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The Journal of Immunology, 1999, 162: 639-642.
Copyright © 1999 by The American Association of Immunologists

Cutting Edge

Cutting Edge: Induction of Enhanced CTL-Dependent Protective Immunity In Vivo by N-End Rule Targeting of a Model Tumor Antigen1

Timothy Tobery and Robert F. Siliciano2

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore MD 21205


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
There is much interest in vaccines that will enhance the induction of CTL. One mechanism to enhance Ag-specific CTL responses involves targeting Ag to undergo rapid cytoplasmic degradation by the N-end rule pathway. We have analyzed the ability of N-end rule targeting to confer protection in an immunization-challenge setting. Using the HIV-1 nef protein as a model tumor Ag, we found that in mice immunized with a vaccinia vector expressing a form of nef that is targeted for rapid cytoplasmic degradation, there was enhanced induction of nef-specific CTL and protection from a lethal challenge with the syngeneic CT26 tumor cells that had been transfected with nef. Protection from tumor challenge correlated with the magnitude of the CTL response. Thus, the targeting of tumor or viral Ags for rapid cytoplasmic degradation by the N-end rule pathway may represent a strategy for the induction of protective Ag-specific CTL responses in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
There is now considerable interest in vaccine strategies designed to enhance the induction of Ag-specific CTL responses. One such strategy involves targeting vaccine Ags directly into the MHC class I Ag-processing pathway, thereby providing more of the peptide epitopes that trigger the CTL response (1, 2, 3, 4, 5, 6, 7). The best understood signal that targets proteins for proteasomal degradation is the assembly of a polyubiquitin chain attached to an accessible Lys residue in the target protein. One factor that influences the rate at which polyubiquitination occurs is the identity of the N-terminal residue of the target protein, as certain non-Met N-termini target proteins for rapid degradation by the 26S proteasome (8, 9). Townsend and others (1, 5, 10) have shown that such N-end rule targeting of Ags can enhance their processing and presentation by the class I pathway in an in vitro setting, and we recently demonstrated that immunization of mice with an N-end rule targeted form of the HIV-1 nef protein induces a more vigorous nef-specific CTL response (5). This enhanced CTL response supports the idea that targeting Ags to undergo rapid cytoplasmic degradation might be a useful vaccine strategy in cases in which the induction of CD8+ CTL is the desired outcome. However, other studies have questioned the importance of this ubiquitin (Ub)3-dependent pathway (11, 12), and it is unclear whether the enhancement of CTL responses observed in in vitro assays of cytolytic function is of sufficient magnitude to confer protection against pathogenic viruses or tumors in vivo. To provide evidence that N-end rule targeting can lead to a functionally significant improvement of vaccine-induced protection in vivo, we have used a lethal tumor challenge model to study tumor Ag-specific immune responses induced by vaccination.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Generation of Ub-nef fusion constructs

Ub-nef fusion constructs with stabilizing Met or destabilizing Arg at the N terminus of nef (UbMNef or UbRNef, respectively) were generated as described (5). The recombinant viral vectors expressing ß-galactosidase (ß-gal; vac), wild-type nef (vVnef), UbMNef (vVUbMNef), or UbRNef (vVUbRNef) were generated using standard methods (13).

Cell lines

CT26nef cells were generated by transfecting CT26 tumor cells with a nef expression vector (pcDNA3nef) (5) using the mammalian transfection kit (Stratagene, La Jolla, CA). Stable transfectants were selected by neomycin resistance and cloned by limiting dilution culture under selection in the presence of 400 µg/ml G418. Expression of nef in stable transfectants was verified by Western blot analysis using anti-nef Abs (National Institutes of Health AIDS Research and Reference Reagent Program, Rockville, MD).

Generation of nef-specific CTL in vivo by immunization

BALB/c mice were immunized i.p. with 107 pfu (plaque forming units as determined by titering on BSC-1 cells) of the indicated recombinant vaccinia vectors expressing either ß-gal (control), UbMNef, or UbRNef. At the indicated time after immunization, spleens were harvested and splenocytes were incubated in the presence of IL-2 and psoralen/UV-treated vVnef infected syngeneic splenocyte stimulator cells for 5 days. Stimulated splenocytes were then used as effectors in a standard 51Cr-release assay as previously described (5). The percent-specific lysis was calculated according to the standard formula as previously described (5). Each determination was performed in quadruplicate. The SEM of the percent-specific lysis was almost invariably <5%.

