their respective in vivo t1/2 (Fig. 5c).


Figure 5
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  		FIGURE 5. Ag immunogenicity correlates with protein t1/2. a, In vivo kinetics of luciferase activity upon vaccination with Ub-X-lucNP variants. Mice were tattooed once with 20 µg of DNA vaccine, encoding Ub-S-lucNP (•), Ub-M-lucNP ({diamondsuit}), Ub-G-lucNP (–), Ub-F-lucNP ({blacksquare}), or Ub-R-lucNP ({blacktriangleup}). At indicated time points postvaccination, luciferase expression was determined by intravital imaging. b, CD8+ T cell responses upon vaccination with Ub-X-lucNP variants. Mice were vaccinated on days 0, 3, and 6 by DNA tattoo with 20 µg of the Ub-S-lucNP (•), Ub-M-lucNP ({diamondsuit}), Ub-G-lucNP (–), Ub-F-lucNP ({blacksquare}), or Ub-R-lucNP ({blacktriangleup}) DNA vaccines. At the indicated time points, peripheral blood was drawn and Ag-specific CD8+ T cell responses were determined ex vivo by Db-NP366 tetramer staining. The percentage of NP366-specific CD8+ T cells in each mouse at the peak of the response is plotted; bars depict group averages. c, The in vivo t1/2 calculated from the data presented in a and the average peak Db-NP366 CD8+ T cell response for each stability variant plotted against each other. Error bars represent the SD of Ag-specific CD8+ T cell responses. d, Mice were vaccinated on days 0, 14, and 28 by i.m. injection of 50 µl (100 µg) of DNA, encoding Ub-M-lucNP (224) or Ub-R-lucNP ({Delta}). Because Ag expression upon i.m. vaccination is highly variable, presumably due to variability in delivery of the DNA vaccine to the target site, luciferase expression was determined by intravital imaging at day 7 postvaccination, and three mice within each group with the lowest luciferase expression were excluded from the experiment. At day 7 postsecond vaccination, one additional mouse was excluded from each group in a similar fashion. Of the five remaining mice in each group, NP366-specific CD8+ T cell responses were determined on days 5, 6, 7, and 8 after the third vaccination by Db-NP366 tetramer staining. The peak response of individual mice is shown. Result representative of two independent experiments.

 
Effect of Ag stability on i.m. DNA vaccination

To investigate the possible role of Ag stability upon administration of DNA vaccines via other routes, we tested the Ub-M-lucNP and Ub-R-lucNP DNA vaccines for their efficiency in the induction of T cell responses upon i.m. DNA vaccination. Analogous to the results obtained after i.d. tattoo vaccination, the magnitude of the CD8+ T cell responses induced by i.m. vaccination with the stable Ub-M-lucNP variant exceeded that induced by the unstable Ub-R-lucNP variant (average CD8+ T cell responses of 1.2 and 0.2%, respectively, p = 0.07), with detectable NP366-specific CD8+ T cell responses in four of five, and one of five mice, respectively (Fig. 5d). This finding suggests that the mechanisms that govern the efficiency of presentation of i.m. expressed Ags are similar to those involved in i.d. Ag presentation.

The effect of transgene design on vaccine immunogenicity

Most DNA vaccines that enter clinical trials encode a modified version of the Ag of interest. Depending on native biological function, viral Ags have been shuffled to prevent cellular transformation (e.g., human papillomavirus E6 and E7) (32), or to prevent other protein functions, such as reverse-transcriptase activity or protease activity (e.g., HIV) (33). Alternatively, fusion proteins have been generated with the aim to incorporate additional immunostimulatory signals (34), or strings of T cell epitopes (35).

