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Recombinant Virus Vaccination against "Self" Antigens UsingAnchor-fixed Immunogens

Surgery Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892 [K. R. I., M. R. P., E. P. S., J. P. T., M. C., C. E. T., P. F. R., S. A. R., N. P. R.]; Therion Biologics Corporation, Cambridge, Massachusetts 02142 [A. G. Y., P. G.]; and Leiden University Medical Center, Leiden, The Netherlands, 2333 ZA [R. P. M. S., R. O.]

	ABSTRACT

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To study the induction of anti-"self" CD8⁺ T-cell reactivity against the tumor antigen gp100, we used a mouse transgenic for a chimeric HLA-A*0201/H-2 K^b molecule (A2/K^b). We immunized the mice with a recombinant vaccinia virus encoding a form of gp100 that had been modified at position 210 (from a threonine to a methionine) to increase epitope binding to the restricting class I molecule. Immunogens containing the "anchor-fixed" modification elicited anti-self CD8⁺ T cells specific for the wild-type gp100_209–217 peptide pulsed onto target cells. More important, these cells specifically recognized the naturally presented epitope on the surface of an A2/K^b-expressing murine melanoma, B16. These data indicate that anchor-fixing epitopes could enhance the function of recombinant virus-based immunogens.

	Introduction

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The recent cloning of TAAs² recognized by T lymphocytes has led to the development of recombinant and synthetic anticancer vaccines; the first of these candidate vaccines are currently being tested in clinical trials (1, 2, 3, 4) . However, recent clinical trials using recombinant virus-based immunogens (such as vaccinia virus, fowlpox virus, or adenovirus) encoding tumor rejection antigens have not yet demonstrated consistent antitumor immune responses or tumor reduction (Ref. 5 and Rosenberg et al.).³ These negative results may be attributed, in part, to preexisting antivector antibodies that neutralize the viral vaccines (5) . However, no CD8⁺ T-cell reactivity was induced in individuals who received the recombinant avipox virus, fowlpox, which has little-to-no antibody cross-reactivity with vaccinia virus (Rosenberg et al.³ and Ref. 6 ). Therefore, factors other than neutralizing antibodies may explain why it is difficult to induce antitumor immunity using recombinant viral vectors. Among the possible factors are the regulatory effects of central or peripheral tolerance on the T-cell repertoire (7) . In addition, the TAA epitopes may not be efficiently presented to the immune system because of interdeterminant competition with peptides derived from the vector (8) . In an attempt to augment the immunogenicity of TAA expressed by recombinant viral vaccines, we modified an anchor residue of a self-epitope within the full-length melanoma-associated antigen, gp100, to increase peptide binding to class I molecules without compromising the interaction of the MHC/peptide complex with the T-cell receptor (9) .

Human gp100, a nonmutated "self"-antigen expressed by melanocytes, pigmented retinal cells, and most melanoma lesions, is not expressed in other normal tissues or nonmelanoma tumors (10 , 11) . Parkhurst et al. (12) have shown that the engineering of a key anchor residue at position 2 of the HLA-A*0201-restricted peptide gp100_209–217 [threonine (T) to an optimal anchor methionine (M), gp100_{209–217(210M)}] yields a peptide with a 9-fold better binding to HLA-A*0201 and an increased ability to sensitize PBL from HLA-A*0201-positive melanoma patients in vitro. Furthermore, when the modified peptide was administered with interleukin 2 as an adjuvant, over 40% of patients with metastatic melanoma experienced tumor regression (4) . In this study, we show that the immunization of A2/K^b transgenic mice with recombinant viral vectors expressing "anchor-fixed" peptides, engineered to have enhanced binding to MHC, induced immune responses against a naturally processed, self-peptide of gp100. Thus, this approach may overcome the negative effects of both immune tolerance and intramolecular competition.

