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The Journal of Immunology, 2003, 171: 2548-2555.
Copyright © 2003 by The American Association of Immunologists

Epitope-Enhanced Conserved HIV-1 Peptide Protects HLA-A2-Transgenic Mice Against Virus Expressing HIV-1 Antigen

Takahiro Okazaki1,*, C. David Pendleton*, François Lemonnier{dagger} and Jay A. Berzofsky2,*

* Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and {dagger} Unité d’Immunité Cellulaire Antivirale, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
HIV epitopes may have developed to be poor immunogens. As a counterapproach HIV vaccine strategy, we used epitope enhancement of a conserved HIV reverse transcriptase (RT) epitope for induction of antiviral protection in HLA-A2-transgenic mice mediated by human HLA-A2-restricted CTLs. We designed two epitope-enhanced peptides based on affinity for HLA-A2, one substituted in anchor residues (RT-2L9V) and the other also with tyrosine at position 1 (RT-1Y2L9V), and examined the balance between HLA binding and T cell recognition. CTL lines and bulk cultures in two HLA-A2-transgenic mouse strains showed that RT-2L9V was more effective in inducing CTL reactive with wild-type Ag than RT-1Y2L9V, despite the higher affinity of the latter, because the 1Y substitution unexpectedly altered T cell recognition. Accordingly, RT-2L9V afforded the greatest protection in vivo against a surrogate virus expressing HIV-1 RT mediated by HLA-A2-restricted CTL in a mouse in which all CTL are restricted to only the human HLA molecule. Such antiviral protection has not been previously achieved with an HLA epitope-enhanced vaccine. These findings define a critical balance between MHC affinity and receptor cross-reactivity required for effective epitope enhancement and also demonstrate construction and efficacy of such a component of a new generation vaccine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In protection against HIV or SIV virus (1, 2, 3), CD8 CTL play a major role. Nevertheless, the natural immune response to HIV is often unable to clear the infection. Although a number of Ags that induce CTL responses and can help to eliminate or reduce virus production by killing viral producer cells have been reported thus far, these do not seem to be sufficient to eliminate infection in most cases. There is no reason to expect that the HIV sequence would have evolved to have optimal CTL epitopes to allow eradication of the virus. Thus, in principle it should be possible to improve the immunogenicity of epitopes, a process called "epitope enhancement," to develop a more highly effective HIV vaccine (4, 5).

A first approach to enhance peptide immunogenicity is to improve the affinity of CTL epitopes for HLA class I molecules. For this reason, we decided to focus on a peptide from a conserved region of the HIV reverse transcriptase (RT) 3 designated RT179–187, VIYQYMDDL. This epitope is endogenously processed and presented and recognized by HLA-A2.1-restricted CTL in HIV-infected patients (6) and has been described as a binder with weak affinity to HLA-A2.1 molecule (7). This weak binding affinity allowed us to introduce modifications aimed at improving binding, compared with other HIV epitopes described as high affinity binders and less in need of improvement. The advantage of this epitope is that it is also strongly conserved because its amino acid sequence, YMDD, is part of an active site of HIV RT. Such a conserved epitope may be more valuable in a vaccine than a higher affinity but more mutable one subject to viral escape. The VIYQYMDDL epitope is found in the vast majority of HIV strains and may be harder for the virus to mutate without loss of fitness.

We have previously succeeded in improving the affinity of a hepatitis C core epitope for HLA-A2.1 (8) and of a helper epitope for murine class II MHC (9, 10), and an epitope-enhanced melanoma peptide has shown efficacy in human clinical trials (11). Other complementary approaches to improve affinity for TCRs have been devised (12, 13, 14). Although one substitution resulting in higher affinity HLA binding of another HIV peptide has been reported (15), no rational strategy to improve epitopes of HIV has been conducted. In particular, no systematic analysis of the competing effects of substitutions on HIV peptide binding to the HLA class I molecule vs peptide-HLA complex binding to the TCR has been reported.

Further, to our knowledge, protection against viral infection in vivo by an epitope-enhanced vaccine mediated by CTL restricted by a human HLA molecule has not previously been demonstrated. To study such protection, we have taken advantage of a novel strain of mice, HHD-2, that is transgenic for human HLA-A2.1 with a covalent human {beta}2-microglobulin and lacks any murine class I molecules because it is deficient in murine {beta}2-microglobulin and murine H-2Db. Thus, in this strain, all CTL are restricted only to the human class I HLA molecule, and any protection cannot be mediated by CTL restricted to murine class I MHC molecules (7, 16). Because of the importance of HIV and AIDS and the critical need for an effective vaccine that is more effective than the natural virus for inducing protective responses, we have now undertaken a systematic program to enhance conserved epitopes of HIV. Here, we show not only such HLA-restricted CTL-mediated antiviral protection but also the design and construction of an enhanced conserved HIV epitope based on balancing effects of binding to an HLA molecule and binding to the TCR that may be a useful component of a second-generation human HIV vaccine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthetic peptides

Peptides were prepared in an automated multiple peptide synthesizer (Symphony; Protein Technologies, Tucson, AZ) using fluorenylmethoxycarbonyl chemistry. They were purified by reverse phase HPLC, and their sequences were confirmed on an automated sequencer (477A; Applied Biosystems, Foster City, CA). Some peptides were also purchased from Multiple Peptide Systems (San Diego, CA).

