JVI Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
JVI.01351-05v1
80/21/10645    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Okazaki, T.
Right arrow Articles by Berzofsky, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Okazaki, T.
Right arrow Articles by Berzofsky, J. A.

 Previous Article  |  Next Article 

Journal of Virology, November 2006, p. 10645-10651, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01351-05

Possible Therapeutic Vaccine Strategy against Human Immunodeficiency Virus Escape from Reverse Transcriptase Inhibitors Studied in HLA-A2 Transgenic Mice{triangledown}

Takahiro Okazaki,1,{dagger} Masaki Terabe,1 Andrew T. Catanzaro,2,{ddagger} C. David Pendleton,1 Robert Yarchoan,2 and Jay A. Berzofsky1*

Vaccine Branch,1 HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-15782

Received 29 June 2005/ Accepted 8 August 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mutation of human immunodeficiency virus (HIV) leading to escape from anti-HIV drugs is the greatest challenge to the treatment of HIV infection. High-grade resistance to the nucleoside reverse transcriptase (RT) inhibitor lamivudine (also known as 3TC) is associated with a substitution of valine for methionine at position 184 of RT. This amino acid residue is contained within the HLA-A2-restricted epitope VIYQYMDDL (RT-WT). Here, we sought to determine whether a peptide vaccine could be developed using an epitope enhancement strategy that could induce a cytotoxic T-lymphocyte (CTL) response specific for an epitope containing the drug resistance mutation M184V to exert an opposing selective pressure. RT-WT-specific CTLs developed from HLA-A2 transgenic mice did not recognize the M184V mutation of RT-WT (RT-M184V). However, RT-M184V exhibited higher binding affinity for HLA-A2 than RT-WT. Also, both anchor-enhanced RT-WT (RT-2L9V) and RT-2L9V-M184V-specific CTLs recognized RT-M184V and displayed cross-reactivity to RT-WT. Nevertheless, the CTL repertoire elicited by the epitope-enhanced RT-2L9V-M184V appeared more selective for the RT inhibitor-induced M184V mutation. Peptide vaccines based on such strategies may be worth testing for their ability to exert selective pressure against drug-resistant strains and thus delay or prevent the development of HIV with the M184V resistance mutation.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Vaccines are among the most effective strategies for preventing and controlling viral infections. However, generally, in chronic viral infections such as human immunodeficiency virus (HIV) or hepatitis C virus infection, the virus does not elicit an immune response sufficient to clear the infection (1, 2). As a possible solution for eliciting an immune response against HIV, we have previously investigated an epitope enhancement strategy, involving modification of the sequence to improve binding to HLA molecules, to a conserved cytotoxic T-lymphocyte (CTL) epitope in HIV reverse transcriptase (RT), VIYQYMDDL RT(179 -187), which is restricted to the most common human class I molecule, HLA-A2 (12). This study demonstrated that the enhanced CTL epitope, in which the anchor residues were modified for enhanced binding to the HLA-A2 molecule, can induce CTLs more efficiently while maintaining full cross-reactivity to the original viral epitope.

In the case of HIV, antiretroviral therapy has been utilized successfully to control viral replication. However, a major barrier to the antiretroviral drug treatment of HIV infections is that the high degree of genetic variation and high levels of viral replication often lead to the emergence of drug-resistant variants during treatment. A common target for therapy is the RT of HIV. However, when employing the nucleoside reverse transcriptase inhibitor lamivudine, [(–)-2',3'-deoxy-3'-thiacytidine, or 3TC], resistant variants containing an M184I alteration in the RT sequence appear transiently and are rapidly replaced by ones characterized by the M184V substitution (3, 6, 9, 17, 20, 22). The selection of high-level resistance to 3TC can occur within weeks in patients with incomplete HIV suppression. We hypothesized that if CTLs could be induced that were specific for this mutant epitope and were able to kill cells infected with the escape variant virus, one could develop a vaccine to apply selective pressure against such drug-resistant virus and prolong and extend the efficacy of the antiretroviral drug. Indeed, CTLs specific for RT-M184V VIYQYVDDL have been reported in an HIV-infected patient (19). It was also reported that the plasma HIV viral load remained stable at low levels and even declined gradually over time without a change in antiretroviral therapy in a patient exhibiting CTLs specific for RT-M184V (19). These findings supported the feasibility of our proposed approach.