	Materials and Methods

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Animals and Cell Lines.
The HLA-A*0201/K^b (A2/K^b) transgenic mice used in this study were a kind gift of L. Sherman (The Scripps Research Institute, La Jolla, CA) and have been described previously (13) . These animals express a chimeric class I molecule composed of the 1 and 2 domains of the HLA-A*0201 class I antigen and the 3, cytoplasmic, and transmembrane domains of the mouse H-2K^b class I molecule. We transfected B16 with a plasmid expressing the A2/K^b molecule driven by the SV40 promoter (B16/A2/K^b) and selected this cell line in 1 µg/ml puromycin (Life Technologies, Inc., Grand Island, NY). We maintained B16/A2/K^b, B16.WT, and the T2 cell line (HLA-A*0201⁺, TAP-deficient, T-B cell hybrid) in CM containing RPMI 1640, 10% heat-inactivated FCS (both obtained from Biofluids, Rockville, MD), 0.03% fresh L-glutamine, 100 µg/ml streptomycin, 100 units/ml penicillin (all from NIH, Media Unit, Bethesda, MD) and 50 µg/ml gentamicin sulfate.

Peptides.
Each of the peptides used in this study was prepared under Good Manufacturing Practice (GMP) conditions by Multiple Peptide Systems (San Diego, CA): (a) gp100_154–162, KTWGQYWQV; (b) gp100_209–217, ITDQVPFSV; and (c) gp100_{209–217(210M)}, IMDQVPFSV. The identity of each of the peptides was confirmed by mass spectral analysis. The peptides were >98% pure as assessed by high pressure liquid chromatography.

Recombinant Virus Construction.
Recombinant viruses were constructed by the insertion of foreign sequences into the genome of a plaque-purified isolate from the Wyeth strain of vaccinia virus as described previously (14) . In each virus, the foreign gene is under the control of the vaccinia 40K (H5R) promoter (15) . Each virus also contains the Escherichia coli lacZ gene under the control of the fowlpox virus C1 promoter used for the selection of recombinant virions (16) . The full-length (FL) gp100 gene was altered by site-directed mutagenesis at codon 210, changing a threonine codon to methionine, and at codon 288, changing an alanine codon to valine. This modified gene was inserted into the HindIII M region of the vaccinia virus genome to produce rVV-FLgp100(210M). The native form of the human gp100 gene was inserted into the HindIII M region of the vaccinia genome to produce rVV-FLgp100. The gp100 minigenes were constructed using synthetic oligonucleotides encoding the leader sequence from the adenovirus type 5 E3/19K gene joined to gp100 codons 209 to 217, with either methionine or the native threonine at codon 210 (17) . The minigenes were inserted into the HindIII J region of the vaccinia genome and were designated rVV-ESgp100_{209–217(210M)} and rVV-ESgp100_209–217, respectively. rVV expressing the full-length tyrosinase gene (rVV-Tyr) was generated in a similar manner.

⁵¹Chromium (⁵¹Cr) and Cytokine Release Assays.
Secondary in vitro lymphocyte populations were generated by harvesting spleens of mice 3–6 weeks after immunization and culturing single-cell suspensions of splenocytes in T-25 flasks (Nunc, Roskilde, Denmark) at a density of 6 x 10⁶ cells/ml with 2 µg/ml antigenic peptide at a total volume of 10 ml of CM containing 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate (both from Biofluids), and 5 x 10^-2 ME (Life Technologies, Inc., Rockville, MD) in the absence of interleukin 2. Splenocytes were harvested and washed in CM before testing in a ⁵¹Cr release or cytokine release assay. Six-h ⁵¹Cr release assays were performed as described previously (6) . Briefly, 2 x 10⁶ target cells were incubated in 0.2 ml of CM labeled with 200 µCi of Na⁵¹CrO₄ for 90 min. Peptide-pulsed T2 cells were incubated for 1 h with 1 µg/ml (approximately 1 µM) antigenic peptide during labeling. Target cells were mixed with effector cells for 6 h at 37°C at 25:1 E:T ratio. The amount of ⁵¹Cr released was determined by gamma counting, and the percentage of specific lysis was calculated as follows:

For cytokine release assays, 10⁵ effector cells were coincubated with 10⁵ target cells in a total of 200 µl for 18–24 h in a 96-well plate. At this time, 150 µl of supernatant were harvested and tested for the presence of murine IFN- or GM-CSF by ELISA as described previously (18) .