Cells

The Jurkat-A2Kb cell line, a gift from Dr. L. Sherman (Scripps Research Institute, La Jolla, CA), is transfected with the HLA chimeric molecule containing the {alpha}1 and {alpha}2 domains from human HLA-A2.1 and {alpha}3 from mouse H-2Kb. C1R.AAD cell line (HMYC1R transfected with the HLA chimeric molecule containing {alpha}1 and {alpha}2 domains from human HLA-A2.1 and {alpha}3 from mouse H-2Dd), a gift from Dr. V. Engelhard (University of Virginia, Charlottesville, VA), has been previously described (8). Cell lines were maintained in 10% FCS-RPMI containing 1 mM sodium pyruvate, nonessential amino acids (Biofluids, Rockville, MD), 4 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME.

Mice

Transgenic A2Kb mice (17) (a gift from Dr. L. Sherman) and transgenic HHD-2 mice (7, 16) were bred in our colony at BioCon (Rockville, MD). HHD-2 mice have the murine {beta}2-microglobulin gene knocked out, as well as murine H-2Db knocked out, and are transgenic for a chimeric human HLA-A2.1 expressing the {alpha}1 and {alpha}2 domains of HLA-A*0201 and a murine Db-derived {alpha}3 domain to allow interaction with mouse CD8 and also have a covalently linked human {beta}2-microglobulin to compensate for lack of any free {beta}2-microglobulin. As a result of this lack of any free {beta}2-microglobulin, even though the H-2Kb gene is not knocked out, the only class I MHC molecule they express is the chimeric human HLA-A2.1 with the covalent human {beta}2-microglobulin, not any murine class I molecule.

Binding assays

Peptide binding to HLA molecules was measured using the T2 mutant cell line as described (8, 18). T2 cells (3 x 105/well) were incubated overnight in 96-well plates with culture medium (a 1:1 mixture of RPMI 1640-Eagle-Hank’s amino acid (EHAA) containing 2.5% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin) with 10 µg/ml human {beta}2-microglobulin (Sigma-Aldrich, St. Louis, MO) and different peptide concentrations. The next day, cells were washed twice with cold PBS containing 2% FBS and incubated for 30 min at 4°C with anti-HLA-A2.1 BB7.2 mAb (1/100 dilution of hybridoma supernatant) and 5 µg/ml FITC-labeled goat anti-mouse Ig (BD PharMingen, San Diego, CA). Cells were washed twice after each incubation, and HLA-A2.1 expression was measured by flow cytometry (FACScan; BD Biosciences, Mountain View, CA). HLA-A2.1 expression was quantified as fluorescence index (FI) according to the formula: FI = [(mean fluorescence with peptide - mean fluorescence without peptide)/mean fluorescence without peptide]. Background fluorescence without BB7.2 was subtracted for each individual value. To compare the different peptides, FI0.5, the peptide concentration that increases HLA-A2.1 expression by 50% over no peptide control background, was calculated from the titration curve for each peptide.

CTL generation in A2Kb and HHD-2-transgenic mice

Mice more than 8 wk old were immunized s.c. in the base of the tail with 100 µl of an emulsion containing 1:1 IFA and PBS solution with Ags and cytokines (50 nmol of CTL epitope, 50 nmol of hepatitis B virus core 128–140 (HBVc128–140) helper epitope, 5 µg of IL-12, and 5 µg of GM-CSF. Mice were boosted 2 wk later, and spleens were removed 10–14 days after the boost. Immune spleen cells (2.5 x 106/well) were stimulated in 24-well plates with autologous spleen cells (5 x 106/well) pulsed for 2 h with 10 µM CTL epitope peptide in complete T cell medium (RPMI 1640:EHAA with additives as under Cells) with 10% T-Stim (Collaborative Biochemical Products, Bedford, MA). After more than four in vitro stimulations with peptide-pulsed syngeneic spleen cells, CTL lines were maintained by weekly restimulation of 1 x 106 CTL/well with 4 x 106 peptide pulsed irradiated (3300 rads) syngeneic spleen cells as feeders, or by weekly stimulation of 1 x 106 CTL/well with 3.8 x 106 peptide pulsed irradiated C57BL/6 spleen cells and 1–3 x 105 peptide pulsed and irradiated (15,000 rad) Jurkat-A2Kb transfectant cells.