CT26 lethal tumor challenge

BALB/c mice were immunized with 107 pfu of recombinant vaccinia vectors expressing either ß-gal, UbMNef, or UbRNef i.p. At 2–3 wk postimmunization, mice were challenged with wild-type CT26 adenocarcinoma cells (CT26wt) or with CT26 cells transfected with the HIV-1 nef gene (CT26nef) at doses of 104 or 105 cells/mouse s.c. on the flank. Mice were then monitored for the formation of solid tumor and were scored as tumor positive if they had a solid tumor of >5 mm in diameter.

Tumor explants

BALB/c mice that were positive for CT26nef tumor were euthanized, and their tumors were surgically explanted into DMEM containing 0.15% collagenase A and 5 mg/ml dispase II. The tumor was then digested by shaking at 37°C for 1 h. Tumor cells were dispersed using trypsin. Cells were then assayed for the expression of HIV-1 nef by Western blot analysis.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Rationale

In this study, a viral protein (HIV-1 nef) has been used as a model tumor-associated Ag to evaluate the in vivo efficacy of the N-end rule targeting strategy for enhancing the induction of de novo CTL responses. Proteins with non-Met N termini can be expressed in cells using fusion constructs in which the coding sequence of the target protein is fused in-frame to the C terminus of the coding sequence of Ub. Ub is normally made in the cell as a polyprotein that is cleaved by Ub hydrolases at the C-terminus of each Ub subunit, giving rise to individual Ub molecules (reviewed in Refs. 14 and 15). These same Ub hydrolases will also cleave the Ub target fusion protein at the C terminus of Ub, exposing the N terminus of the target. In a previous study, we generated Ub fusions to HIV-1 nef with either Met or Arg as at the N terminus of nef (UbMNef and UbRNef, respectively) (5). In in vitro experiments using vaccinia vectors to express UbMNef and UbRNef, it was shown that although both vectors induced expression of comparable amounts of nef, the form of nef with an Arg residue at the N terminus had a much shorter half-life (t1/2 = 15 min vs 10 h). Immunization of mice with a vaccinia vector expressing the rapidly degraded UbRNef resulted in the induction of a more vigorous nef-specific CTL response than did immunization with a vaccinia vector expressing the stable UbMNef.

Resistance to a lethal tumor challenge

To evaluate the functional significance of these vaccine-induced CTL, we used a challenge model involving the CT26 tumor cell line transfected with nef (CT26nef). The minimal dose of CT26nef tumor cells required to induce progressively growing tumors in 100% of naive syngeneic mice was 104 CT26nef cells (data not shown). BALB/c mice (30/group) were immunized once with a control vaccinia vector (vac) or with vaccinia vectors expressing UbMNef or UbRNef (vVUbMNef and vVUbRNef, respectively). Mice were challenged with 104 CT26nef cells s.c. 2.5 wk after immunization and then monitored for tumor growth. On day 20, 10 mice in each group were sacrificed and analyzed for CTL activity (see below). The remaining mice (20/group) were followed and examined for tumor growth. As shown in Fig. 1Go, all mice immunized with the control vac vector developed tumors by day 16 postchallenge with a mean time to tumor positivity of 12.2 days. All mice immunized with vVUbMNef also developed tumors, with all mice positive for tumor growth by day 18 postchallenge and a mean time to tumor positivity of 12.4 days for the entire group. These results suggest that the weak nef-specific CTL response induced by the vVUbMNef vector, which expresses a stable form of nef, was not sufficient to protect immunized mice from the CT26nef challenge. In sharp contrast, only 30% of the mice immunized with vVUbRNef developed tumors, with a mean time to tumor positivity for those mice of 14 days. Significantly (p < 0.0001), the remaining 70% of mice immunized with vVUbRNef remained tumor-free for at least 42 days postchallenge, at which time the surviving mice were sacrificed. These data suggest that immunization with the vVUbRNef vector that expresses a rapidly degraded form of nef was able to protect these mice from challenge with CT26nef tumor cells. When immunized mice were challenged with 104 wild type CT26 tumor cells, all mice developed tumors with similar kinetics regardless of the vaccinia vector used for immunization. The mean time to tumor positivity was 14 days for all groups and all mice were positive for tumor by day 17 (data not shown). Thus the protection induced by vVUbRNef depends on expression of nef in the challenge tumor. Taken together, these results indicate that a single immunization with a viral vector that expresses an Ag targeted into the class I-restricted Ag processing pathway can have a dramatic protective effect in vivo under conditions in which a similar immunization with a wild type form of the Ag has no protective effect.