	Results

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Comparison of Human and Murine gp100 Epitope Sequences.
To explore the relevance of immunizing A2/K^b transgenic mice with human gp100, we compared the human and murine sequences from two well-characterized HLA-A*0201-restricted epitopes from gp100 (19) . Human gp100_154–162 differs from the mouse sequence by one amino acid at the nonanchor position 5, where glutamine (KTWGQYWQV) replaces lysine (KTWGKYWQV). In contrast, the sequence for gp100_209–217 is identical in mouse and human gp100 (ITDQVPFSV). Therefore, for studies in the A2/K^b transgenic mouse model, we focused our efforts on the induction of self-reactive, gp100_209–217-specific CD8⁺ T-lymphocyte reactivity.

No Induction of Self-Reactive CD8⁺ T Cells after Immunization with rVV Encoding Wild-Type Full-Length Human gp100 (FLgp100).
To study the induction of anti-gp100 immune responses in A2/K^b transgenic mice, we cultured splenocytes from mice immunized with rVV-FLgp100 with each of the human gp100 epitopes, gp100_154–162 or gp100_209–217. There were high levels of gp100_154–162-specific lysis from rVV-FLgp100 cultures stimulated with gp100_154–162 (Fig. 1) . In contrast, we could not detect gp100_209–217-reactive CD8⁺ T cells after stimulation of the rVV-FLgp100-immune splenocytes with the gp100_209–217 peptide (Fig. 1) . Furthermore, in vitro stimulation of rVV-FLgp100 immune splenocytes with an anchor-fixed modified peptide, gp100_{209–217(210M)}, did not lead to the induction of gp100_209–217-specific lysis (Fig. 1) . Thus, we could not detect specific reactivity against the self-epitope, gp100_209–217, using the unmodified, full-length gp100, despite the ability of this recombinant viral vaccine to elicit powerful immunoreactivity against the "nonself," gp100_154–162 epitope.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Immunization with recombinant poxviruses expressing gp100 induced CD8+ T-cell lytic responses against gp100154–162 but not against gp100209–217. A2/Kb transgenic mice were immunized once i.v. with 107 pfu of rVV-FLgp100. Three to 6 weeks later, pooled splenocytes (two mice per group) were harvested and stimulated for 6 days with 2 µg of the indicated peptides per ml. Cultures were then assayed for specific lytic activity in a 51Cr-release assay against either T2 cells alone () or T2 cells pulsed with the relevant native peptides, gp100154–162 or gp100209–217 (). A 100:1 E:T ratio is shown. This experiment was repeated two times with similar findings.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. T-cell recognition of gp100-specific epitopes of rVV-infected HLA-A*0201+, gp100- breast cancer cell line. The MDA 231 HLA-A*0201+, gp100- breast cancer cell line was infected at an MOI of 10 for 1 h at 37°C with rVV-Tyrosinase (rVV-Tyr), rVV-FLgp100, rVV-FLgp100(210M), rVV-ESgp100209–217, or rVV-ESgp100209–217(210M). The infected MDA 231 cells were washed and plated at 105/well and coincubated with 2.5 x 104 human T-cell clones for 18 h. Clone 70 specifically recognizes gp100154–162, and clone DW2C8 recognizes either gp100209–217 or gp100209–217(210M). Supernatants were assayed for human IFN- release by ELISA.

View this table: [in this window] [in a new window]   Table 1 Induction of gp100209–217-specific IFN- release after immunization with rVV expressing the anchor-fixed modification

Enhanced Recognition of rVVs Expressing Minimal Determinant Epitopes of gp100.
We have reported previously (17) that facilitating antigen processing enhances the immunogenicity of some antigens. In these cases, we used methods that eliminate the need for protease-mediated cleavage and for TAP-mediated transport by using minimal determinant epitopes preceded by endoplasmic reticulum insertion signal sequences (ES). Thus, this approach increases the amount of peptide complexed with MHC class I on the cell surface (17 , 20) . To determine whether this increased amount of peptide/MHC complexes would break tolerance to the self-gp100_209–217 epitope, we generated rVV constructs expressing either the native gp100_209–217 (rVV-ESgp100_209–217) or the anchor-fixed gp100_{209–217(210M)} (rVV-ESgp100_{209–217(210M)}) minimal determinant epitopes preceded by the ES leader sequence.