Cytotoxicity assay

CTL activity was measured using a 4-h assay with 51Cr-labeled target cells. Target cells (106) were pulsed in 100 µl of complete T cell medium and 150 µCi of 51Cr for 1.5 h, washed three times, and added at 3000 cells/well to the 96-well round-bottom plates with different peptide concentrations. Effector cells were added 2 h later, and the supernatants were harvested and counted after an additional 4 h of incubation. The percentage of specific 51Cr release was calculated as 100 x [(experimental release - spontaneous release)/(maximum release - spontaneous release)]. Spontaneous release was determined from target cells incubated without effector cells, and maximum release was determined in the presence of 0.1 M HCl. Jurkat-A2Kb lines or C1R.AAD cell lines were used as targets.

IFN-{gamma} and RANTES assay

IFN-{gamma} and RANTES in the culture supernatant were determined by ELISA kit (R&D, Minneapolis, MA) according to the manufacturer’s instructions. All samples were analyzed in triplicate.

Protection assay from viral challenge

Female mice were immunized with the same protocol as in the CTL generation protocol described above, boosted i.p. 2 weeks after primary immunization, and challenged i.p. 30 days later with recombinant vaccinia virus (2 x 107 PFU/mouse) expressing HIV RT (vCF21) or {beta}-galactosidase (vSC8) (gifts from Dr. B. Moss, National Institute of Allergy and Infectious Diseases, Bethesda, MD). Five days later, virus titers in the ovaries of individual mice were determined on BSC-1 indicator cells as previously described (19).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RT alanine-substituted peptides binding to HLA-A2.1 molecules

We evaluated the binding affinity of wild-type RT179–187 (RT-WT) with the T2 binding assay, measuring the cell surface stabilization of HLA-A2.1 molecules after incubation with peptide. To compare the different peptides, FI0.5 was chosen as a way to compare their titration and relative affinity for MHC molecules. Using this method, an FI0.5 of 41.9 µM was calculated for RT-WT. This binding affinity was much weaker than that of other nonamer peptides tested in our laboratory such as hepatitis C virus peptide C7A2 (8), flu matrix peptide 58–66 (20), and HIV-gag peptide SLYNTVATL (21) (Table I).


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  		Table I. Binding of RT179–187-WT and -substituted peptide to HLA-A2

 
Besides the primary anchor residues at positions 2 and 9 in the peptide, other residues can play an important role in binding either as secondary anchors, or as residues that interfere with binding by introducing adverse interactions (22, 23). Alanine substitutions replace a functional side chain with a methyl group, allowing one to ascertain the requirement for, or adverse effect of, such functional side chains. Therefore, to define key functional residues, peptides with alanine substitutions at each one of the positions were synthesized and tested in binding assays (Table I). Binding experiments with these alanine-substituted peptides showed (Table I) that peptide 2A and 7A had lost almost all binding ability, in accordance with the anchor character of 2nd position of peptides restricted to HLA-A2, and also 9A had impaired binding. Other substitutions that decreased binding were 5A and 7A, suggesting the importance of these residues as critical anchors secondary to the 2nd and 9th residues. However, peptide 6A had a higher affinity with FI0.5 ~17 µM. Finally, alanine substitutions at positions 1, 3, 4, and 8 had no effect on peptide binding.

Leucine and/or valine-substitution at anchor regions in RT-WT

In a second set of experiments to define key functional residues, peptides with leucine and/or valine substitution at the 2nd and 9th anchor position were synthesized and tested in the binding assay because RT-WT does not have the optimal anchor residues for binding to HLA-A2.1, namely L and V at positions 2 and 9, respectively. Substitution for the typical anchor amino acid at either anchor residue (24), position 2 for L or position 9 for V, had ~2-fold higher affinity of the RT-WT. Moreover, the peptide with substitution at both anchor residues, RT-2L9V, had a ~8-fold higher binding affinity than the RT-WT. This affinity was higher than that of any other alanine-substituted peptides of RT179–187 tested in Table I.

Comparison of the binding affinity between substitutions in anchor region and tyrosine substitution in position 1

Recent studies reported that a tyrosine substitution in the first position (P1Y) can increase peptide-MHC binding stability without altering antigenic specificity (15, 25). On the basis of these studies, we attempted to compare the peptide-MHC binding among the following four derivative peptides, RT-WT, RT-2L9V, RT-1Y (YIYQYMDDL), and RT-1Y2L9V (YLYQYMDDV), in the T2 binding assay. As shown in Fig. 1a, the 2L9V substitution showed much better binding ability than the RT-1Y substitution, whereas both substituted peptides had higher affinity than RT-WT. However, the peptide (RT-1Y2L9V) with the combination of both P1Y and 2L9V substitutions displayed the highest affinity of all the peptides. The binding ability of RT-1Y2L9V in the T2 binding assay was almost as good as that of FMP (Fig. 1b). According to these data, we focused on two kinds of substituted peptides, RT-2L9V and RT-1Y2L9V, as candidates for the epitope enhanced vaccine.