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  		FIGURE 1. Protection against a lethal tumor challenge by an N-end rule targeted immunization strategy. Twenty BALB/c mice per group were immunized i.p. with 107 pfu of the control recombinant vaccinia vector vac that expresses ß-gal (X’s), or with 107 pfu of vVUbMNef (circles), or vVUbRNef (diamonds). Mice were challenged s.c. with 104 CT26nef cells and monitored for the formation of solid tumor 2.5 wk after immunization. Mice were scored as tumor positive if they had a solid tumor of >5 mm in diameter.

 
Relationship between nef-specific CTL activity and protection

To examine the relationship between nef-specific CTL activity and protection, 10 of the immunized mice in each group were sacrificed on day 20 postchallenge and splenocytes were assayed for CTL activity. Among these mice, 10/10 mice immunized with the control vaccinia vector and 10/10 mice immunized with vVUbMNef had tumors, whereas only 3/10 mice immunized with vVUbRNef had tumors. As shown in Table IGo, minimal nef-specific CTL activity was detected in stimulated splenocytes from mice immunized with the control vaccinia vector. In the case of mice immunized with vVUbMNef, significant nef-specific CTL activity was seen in only 1 of 10 mice, and this mouse was the last mouse to develop tumor in this group (day 18 postchallenge compared with a mean of 12.4 days for the entire group), suggesting that the nef-specific CTL response in this animal may have delayed the tumor growth. In mice immunized with vVUbRNef, there was a relatively high level of nef-specific CTL activity in stimulated splenocytes from 8 of 10 mice. The two mice that failed to mount a nef-specific CTL response both developed tumors after challenge with CT26nef. The third mouse that was tumor positive had a moderate nef-specific CTL response, but this animal developed tumor on day 16 postchallenge, compared with a mean of day 12.2 for mice immunized with the control vac vector, again suggesting that the CTL response delayed the onset of tumor. These data confirm that immunization with a vector expressing a rapidly degraded form of nef enhances the nef-specific CTL response. More importantly, these data show that the frequency of tumor growth in challenged mice is inversely related to the magnitude of the CTL response induced by vaccination. Previous studies have shown that the lysis detected in this system is mediated by CD8+ CTL and is MHC class I-restricted (5). In the present study, class II-negative P815 targets were used to ensure the detection of only class I-restricted lytic activity.


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  		Table I. Relationship between CTL activity and degree of protection against a tumor challenge

 
Expression of nef by explanted tumors

To provide further evidence for the relationship between vaccine-induced protection and nef-specific CTL activity, tumors were explanted from tumor-bearing mice on day 20 postchallenge and assayed for the expression of the transduced HIV-1 nef gene by Western blot analysis. All of the tumors explanted from mice immunized with the control vaccinia vector retained the expression of HIV-1 nef as evidenced by the presence of a specific band at 27 kDa (Table IGo). This result demonstrates that the expression of this model tumor Ag was stable in the mice. From mice that were immunized with vVUbMNef, all of the explanted tumors expressed nef with the exception of tumor from one mouse (Table IGo). This mouse showed a delay in tumor growth (18 days to tumor positivity compared with the mean 12.4 days in this group, Fig. 1Go) and had a nef-specific CTL response. One possible explanation is that in this mouse the nef-specific CTL response exerted immunologic pressure upon the tumor cells that led to the loss of expression of the nef gene. After a delay, the resulting nef- tumor cells grew out in the face of this nef-specific CTL response. It is also possible that the results reflect heterogeneity in the level of nef expression, although cloned CT26nef cells were used in this experiment. A total of 3 of 10 mice immunized with vVUbRNef developed tumors, all of which expressed nef (Table IGo). However, two of these mice had no significant nef-specific CTL response whereas the third had only a moderate nef-specific CTL response. Thus, the nef-specific immune response in these mice was insufficient to confer either protection from CT26nef challenge or the loss of nef expression by the tumor cells due to immunologic pressure.