These types of manipulations that alter the three-dimensional structure of the resulting proteins are likely to result in aberrant folding and thereby affect protein t1/2. Based on the data presented above, vaccines encoding such rearranged gene products may be inferior in T cell induction. To directly test this hypothesis, we constructed a shuffled version of lucNP (hereafter referred to as ulcNP), in which the N-terminal 50 aa have been removed from the N terminus and reincorporated into the protein 114 aa downstream within the sequence. Based on the luciferase x-ray structure, this change is expected to disrupt luciferase domain architecture and thereby affect protein stability. Consistent with this, immunoprecipitation of ulcNP and lucNP after pulse-chase labeling indicates that the in vitro t1/2 of the scrambled ulcNP Ag is decreased as compared with the wild-type lucNP (Fig. 6, a and b). DNA vaccines encoding lucNP and ulcNP were then compared with regard to immunogenicity upon i.d. vaccination. Consistent with the notion that manipulations that affect Ag t1/2 influence DNA vaccine immunogenicity, NP366-specific CD8+ T cell responses induced by DNA vaccination with the scrambled ulcNP variant were inferior to those induced by vaccination with the unaltered lucNP vaccine (1.0 vs 4.0% on average at the peak of the response, p = 0.016). This indicates that modification of vaccine-encoded Ags by gene scrambling can substantially affect the ability of DNA vaccines to induce Ag-specific CD8+ T cell responses (Fig. 6c).


Figure 6
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  		FIGURE 6. Adverse effect of gene shuffling on DNA vaccine immunogenicity. a, SDS-PAGE analysis of antiluciferase immunoprecipitates of COS cells that were transiently transfected with pVAX encoding either lucNP or ulcNP. b, Graphical representation of an automated quantification of lucNP (•) and ulcNP ({blacktriangleup}) from a. c, Immunogenicity of lucNP and ulcNP DNA vaccines. Mice were vaccinated with 20 µg of lucNP (•) and ulcNP ({blacktriangleup}) on days 0, 3, and 6. At the indicated time points, peripheral blood was drawn and Ag-specific CD8+ T cell responses were determined ex vivo by Db-NP366 tetramer staining. Symbols represent CD8+ T cell responses of individual mice; bars indicate group averages.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have generated DNA vaccine variants by three types of design to assess whether Ag t1/2 affects the immunogenicity of DNA vaccines. Using these constructs for DNA vaccination, we have shown the following: 1) CD8+ T cell responses against two signal peptide-encoded epitopes were highly reduced as compared with those against the same epitopes contained within the mature part of the same carrier protein; 2) five different Ub-luciferase fusion proteins induced CD8+ T cell responses that correlated fully with their relative in vivo stability, and the correlation between Ag stability and immunogenicity was observed for both i.d. and i.m. DNA vaccination; and 3) CD8+ T cell responses against a shuffled and as a result instable variant of luciferase were substantially reduced as compared with those against the unmodified luciferase gene product. Of these experiments, the first and third line of experiments provide indirect evidence for an effect of Ag t1/2 on immunogenicity, and the second line of experiments provides direct proof for this link.

Elegant work by Trombetta and colleagues (36) has recently demonstrated how the induction of Th cell responses by protein vaccination is correlated with resistance of the Ag to degradation within the lysosomal pathway of the APC. In this work, we complement this study by demonstrating that the induction of cytotoxic T cell responses by DNA vaccines is correlated with Ag stability within the cytosol. Given the fact that efficient T cell priming induced by DNA vaccines depends on CD4+ T cell help (9, 30), it seems plausible that optimal immunogens may in fact require a high stability in both the cytosol and within the lysosomal pathway, a notion that may be tested in further studies.

The detrimental effect of low Ag stability on DNA vaccine immunogenicity that was observed in this study correlates with a reduced cross-presentation of epitopes derived from instable DNA vaccine-encoded Ags, and was also observed in a setting in which direct presentation was precluded. It is therefore interesting to speculate how the results from these experiments fit with existing models for Ag transfer from the Ag-donating cell to APC (10, 11, 12, 13, 14, 15, 16), and in particular whether the transferred material consists of peptides or full-length protein. For such an analysis, it may be more informative to use the data from i.m. DNA vaccination than those from i.d. DNA vaccination for the following reason. Two lines of evidence suggest that the in vivo t1/2 of free peptide epitopes in cells is very short. Specifically, work from Rammensee and colleagues (37) has demonstrated that T cell epitopes can only be recovered in appreciable levels from cells that express the relevant restriction element. Furthermore, it has been shown by Neefjes and coworkers (25) that fluorescently labeled peptide fragments have an extremely short t1/2 when introduced into cells. Because of this presumed short t1/2 of free intracellular peptides, a rapid decay in synthesis rate of an instable protein is most likely to be mirrored in an equally rapid decay in peptide levels. In situations in which de novo production of the vaccine-encoded protein is transient, as is the case for i.d. DNA vaccination (19), inefficient induction of Ag-specific T cell responses could therefore be explained by either a low pool of protein or a low pool of peptides in the Ag- donating cell.