Next, we infected the MDA 231 HLA-A*0201⁺, gp100^- cell line with each of the recombinant viruses and tested for recognition by the gp100_209–217-specific, human T-cell clone DW2C8 to confirm that they expressed either the gp100_209–217 or gp100_{209–217(210M)} epitopes (Fig. 2) . Large quantities of human IFN- were present after coincubation of the gp100_209–217-specific T-cell clone (DW2C8) admixed with MDA 231 cells infected with either rVV-ESgp100_209–217 or rVV-ESgp100_{209–217(210M)}. DW2C8 released 2–3 times more IFN- when target cells were infected with minigene-containing viruses than when they were infected with the rVV expressing the full-length, anchor-fixed gp100 construct, rVV-FLgp100(210M), and about 40 times as much as with the native, full-length gp100 construct, rVV-FLgp100. These data suggest that the use of minigene constructs enhances the level of peptide complexed with MHC class I for recognition by CD8⁺ T cells.

Induction of Self-Reactive, gp100_209–217-specific CD8⁺ T Cells with rVV Expressing the Anchor-fixed Minimal Determinant.
To determine whether we could induce native gp100_209–217 CD8⁺ T-cell reactivity using vaccinia viruses encoding minimal determinant epitopes preceded by endoplasmic reticulum leader sequences, we vaccinated A2/K^b Tg mice with either rVV-ESgp100_209–217 or rVV-ESgp100_{209–217(210M)} (Table 1B) . There was no gp100_209–217-specific cytokine release from the rVV-ESgp100_209–217 immune splenocyte cultures after restimulating with either the wild-type gp100_209–217 or the anchor-fixed gp100_{209–217(210M)} peptides (Table 1B) . However, there was IFN- release from the rVV-ESgp100_{209–217(210M)} splenocyte culture stimulated in vitro with the anchor-fixed gp100_{209–217(210M)} peptide against T2 pulsed with either the wild-type or anchor-fixed peptide but not against T2 pulsed with gp100_154–162. We, therefore, conclude that even with the enhanced presentation of the gp100_209–217 using minigene constructs, we still needed the anchor-fixed modification to induce CD8⁺ T-cell immunoreactivity against the native gp100_209–217 epitope.