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  		FIGURE 1. a, Comparison of the HLA-A2 binding curves among the wild-type RT179–187, VIYQYMDDL, RT-1Y (YIYQYMDDL), RT-2L9V (VLYQYMDDV), and RT-1Y2L9V (YLYQYMDDV) in the T2-binding assay. b, Comparison of the HLA-A2 binding curves among the RT-2L9V, p17-WT (SLYNTVATL), RT-1Y2L9V, and flu matrix peptide 58–66 (FMP) (GILGFVFTL).

 
Recognition of RT-variant peptides by CTL lines from A2Kb- and HHD-2-transgenic mice

To determine residues involved in CTL recognition, we immunized HLA-A2-transgenic mice, using two different strains. A2Kb (from Dr. L. Sherman) expresses a chimeric class I molecule consisting of the HLA-A2.1 {alpha}1 and {alpha}2 domains with the {alpha}3 domain from murine H-2Kb, allowing better binding of the murine CD8 molecule, on the C57BL/6 background (17). HHD-2 mice express a chimeric HLA-A2.1 molecule in which the {alpha}3 domain is replaced by that of murine H-2Db for the same reason, along with a covalently attached human {beta}2-microglobulin chain, but also express no murine class I MHC molecules because they have been knocked out for the murine {beta}2-microglobulin and H-2Db genes (7, 16). We separately developed RT-WT and -2L9V specific CTL lines from both A2Kb- and HHD-2-transgenic mice and developed an RT-1Y2L9V-specific CTL line from HHD-2 mice immunized with each peptide after several rounds of stimulation with each peptide. In fact, these CTL lines we developed had almost completely nonoverlapping V{beta} repertoires. (In the case of A2Kb mice, the RT-WT-specific CTL line had V{beta}2, 3 and 12, whereas the RT-2L9V-specific CTL line had V{beta}3, -4, -5, -8, and -10b. In the case of HHD-2 mice, the RT-WT-specific CTL had V{beta}8.1 or -8.2, whereas the RT-2L9V-specific CTL had V{beta}4, and that against 1Y2L9V was dominated by V{beta}8.3 but also contained cells expressing V{beta}8.1, -8.2, and -9; data not shown). First, cross-reactivity among RT-WT, -2L9V, and -1Y2L9V was checked using these peptide-specific CTL lines from both A2Kb and HHD-2 mice (Fig. 2). Jurkat-A2Kb transfectant cells or C1R.AAD cells were used as a target. RT-WT-, -2L9V-, and -1Y2L9V-specific CTL lines killed target cells pulsed with adequate Ag concentration in an Ag-specific manner. All these peptide-specific CTL lines were cross-reactive with targets pulsed with the wild-type peptide. In the case of A2Kb-derived CTL lines, RT-2L9V-coated targets were killed at lower concentration than RT-WT-coated targets, consistent with the higher affinity of the RT-2L9V peptide and the cross-reactivity of the CTL lines for the two peptides. However, unexpectedly, the RT-2L9V-specific CTL line killed RT-WT pulsed targets at >1 log lower concentration of peptide than did the line raised against this peptide, indicating that the RT-2L9V peptide also elicited higher avidity CTL. Among the HHD-2-derived CTL lines, all three peptide-specific CTL lines recognized wild-type pulsed targets. 2L9V-specific CTL had the same ability to recognize the wild-type peptide as the RT-WT-specific CTL. However, the recognition pattern against the wild-type peptide by 1Y2L9V-specific CTL was paradoxically weaker than that by the other two CTL lines (Fig. 2b). Conversely, RT-WT-specific CTL did not recognize 1Y- and 1Y2L9V-pulsed targets (Fig. 2b). Furthermore, 1Y2L9V-specific CTL recognized peptides with tyrosine substitution in position 1 preferentially over peptides not mutated in position 1, even though RT-1Y has lower binding affinity to the HLA-A2 molecule than RT-2L9V in the T2-binding assay. Thus, in both directions, the difference between V and Y at position 1 can clearly be distinguished by the TCR, and this specificity for V or Y can override the effect of the higher affinity for MHC. These data suggested that 2L9V-specific CTLs derived from HLA-A2-transgenic mice in vitro have the same avidity as or higher avidity than the RT-WT-specific CTLs and that the amino acid in position 1 of an HLA-A2.1-restricted CD8 epitope could contribute to the specificity for recognition by the TCR, in contrast with the examples studied by Tourdot et al. (25).