However, upon i.m. DNA vaccination, de novo production of proteins is highly constant (23). In this setting, a short t1/2 of the vaccine-encoded protein will affect steady-state protein levels, but is not expected to affect steady-state peptide levels, because these are predicted to correlate with the rate of steady-state protein synthesis. In this situation, the observed beneficial effect of Ag stability on the immunogenicity of i.m. DNA vaccines (Fig. 5d) would not be expected if Ag transfer from Ag-donating cell to APC would involve the transport of peptide Ags. In contrast, if cross-presentation involves the transfer of proteasomal substrates, an increased protein t1/2 would lead to a proportional increase in the pool of Ag available for transfer and could thereby enhance vaccine immunogenicity. Based on this reasoning, the current data seem most consistent with a model in which protein Ags rather than peptides are transferred from Ag-donating cell to APC following DNA vaccination. However, the evidence for this is clearly very indirect, and a final resolution of the issue will most likely only be provided by the direct in vivo visualization of the process of cross-presentation.

Assuming that the observed reduced cross-presentation is the major cause for the reduced immunogenicity of instable Ags, the effect of Ag stability on vaccine immunogenicity shown in this study for DNA vaccines may also hold true for other vaccination strategies in which cross-presentation is involved. For instance, clinical strategies for vaccination with modified vaccinia Ankara (MVA)-based vectors by i.m. injection or skin scarification have been shown to be partially dependent on cross-presentation (7). Likewise, the induction of CD8+ T cell responses upon vaccination with recombinant adenoviral vectors is expected to occur through cross-presentation, because the coxsackievirus and adenovirus receptor (CAR) that is required for efficient infection of cells by commonly used adenoviral vectors such as Ad5 is absent on dendritic cells (8). Thus, T cell induction in both the priming as well as the boosting phase in DNA-modified vaccinia Ankara and DNA-Ad5 prime boost regimens that are currently in clinical trials (38, 39) may well benefit from vaccine designs that take Ag stability into account. In this respect, it is noteworthy that the type of fusion gene vaccines used in current and planned large-scale vaccination trials is expected or has in fact been shown to yield instable protein products (40), and may therefore well form suboptimal immunogens.

The current experiments have used the magnitude of vaccine-induced CD8+ T cell responses as a surrogate parameter for DNA vaccine efficacy. By this criterion, designs of DNA vaccines that disrupt protein folding should be avoided, and the in vivo stability of Ags should form a consideration when multiple candidate Ags are available. For DNA vaccines and for possible other vaccine formats for which Ag stability forms a relevant factor, it may be attractive to develop more generally applicable strategies to promote the accumulation of otherwise instable Ags. For instance, judicious removal of surface-exposed lysine residues may increase protein t1/2 by preventing Ub conjugation and thus proteasomal degradation of the Ag (41, 42). Secondly, incorporation of specific protein domains has previously been shown to lead to high-level protein accumulation, either as endoplasmic reticulum aggregates (43) or as cytosolic proteins, both with the possibility of pharmacological control (44). Based on the data presented in this study, evaluation of such strategies appears useful and may provide novel formats for vaccine transgene design.


   Acknowledgments
 
We thank M. Toebes for technical help, and J. Borst and M. de Witte for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 A.D.B. and M.C.W. contributed equally to this work. Back

2 Current address: La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037. Back

3 Address correspondence and reprint requests to Dr. Ton N. M. Schumacher, Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. E-mail address: t.schumacher{at}nki.nl Back

4 Abbreviations used in this paper: NP, nucleoprotein; DLN, draining lymph node; i.d., intradermal; Ub, ubiquitin. Back

Received for publication February 28, 2007. Accepted for publication May 22, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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