To confirm that the vaccine-induced CTL could actually lyse autologous CT26nef tumor cells, mice were immunized with vVUbRNef and inoculated with 105 CT26nef cells 3 wk later. Three mice that developed tumors were sacrificed and spleen cells were stimulated with psoralen/UV-inactivated, vVnef-infected syngeneic spleen cells. Tumor cells were explanted and cultured. Six days later, the lysis of explanted CT26nef tumor cells and of control CT26wt cells by stimulated splenocytes was measured in a 51Cr release assay. As shown in Table IIGo, there was strong lysis of explanted autologous nef-expressing tumor cells and no lysis of the control CT26 cells.


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  		Table II. Killing of nef-expressing autologous tumor cells by vaccine induced CTL

 
Resistance to high dose tumor challenge

Taken together, the results presented above suggest that the N-end rule targeting strategy can lead to an enhancement in the induction of CTL that is sufficient to confer protection against lethal dose of Ag-expressing tumor cells. Some partial protection was even apparent when immunized mice were challenged with a tumor cell dose that was 10-fold higher that the dose used above. In the case of mice immunized with vac, challenge with the higher dose of 105 CT26nef tumor cells resulted in a mean time to tumor positivity of 13.2 days (Fig. 2Go). No significant protective effect was apparent in mice immunized with vVUbMNef. The mean time to tumor positivity of 13.4 days. Mice immunized with vVUbRNef did develop tumors but showed a substantial delay in the onset of tumor growth, with a mean time to tumor positivity of 16.9 days. A small fraction (2/30) of the mice immunized with vVUbRNef failed to develop tumors when challenged with 105 CT26nef cells. Thus, even after a high tumor challenge dose, the enhanced nef-specific CTL response induced by vVUbRNef was able to delay the onset of tumor and in a few cases to afford complete protection. Based on the level of protection and the time to tumor positivity, the protection afforded by vVUbRNef immunization against a 105 cell challenge dose was superior to that seen in vVUbMNef-immunized mice challenged with a 10-fold lower dose of tumor cells. Taken together, these results show that targeting the HIV-1 nef for rapid cytoplasmic degradation not only enhances the nef-specific CD8+ CTL response, but also results in better protection of immunized mice against challenge with CT26 tumor cells expressing nef.



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  		FIGURE 2. High dose CT26nef tumor challenge. Thirty BALB/c mice per group were immunized i.p. with 107 pfu of recombinant vaccinia vectors expressing either ß-gal (X’s), UbMNef (circles), or UbRNef (diamonds). Three weeks postimmunization, mice were challenged with 105 CT26nef cells and monitored for the formation of solid tumor. Mice were scored as tumor positive if they had a solid tumor of >5 mm in diameter.

 
Although this study addresses the ability of a degradation-targeted vaccine Ag to protect against a tumor challenge, the strategy is also expected to be useful in enhancing vaccine-induced protection against challenge with microbial pathogens for which cell-mediated immunity can be protective, including viruses and intracellular parasites. Although there is some evidence that the rate of degradation of an Ag does not always directly correlate with the generation of class I epitopes (12), other approaches for targeting viral Ags for rapid degradation have recently been shown to be associated with in vivo protection from a viral challenge. Rodriguez et al. (7) have generated a noncleavable Ub conjugate of the lymphocytic choriomeningitis virus nuclear protein that is rapidly degraded and have shown that in the setting of DNA immunization, this construct can enhance the induction of CTL and confer protection against a lethal lymphocytic choriomeningitis virus challenge. The mechanism involved in this case does not depend on the N-end rule, but the results nevertheless support the potential utility of the approach advanced here. Minev et al. (16) targeted OVA peptides for translocation into the endoplasmic reticulum (ER) by generating fusion peptides consisting of an ER-insertion signal sequence fused to the N terminus of OVA-derived peptide epitopes and showed that immunization of mice with vectors expressing these fusion peptides significantly enhanced the CD8+ CTL response against these epitopes and prolonged the survival of mice challenged with the OVA-expressing tumor E.G7OVA. Thus, three different strategies for the targeting of Ags to the class I processing pathway (rapid cytoplasmic degradation of Ags with a non-Met N terminus, rapid cytoplasmic degradation of noncleavable Ub conjugates, and rapid insertion of epitopes in the ER), have resulted in enhanced induction of Ag-specific CTL and protection from challenge in vivo. The targeting of whole Ags for rapid degradation has the advantage that there is no need to know a priori which peptide epitopes will elicit a CTL response because degradation of the intact Ag will naturally produce all potential peptide epitopes. Thus, targeting of Ags for rapid intracellular degradation may represent a very attractive general vaccine strategy for the induction of CD8+ CTL-mediated immunity.