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  		FIGURE 2. Comparison of antigenic potency by RT-WT, -2L9V, and -1Y2L9V CTL lines. a, Recognition of RT-WT and RT-2L9V peptides by RT-WT- and -2L9V-specific CTL lines from A2Kb-transgenic mice as a function of peptide concentration reveals difference in peptide affinity for HLA-A2 and CTL avidity for the same peptide-MHC complexes (E:T ratio, 10:1). b, Recognition of RT-WT, -1Y, -2L9V, and -1Y2L9V peptides by RT-WT-, -2L9V-, and -1Y2L9V-specific CTL lines from HHD-2-transgenic mice as a function of peptide concentration reveals difference in peptide affinity for HLA-A2 and CTL avidity for the same peptide-MHC complexes (E:T ratio, 10:1).

 
IFN-{gamma} and RANTES production from the RT-specific CTL lines stimulated by RT-variant peptides

To compare the inducibility of other forms of T cell activity by these peptides, we also tested the peptide-specific IFN-{gamma} and RANTES production by each CTL line as a function of peptide concentration (Fig. 3), because each can mediate antiviral protection. IFN-{gamma} is known to contribute to clearance of recombinant vaccinia virus in mice (26), and RANTES can inhibit binding of HIV to its coreceptor, CCR5 (27). In A2Kb mice, the RT-2L9V peptide could induce more IFN-{gamma} production by the RT-WT-specific CTL line than the RT-WT peptide itself at low peptide concentration, consistent with the affinities of the peptides for HLA-A2.1. In addition, the CTL raised against RT-2L9V appeared to have higher avidity for the RT-WT peptide than the CTL raised against RT-WT, with a shifted titration curve for RT-WT and a difference in the IFN-{gamma} production of >100-fold at 0.1 µM peptide (Fig. 3a). In HHD mice (Fig. 3b) Ag-specific IFN-{gamma} production by RT-2L9V-specific CTL was better stimulated with RT-2L9V than with RT-1Y2L9V, and importantly, the RT-2L9V-specific CTL line produced ~10 times as much IFN-{gamma} as the RT-1Y2L9V-specific CTL when stimulated with RT-WT.



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  		FIGURE 3. Comparison of IFN-{gamma} and RANTES production induced by RT-WT, -2L9V, and -1Y2L9V peptides. After being pulsed with different peptide concentrations for 2 h, Jurkat-A2Kb cells were irradiated at 15,000 rad and plated at 100,000 cells/well in 96-well round-bottom plates. Into each well 500,000 CTLs were added, and the supernatants were harvested at 48 h. IFN-{gamma} in the culture supernatant was determined by ELISA kit according to the manufacturer’s instructions. All samples were used at 2- to 640-fold dilution and analyzed in triplicate. a, IFN-{gamma} production by RT-WT- and -2L9V-specific CTL line derived from A2Kb-transgenic mice. b, IFN-{gamma} production by RT-2L9V- and -1Y2L9V-specific CTL line derived from HHD-2-transgenic mice. c, RANTES production by RT-WT- and -2L9V-specific CTL line derived from A2Kb-transgenic mice. d, RANTES production by RT-2L9V- and -1Y2L9V-specific CTL line derived from HHD-2-transgenic mice.

 
RANTES, a CC chemokine produced by CTL, has been shown to inhibit HIV infection of human cells (27). In A2Kb mice, the RANTES production by the RT-WT-specific CTL decreased to background level at 0.1 µM, whereas that by the RT-2L9V-specific CTL remained strong, regardless of which peptide was stimulatory (Fig. 3c). In HHD-2 mice, the 2L9V-substituted peptide could induce wild-type specific CTL more effectively, whereas the cross-reactivity of RT-1Y2L9V-specific CTL for wild-type peptide was weaker (Fig. 3d).

In vivo immunogenicity of RT-2L9V and -1Y2L9V peptide in HLA-A2-transgenic mice

Because the goal is a more potent HIV vaccine, after studying the inducibility of CTL activity by RT179–187-derived peptides by using CTL lines in vitro, we tested the in vivo immunogenicity of these peptides in the A2Kb- and HHD-2 transgenic mouse models. First, we checked the induction of CTL immune response against the RT-WT by the RT-WT and -2L9V peptides in A2Kb mice. Different groups of animals were immunized with the wild-type or 2L9V-substituted CTL epitope in conjunction with a helper epitope and cytokines as described in Materials and Methods, and their ability to induce an immune response was tested in CTL assays after stimulation twice with an adequate concentration of peptide (Fig. 4a). Both the RT-WT and -2L9V peptide could induce an immune response after stimulation with the higher peptide concentrations (10 and 0.3 µM), but the RT-2L9V induced higher CTL immune responses than the RT-WT. The CTL response with 0.01 µM concentration of the RT-WT stimulation was decreased down to the background level, whereas a CTL response could still be induced by 0.01 µM concentration of the RT-2L9V. These results suggested that the 2L9V-substituted peptide could induce a CTL response against the wild-type peptide-pulsed target stronger than that induced by the RT-WT and that the CTL response induced by this substituted peptide could recognize the wild-type peptide as an Ag more effectively.