As the acquisition of resistance to an RT inhibitor is mediated by a mutation within the HLA-A2-restricted CTL epitope RT(179-187), which we had previously studied, from VIYQYMDDL to VIYQYVDDL, we hypothesized that CTLs specific for the mutant epitope RT(179-187) M184V might be raised by immunization with a mutant sequence M184V peptide that was made more immunogenic by an epitope enhancement strategy and that these CTLs might prevent viral escape from the drug. In the current investigation, we attempted to develop the immunologic foundation for our strategy of using a therapeutic HIV vaccine along with HIV antiretroviral therapy with the aim of developing CTLs specific for a mutation that confers resistance to the antiviral drug in order to prevent such resistant mutants from occurring and of utilizing epitope enhancement to optimize the vaccine.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthetic peptides. Peptides were prepared on an automated multiple peptide synthesizer (Symphony; Protein Technologies, Inc.) using 9-fluorenylmethoxy carbonyl chemistry. Peptides were purified by reverse-phase high-performance liquid chromatography; subsequently, peptide composition and concentration were confirmed by amino acid analysis and, where necessary, 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 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) has been previously described (10, 18). The Jurkat-A2Kb cell line, a gift of Linda Sherman (Scripps Research Institute), 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. Cell lines were maintained in RPMI containing 10% fetal calf serum, 1 mM sodium pyruvate, nonessential amino acids (Biofluid, Rockville, MD), 4 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-mercaptoethanol (henceforth designated complete T cell medium [CTM]).

Mice. Transgenic HHD-2 mice (a gift of François Lemonnier, Institut Pasteur, Paris, France) were bred in our colony at BioCon, Inc. (Rockville, MD). HHD-2 mice are characterized by knockout of the murine ß2-microglobulin gene, as well as murine H-2Db, transgenic expression of human HLA-A2.1 with a covalently linked human ß2-microglobulin, and a murine Db-derived {alpha}3 domain to allow interaction with mouse CD8. Thus, HLA-A2.1 is the only class I major histocompatibility complex (MHC) molecule expressed by this strain (5, 13).

Binding assays. Peptide binding to HLA molecules was measured using the T2 mutant cell line as described previously (11, 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 medium containing 2.5% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin) with 10 µg/ml human ß2-microglobulin (Sigma Chemical Co., St. Louis, MO) and different peptide concentrations. On the following day, cells were washed twice with cold phosphate-buffered saline containing 2% fetal calf serum and incubated for 30 min at 4°C with anti-HLA-A2.1 monoclonal antibody BB7.2 (1/100 dilution of hybridoma supernatant) and 5 µg/ml fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin (Pharmingen, San Diego, CA). Cells were washed twice after each incubation; subsequently, HLA-A2.1 expression was measured by flow cytometry (FACScan; Becton Dickinson, Mountain View, CA). HLA-A2.1 expression was quantified as the fluorescence index (FI) according to the following formula: FI = (mean fluorescence with peptide – mean fluorescence without peptide)/(mean fluorescence without peptide). FI0.5 is the concentration (in µM) required to give an FI of 0.5, meaning a 50% increase in HLA-A2 on the cell surface. Background fluorescence without BB7.2 was subtracted for each individual value. The peptides used in this study are chemically stable at 37°C and are short enough that they have no "native conformation" to denature, and so peptide stability should not be an issue in this assay.

CTL generation in HHD-2 transgenic mice. Mice more than 8 weeks of age were immunized subcutaneously in the base of the tail with 100 µl of an emulsion containing 1:1 incomplete Freund's adjuvant and phosphate-buffered saline solution with antigens and cytokines (50 nmol CTL epitope, 50 nmol hepatitis B virus core 128-140 helper epitope, 5 µg of interleukin-12, and 5 µg of granulocyte-macrophage colony-stimulating factor). Mice were boosted 2 weeks later, and spleens were removed 10 to 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 CTM supplemented with 10% T-Stim (Collaborative Biochemical Products, Bedford, MA). Following more than four in vitro stimulations with peptide-pulsed syngeneic spleen cells, CTL lines were maintained by weekly restimulation of 1 x 106 CTLs/well with 4 x 106 peptide-pulsed irradiated (3,300 rads) syngeneic spleen cells as feeders or by weekly stimulation of 1 x 106 CTLs/well with 3.8 x 106 peptide-pulsed irradiated C57BL/6 spleen cells and 1 x 105 to 3 x 105 peptide-pulsed and irradiated (15,000 rads) 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 CTM and 150 µCi 51Cr for 1.5 h, washed three times, and added at 3,000 cells/well to 96-well round-bottom plates with different peptide concentrations. Effector cells were introduced 2 h later; then, the supernatants were harvested and counted following an additional 4-h 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 in the absence of effector cells, and maximum release was determined in the presence of 0.1 M HCl. C1R.AAD cell lines served as targets.