CD8⁺ T-Cell Recognition of Naturally Processed and Presented gp100_209–217 by Murine Melanoma, B16/A2/K^b.
To determine whether rVV-ESgp100_{209–217(210M)}-induced T cells recognize gp100_209–217 naturally presented with MHC class I on the surface of tumor cells, we assayed splenocyte cultures derived from mice immunized with rVV-Tyrosinase, rVV-ESgp100_209–217, or rVV-ESgp100_{209–217(210M)} for the ability to recognize and lyse the murine melanoma, B16, transfected with the chimeric A2/K^b molecule (B16/A2/K^b; Fig. 3A ). Only the rVV-ESgp100_{209–217(210M)}-immune splenocytes stimulated in vitro with gp100_{209–217(210M)} peptide specifically lysed murine melanoma, B16/A2/K^b (Fig. 3A) . The rVV-ESgp100_{209–217(210M)}-induced splenocytes cultured in vitro with gp100_{209–217(210M)} also specifically secreted both IFN- and GM-CSF in response to B16/A2/K^b (Fig. 3, B and C) but did not release IFN- or GM-CSF when cocultured with wild-type murine melanoma, B16.WT (HLA-A*0201 negative). This result indicated that the gp100_209–217-specific reactivity was HLA-A*0201-restricted (Fig. 3, B and C) . In addition, this gp100_{209–217(210M)}-specific T-cell culture recognized as little as 10 ng/ml gp100_209–217 peptide pulsed onto T2 cells, as assessed by the release of IFN, which indicated high avidity for wild-type peptide complexed with MHC (data not shown). Despite the induction of the antiself immunoreactivity, we did not observe autoimmune vitiligo in response to rVV-FLgp100 or rVV-FLgp100(210M) vaccination.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Immune recognition of the gp100209–217 epitope naturally processed and presented by B16/A2/Kb. In A, A2/Kb transgenic mice were immunized once i.v. with 107 pfu of rVV-Tyrosinase (Tyr), rVV-ESgp100209–217 (ES209), or rVV-ESgp100209–217(210M) (ES210M). Three to 6 weeks later, pooled splenocytes (two mice per group) were harvested and stimulated in vitro for 7 days with 2 µg of gp100154–162 (154–162), gp100209–217 (209–217), or gp100210–217(210M) (210M) peptides per ml. Cultures were restimulated once with the same peptide. After 7 days in culture, splenocytes were assayed at a 25:1 E:T ratio for specific lytic activity in a 51Cr-release assay against T2 cells alone, T2 pulsed with the relevant gp100209–217, or a murine melanoma stably transfected with the chimeric A2/Kb molecule, B16/A2/Kb. In B and C, the rVV-ESgp100209–217(210M)-immune splenocyte culture stimulated in vitro with gp100209–217(210M) was further assayed for cytokine release in response to a panel of target cells. Specifically, 105 effector cells were admixed with 105 target cells in each well of 96-well plates. Eighteen to 24 h later, supernatants were harvested and assayed by ELISA for the presence of either murine IFN- (B) or murine GM-CSF (C).

	Discussion

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Peptides can be modified at the anchor residues in a way that increases their binding to class I molecules without compromising the interaction of this complex with the T-cell receptor. Furthermore, by stabilizing peptide/MHC complexes, specific CD8⁺ T-lymphocyte responses can be enhanced, and tolerance can be broken. In the clinic, immunization with viral vectors encoding full-length gp100 have not consistently elicited immunoreactivity against gp100_209–217, an epitope with low binding affinity to HLA-A*0201 (Ref. 5 and Rosenberg et al.).³ gp100_209–217 may compete for presentation by MHC class I molecules with other epitopes from gp100, or even more likely, with immunodominant epitopes derived from the recombinant virus (8) . Alternatively, mechanisms of central or peripheral tolerance having negative regulatory effects on the T-cell repertoire may also explain the lack of immunoreactivity against the self-epitope, gp100_209–217 (7) . In this study, "anchor-fixing" the gp100_209–217 epitope within the full-length gp100 molecule not only increased the amount of peptide presented complexed with MHC class I but also induced in vitro epitope-specific CD8⁺ T-cell reactivity (Fig. 2 and Table 1A ). The gp100_209–217-specific CD8⁺ T cells were not merely low-affinity CTLs that recognized exogenously loaded peptides at nonphysiologically high concentrations, but they also recognized the wild-type epitope naturally expressed by the murine melanoma, B16 transfected with A2/K^b.

Peptide elution studies that measured approximate numbers of peptides presented by cells that were infected with rVV that expressed different molecular forms of antigen showed an enhancement of peptide presentation in cells infected with rVV minigenes compared with rVV-expressing full-length proteins (20) . Specifically, although only 30 peptides per cell were presented by cells infected with rVV encoding the full-length form of nucleoprotein from influenza virus, up to 55,000 copies of peptides were recovered from cells expressing nucleoprotein minigene products (20) . Furthermore, we have previously demonstrated that facilitating antigen presentation by vaccinating with minigene constructs enhanced immunogenicity compared with immunizing with full-length antigens (17) . rVV encoding the gp100_209–217 minigene preceded by an endoplasmic reticulum insertion signal sequence expressed increased amounts of gp100_209–217 compared with the rVV encoding the full-length gp100, as assessed by T-cell recognition (Fig. 2) . Despite enhanced presentation, rVV-ESgp100_209–217 failed to elicit CD8⁺ T cells reactive against the gp100_209–217 native epitope. Only the rVV expressing the anchor-fixed minigene induced immunity against the wild-type gp100_209–217. This result suggests that the stability of peptides associated with MHC, not simply the peptide levels, was critical to induce reactivity against the weak immunogen, gp100. One explanation for this may be that anchor-fixing increased the binding stability to MHC class I by potentially decreasing the rate of dissociation of the peptide from the MHC class I molecule (21) .