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  		FIGURE 4. a, Induction of CTL immune response and comparison of CTL avidity against RT-WT in A2Kb-transgenic mice using different RT peptide variants. A2Kb-transgenic mice were immunized with 50 nmol of CTL epitope RT-WT or -2L9V plus 50 nmol of HBVc128–140 helper epitope and 5 µg of IL-12 and GM-CSF in IFA. After 2 wk, they were boosted under the same conditions; 10–14 days after the boost, spleen cells were removed and stimulated separately in vitro with 10, 0.3, and 0.01 µM CTL peptide-pulsed spleen cells. One week after the second stimulation in culture, a cytotoxic assay was performed with each concentration of RT-WT (WT) peptide. b, Induction of Ag-specific IFN-{gamma} production by peptide-specific culture line. HHD-2-transgenic mice were immunized with 50 nmol of CTL epitope RT-WT, -2L9V, or -1T2L9V plus 50 nmol of HBVc128–140 helper epitope and 5 µg of IL-12 and GM-CSF in IFA. After 2 wk, they were boosted under the same conditions; 10–14 days after the boost, spleen cells were removed and stimulated separately in vitro with irradiated spleen cells pulsed with the optimum concentration of each peptide. Nine days after the stimulation, the cultured cells were restimulated with each peptide. Each supernatant was assayed for IFN-{gamma} by ELISA 48 hr later.

 
Second, we tested the induction of Ag-specific IFN-{gamma} production by using the bulk cultures only one stimulation after the immunization with each peptide in HHD-2 mice. We immunized mice with 50 nmol of peptide and cytokines in IFA twice as described and stimulated immune spleen cells with 10 µM pulsed and irradiated syngeneic spleen cells. After 1 wk, we set up a short term stimulated culture with each peptide and measured IFN-{gamma} production of each supernatant after 48 h. No Ag-specific IFN-{gamma} production could be detected in the RT-WT-immunized bulk culture using this system. However, surprisingly, both RT-2L9V- and -1Y2L9V-immunized bulk culture induced much higher IFN-{gamma} production (Fig. 4b). Each culture responded best to its cognate Ag. Furthermore, IFN-{gamma} produced by the RT-2L9V-immunized bulk culture against the RT-WT was ~70-fold higher than that by the RT-1Y2L9V culture, even though the RT-1Y2L9V was much stronger in the binding to HLA-A2 than the RT-2L9V. These data suggested that the tyrosine substitution in position 1 adversely affects cross-reactivity and induces CTL that do not fully cross-react with the wild peptide, although both peptides have strong CTL-inducing ability against themselves.

Protection ability of the epitope-enhanced peptides in vivo

To test antiviral vaccine efficacy in the HLA-A2.1-transgenic mice, which cannot be infected with HIV-1 itself, we tested the protection ability of each RT peptide against the vaccinia virus expressing RT protein as a surrogate challenge virus in vivo. We specifically used HHD-2 mice because the only class I molecule they express is HLA-A2.1 (16); therefore, protection cannot be mediated by CTL restricted to murine MHC molecules. In both protection assays in Fig. 5, RT-2L9V-immunized mice were protected against vCF21, which replicates in ovaries, resulting in a 4- to 5-log reduction in virus titer (experiment 1) or complete protection (6-log reduction) (experiment 2) compared to unimmunized animals (p < 0.01). As a control, there was no protection against vSC8 that does not express RT. On the other hand, RT-1Y2L9V-immunized mice were only partially protected. These data confirm that the epitope-enhanced peptide, RT-2L9V, is more effective as an improved vaccine candidate than the wild-type Ag, and, because it is more cross-reactive with WT than the peptide with tyrosine substitution in position 1, RT-1Y2L9V, RT-2L9V is a more effective immunogen than RT-1Y2L9V even though RT-1Y2L9V has much higher binding affinity to HLA-A2 molecule than RT-2L9V.