   Acknowledgments
 
We thank Dr. Hyam Levitsky for advice on the CT26 model.


   Footnotes
 
1 The work was supported by National Institutes of Health Grants AI28108 and AI37924. Back

2 Address correspondence and reprint requests to Dr. Robert F. Siliciano, Johns Hopkins University School of Medicine, 1049 Ross Building, 720 Rutland Avenue, Baltimore, MD 21205. E-mail address: Back

3 Abbreviations used in this paper: Ub, ubiquitin; pfu, plaque forming units; ER; endoplasmic reticulum; ß-gal; ß-galactosidase. Back

Received for publication August 26, 1998. Accepted for publication November 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Townsend, A., J. Bastin, K. Gould, G. Brownlee, M. Andrew, B. Coupar, D. Boyle, S. Chan, G. Smith. 1988. Defective presentation to class I-restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen. J. Exp. Med. 168:1211.[Abstract/Free Full Text]
  2. Bacik, I., J. H. Cox, R. Anderson, J. W. Yewdell, J. R. Bennink. 1994. TAP (transporter associated with antigen processing)-independent presentation of endogenously synthesized peptides is enhanced by endoplasmic reticulum insertion sequences located at the amino- but not carboxyl-terminus of the peptide. J. Immunol. 152:381.[Abstract]
  3. Restifo, N. P., I. Bacik, K. R. Irvine, J. W. Yewdell, B. J. McCabe, R. W. Anderson, L. C. Eisenlohr, S. A. Rosenberg, J. R. Bennink. 1995. Antigen processing in vivo and the elicitation of primary CTL responses. J. Immunol. 154:4414.[Abstract]
  4. Vitiello, A., G. Ishioka, H. M. Grey, R. Rose, P. Farness, R. LaFond, L. Yuan, F. V. Chisari, J. Furze, R. Bartholomeuz. 1995. Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection. I. Induction of a primary cytotoxic T lymphocyte response in humans. J. Clin. Invest. 95:341.
  5. Tobery, T., R. F. Siliciano. 1997. Targeting of HIV-1 antigens for rapid intracellular degradation enhances CTL recognition and the induction of de novo CTL responses in vivo following immunization. J. Exp. Med. 185:909.[Abstract/Free Full Text]
  6. Wu, Y., T. J. Kipps. 1997. Deoxyribonucleic acid vaccines encoding antigens with rapid proteasome-dependent degradation are highly efficient inducers of cytolytic T lymphocytes. J. Immunol. 159:6037.[Abstract]
  7. Rodriguez, F., J. Zhang, J. L. Whitton. 1997. DNA immunization: ubiquitinization of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody formation. J. Virol. 71:8497.[Abstract]
  8. Bachmair, A., D. Finley, A. Varshavsky. 1986. In vivo half-life of a protein is a function of its amino-terminal residue. Science 234:179.[Abstract/Free Full Text]
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  10. Michalek, M. T., E. P. Grant, K. L. Rock. 1994. Ubiquitin dependent and independent proteolytic pathways for MHC class I-restricted presentation of ovalbumin. FASEB J. 8:A747. (abstr.).
  11. Cox, J. H., J. R. Bennink, J. W. Yewdell. 1994. The ubiquitin-dependent proeolytic pathway is not involved in processing of viral antigens for recognition by MHC class I-restricted T cells. FASEB J. 8:A747. (abstr.).
  12. Goth, S., V. Nguyen, N. Shastri. 1996. Generation of naturally processed peptide/MHC class I complexes is independent of the stability of endogenously synthesized precursors. J. Immunol. 157:1894.[Abstract]
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