The amount of peptide presented by the MDA 231 cells infected with rVV-ESgp100_209–217 was apparently similar to those infected with rVV-ESgp100_{209–217(210M)} (Fig. 2) . We have repeated this experiment using cells infected at different MOI in an attempt to detect differences between the wild-type and anchor-fixed minigene forms of the immunogens. We, nevertheless, could not detect clear differences in the in vitro recognition of vaccinia infected target cells. The in vivo results, on the other hand, are highly reproducible. Only the anchor-fixed immunogen is effective at eliciting self-reactive T cells. What can account for this apparent discrepancy? It seems that the viruses produce high levels of these peptides even at lower MOI, and that the levels produced are not limiting for recognition by CD8⁺ T cells. This situation parallels the in vitro recognition assays using T2 cells pulsed with either 1 µg/ml gp100_209–217 peptide or 1 µg/ml gp100_{209–217(210M)} peptide. Both are recognized similarly, an observation likely due to the saturation of the MHC class I on the cell surface.

Several papers have also reported the use of anchor-fixing modifications to enhance cytotoxic T-cell responses against self-molecules (4 , 18 , 22) . Recently, in a mouse model, we elicited CD8⁺ T cells against the murine homologue of gp100 using the human sequence gp100_25–33, which was shown to bind to MHC H-2 D^b 100-fold better than the murine gp100_25–33 sequence. This fortuitous difference between species altered the predicted MHC binding rank from number 14 to number 1 (18) . In addition, a peptide from the nonmutated murine tyrosinase-related protein-1 could be anchor-fixed, which enabled it to elicit tumor-reactive CD8⁺ T cells as well as tumor protection (22) . In human clinical trials using peptides as immunogens, 10 (91%) of 11 of patients vaccinated with the gp100_{209–217(210M)} peptide successfully induced CD8⁺ T cells reactive with native gp100_209–217, whereas only 2 of 8 patients who received the gp100_209–217 peptide exhibited gp100_209–217-specific reactivity (4) .

The similarity of these results to previous findings in melanoma patients documents the potential utility of the HLA/A2/K^b transgenic model system to predict the immunogenicity of other self-antigens (4) . Peptides can also be engineered to optimize immunity by modifying secondary anchor residues, by removing amino acids that may have deleterious effects on MHC/peptide contacts or by altering T cell receptor contact residues (9) . In addition, with the creation of peptide libraries, a new range of T-cell epitopes can be identified for testing in vivo to explore their relative immunogenicity (23) . This study suggests that anchor-fixed modifications of peptides may be a key to inducing reactivity against nonmutated "self"-TAAs expressed by recombinant virus-based immunogens.

	ACKNOWLEDGMENTS

 
We thank W. Overwijk for helpful discussions. We also thank M. Blalock for assistance with the graphics and L. Gritz and G. Mazzara for provision of the viruses and critical review of this manuscript.

	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 To whom requests for reprints should be addressed, at Surgery Branch, National Cancer Institute, NIH, Building 10, Room 2B42, Bethesda, MD 20892. Phone: (301) 496-4904; Fax: (301) 496-0011; E-mail: restifo{at}nih.gov

² The abbreviations used are: TAA, tumor-associated antigen; rVV, recombinant vaccinia virus; A2/K^b, HLA-A*0201/H-2 K^b chimeric molecule; HLA, human leukocyte antigen; CM, complete media; GM-CSF, granulocyte macrophage colony-stimulating factor; MOI, multiplicity/multiplicities of infection; pfu, plaque-forming unit(s); FLgp100, full-length human gp100; TAP, transporter associated with antigen processing; ES, endoplasmic reticulum insertion signal sequence.

³ S. A. Rosenberg et al., unpublished data.

Received 1/22/99. Accepted 4/14/99.

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