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  		FIGURE 5. Protection induced by immunization with RT-peptides. On day 30 after the last immunization, female HHD-2 mice, expressing only the human HLA-A2.1 class I molecule and no murine class I molecules, were challenged i.p. with 2 x 107 PFU of vaccinia virus expressing a reverse transcriptase protein of HIV (vCF21) or a {beta}-galactosidase protein (vSC8). Five days later, virus titers in the ovaries were determined.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Protection by classical vaccines such as poliovaccine is mediated mostly by neutralizing Abs, but such Ab-inducing vaccines have been ineffective against viruses causing chronic infection such as HIV or hepatitis C virus. Rather, in this case, T cell immunity might be crucial as has been confirmed by CD8 cell depletion in AIDS virus infection of macaques (2, 3). Further, viral sequences evolving under immune selective pressure would not likely have optimal HLA molecule-binding epitopes. Thus, modifying epitope sequence to improve the CTL response could be one effective strategy toward development of new generations of vaccines (4, 5). We have previously enhanced a murine helper epitope (9, 10) and an HLA-A2-restricted epitope of HCV (8). Others have shown enhancement of an HIV epitope (15) and cancer epitopes (11, 28). However, to our knowledge, no previous study has shown improved protection against virus challenge in vivo by an epitope-enhanced vaccine mediated by human HLA-restricted CTL or systematically examined the competing effects of substitutions in HIV peptides on binding to HLA class I molecules vs binding of the peptide-MHC complex to the TCR.

For this reason, we first focused on the optimization of the anchor residues of the HLA-A2-restricted peptide and compared the response of CTL lines specific for both the wild-type- and the 2L9V-substituted epitope of RT in HIV using HLA-A2-transgenic mice. The peptide VIYQYMDDL is a known HIV epitope restricted to HLA-A2 but does not have any optimum amino acid in the anchor positions. The most optimum anchor residues for peptide binding to HLA-A2 are leucine and valine at the 2nd and 9th positions of peptide, respectively (24). As shown in Table I, the singly-substituted peptides at either the 2nd or 9th position could induce ~2-fold better binding capacity to HLA-A2 than the wild type. Moreover, the substituted peptide optimized at both the 2nd and 9th positions showed ~8-fold better binding than the wild type.

Some investigators reported that tyrosine substitution in position 1 of an HLA-A2-restricted CTL epitope could produce a high affinity epitope as a better vaccine strategy that was reported not to interfere with TCR interaction (15, 25, 29). On the basis of these reports, we also investigated tyrosine substitution at position 1. In contrast to these findings for other peptides, in our case, the combination of 1Y and these optimum anchors increases the binding ability to HLA-A2 up to ~100-fold over that of wild type but at the expense of some TCR cross-reactivity. Thus, we had two candidate epitope-enhanced peptides for the RT-WT peptide.

However, what is important in an improved vaccine is not only the binding affinity to the MHC molecule. Also the CTL induced by the improved peptide must have equal or better cross-reactivity to the wild-type epitope of a pathogen. To test the strict cross-reactivity to the wild-type epitope by CTL induced by the epitope-enhanced peptides, we developed RT-WT-, -2L9V-, and -1Y2L9V-specific CTL lines, respectively, from HLA-A2-transgenic mice. As shown in Fig. 2, both RT-1Y2L9V and -2L9V-specific CTL lines recognized the wild-type-pulsed target. However, RT-1Y2L9V-specific CTL did not have higher avidity than RT-WT-specific CTL, whereas 2L9V-specific CTL lines had an avidity equal to or higher than that of RT-WT-specific CTL lines. In addition, RT-WT-specific CTL did not recognize the tyrosine-substituted peptide. Furthermore, RT-1Y2L9V-specific CTL preferentially recognized these peptides with tyrosine substituted in position 1. These facts strongly suggest that the tyrosine in position 1 does affect the interaction with the T cell receptor, contrary to the examples examined in previous reports (25). Also in the IFN-{gamma} and RANTES production assays, the 1Y2L9V-specific responses by the RT-2L9V- and -WT-specific CTLs were weak or abrogated (Figs. 3 and 4b). A role for the amino acid residue in position 1 of an HLA-A2.1-binding peptide in interaction with the TCR, as demonstrated functionally here, is consistent with the crystallographic data for two TCRs crystallized with the complex of HLA-A2.1 and the Tax11–19 peptide of human T cell leukemia virus-I, in which the N-terminal leucine interacts with glutamine 30 of the A6 TCR or with methionine 28 of the B7 TCR (30, 31, 32).

To confirm the effectiveness of the improved peptide as a vaccine in vivo, we first tested the IFN-{gamma} production by bulk cultures of cells from mice immunized with each peptide and then conducted virus protection experiments in vivo. As shown in Fig. 4, IFN-{gamma} production by the RT-1Y2L9V-stimulated bulk culture against the wild-type Ag was ~90-fold less than that against the cognate Ag, whereas IFN-{gamma} production by the RT-2L9V-stimulated bulk culture against the wild type was only ~3-fold lower than that against the RT-2L9V Ag. Most importantly, RT-2L9V-immunized HLA-A2-transgenic mice were almost completely protected against the virus challenge, whereas RT-1Y2L9V-immunized mice were only partially protected (Fig. 5). The protection could not have been neutralizing Ab mediated because the RT protein is expressed only in the infected cell, not incorporated in the virus particle (33). Also, because the epitope is presented by HLA-A2.1, not murine MHC molecules, and importantly, because HHD-2 mice do not express any murine class I molecules, only the human HLA-A2.1 molecule, the protection must have been mediated by CD8+ T cells restricted to the human HLA-A2.1 class I molecule. Although the murine TCR repertoire is not identical with the human one, both are broad enough that CTL responses in HLA-A2.1-transgenic mice have been found to be predictive of human HLA-A2.1-restricted CTL responses (34), and the HHD-2 strain makes a broader response to HLA-A2.1-restricted epitopes than A2Kb mice (35), possibly because there is no competition from murine class I MHC molecules. Furthermore, because the wild-type sequence of this peptide is presented by HLA-A2.1 on HIV-1-infected human cells and CTL to this epitope can be found in HLA-A2.1-positive HIV-1-infected individuals (6), the successful use of this epitope-enhanced vaccine in mice expressing this HLA molecule as their sole class I MHC molecule should be directly translatable to human vaccines.

Therefore, an epitope-enhanced peptide should have a better binding affinity for an MHC molecule to induce the epitope-reactive CTL repertoires more efficiently. However, these results indicate that the strength of the cross-reactivity to the wild-type epitope is just as critical a criterion in the strategy of designing an epitope-enhanced vaccine as the binding affinity of a CTL epitope for an MHC molecule. Because high avidity CTL are critical in clearance of virus infection (36, 37, 38), the ability of an epitope-enhanced peptide to induce high avidity CTL, as we have seen here and with a hepatitis C virus peptide (8), makes this approach especially attractive. The higher avidity CTL induced by higher affinity peptide in this case and the hepatitis C case may be surprising in view of the fact that higher densities of peptide-MHC complex select for lower avidity CTL (36). Although we have no clear explanation for this favorable outcome, several possible mechanisms might be considered. First, it is possible that the more stable peptide-MHC complexes of the higher affinity peptides result in a longer duration of each TCR-peptide-MHC interaction (and thus of signal) that is more effective at eliciting high avidity CTL. Second, slight alterations in the conformation of the peptide bound in the MHC groove might shift some TCR-binding residues and select for a different, but perhaps overlapping, TCR repertoire and these may oftentimes have higher avidity. The stronger signal might also elicit a broader repertoire, which can allow selection of higher avidity CTL as demonstrated recently in a study of the role of MHC polymorphism in CTL diversity and avidity (39). Consistent with these last two explanations, we have seen differences in V{beta} usage between the CTL lines raised against the variant peptides as described in Results. If this pattern of higher avidity is generalizable in other examples, it would be of interest to explore these potential mechanisms further.

These studies provide a model for the construction of enhanced epitopes that can be used to build second generation vaccines, applicable to all forms of vaccine, peptide, DNA, recombinant viral or bacterial vector, or live attenuated virus. They also define and demonstrate the efficacy of a prototype conserved enhanced epitope that can be incorporated into many candidate vaccines currently under study. Although we show here proof of principle for a single epitope, albeit an important one because of its high degree of conservation, and it will be necessary to conduct similar studies for other epitopes to make an optimized vaccine, the results in this study may encourage such attempts for other epitopes, and the approach may be valuable in making a more effective AIDS vaccine (5).


   Acknowledgments
 
We thank Dr. William E. Biddison and Dr. Pierre Henkart for critical reading of the manuscript and helpful suggestions. We thank Drs. Jeffrey D. Ahlers, Igor Belyakov, Amiran Dzutsev, SangKon Oh, Jong Myun Park, James T. Snyder, Masaki Terabe, and Leon van den Broeke for helpful discussion and Lisa Smith for secretarial assistance.


   Footnotes
 
1 Current address: Division of Rheumatology, Department of Internal Medicine, St. Marrianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki 216-8512, Japan. Back

2 Address correspondence and reprint requests to Dr. Jay A. Berzofsky, Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, National Cancer Institute, Building 10, Room 6B-12 (MSC No. 1578), National Institutes of Health, Bethesda, MD 20892-1578. E-mail address: berzofsk{at}helix.nih.gov Back

3 Abbreviations used in this paper: RT, reverse transcriptase; FI, fluorescence index; FI0.5, the peptide concentration that increases HLA-A2.1 expression by 50% over no peptide control background; RT-WT, wild-type RT; HBVc128–140, hepatitis B core 128–140 helper epitope. Back

Received for publication March 20, 2003. Accepted for publication June 20, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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