IFN-{gamma} assay. Jurkat-A2Kb cells were treated with 100 µg/ml mitomycin C for 20 min. After washing three times, 100,000 cells/well were used as antigen-presenting cells. Five hundred thousand CTLs were added into each well with or without an optimal concentration of peptide (1 µM), and the supernatants were harvested at 48 h. Gamma interferon (IFN-{gamma}) in the culture supernatant was determined by using an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, Minn.) according to the manufacturer's instructions. Concentrations less than 125 pg/ml were below the level of detection and are reported as not detected. IFN-{gamma} production in all wells without any peptide was not detected. All samples were used at a 4- to 32-fold dilution. All samples were analyzed in triplicate.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RT(179-187)-substituted peptides binding to HLA-A2.1 molecules. To develop CTLs to an epitope spanning the drug resistance mutation site that bound to HLA-A2, we first measured binding of wild-type and mutated peptides to HLA-A2. The T2-binding assay (11, 18) was utilized to assess whether the M184V mutation of RT(179-187), RT-M184V, possessed the ability to bind the HLA-A2 molecule; this protocol involved measurement of the cell surface stabilization of HLA-A2.1 molecules on transporter of antigenic peptides (TAP)-deficient T2 cells following incubation with each peptide (Fig. 1). We also studied several candidate peptides using epitope enhancement strategies. Based on our previous results regarding epitope enhancement of RT-WT (12) and the results of Harrer et al. on peptides with the wild-type sequence (8), the following peptides were selected: VIYQYMDDL (RT-WT), VLYQYMDDV (RT-2L9V), IVIYQYMDDL (I-RT-WT), IVIYQYVDDL (I-RT-M184V), VIYQYVDDL (RT-M184V), VLYQYVDDV (2L9V-M184V), YLYQYVDDV (1Y2L9V-M184V), and YLYQYIDDV (1Y2L9V-M184I) (Table 1).


Figure 1
View larger version (20K):
[in this window]
[in a new window]
  		FIG. 1. (a) Comparison of HLA-A2-binding curves among RT-WT (VIYQYMDDL), RT-M184V (VIYQYVDDL), I-RT-WT (IVIYQYMDDL), and I-RT-M184V (IVIYQYVDDL) in the T2-binding assay. (b) Comparison of HLA-A2-binding curves among RT-M184V, 2L9V-M184V (VLYQYVDDV), 1Y2L9V-M184V (YLYQYVDDV), and 1Y2L9V-M184I (YLYQYIDDV). (c) Comparison of HLA-A2-binding curves for 1Y2L9V-M184V and 1Y2L9V-M184I with that of the positive control peptide FMP. Comparable results for each panel were obtained in two similar experiments.

 

View this table:
[in this window]
[in a new window]
  		TABLE 1. Peptides used in this study

 
As previously described (5, 12), RT-WT was observed to bind weakly to the HLA-A2 molecule. We found that peptide RT-M184V binds with somewhat stronger affinity than does RT-WT (Fig. 1a) (FI0.5 = 32 for RT-WT and 17.2 for RT-M184V). This result indicates that RT-M184V could be an antigenic epitope restricted to HLA-A2. However, both 10-mer peptides tested, I-RT-WT and I-RT-M184V, demonstrated weaker binding abilities (FI0.5 = 79 and 55 µM, respectively) to HLA-A2 than the 9-mer peptide RT-WT, consistent with earlier results on recognition of the wild-type sequence 9- and 10-mer peptides by human CTLs (8), suggesting that the HLA-A2-restricted peptide derived from this RT region should be the 9-mer VIYQYMDDL of HIV type 1 (HIV-1) or VIYQYVDDL for the lamivudine resistance mutation. The concordance with the human CTL results also strengthens the relevance to the human immune response.

Recent studies reported that a tyrosine substitution at the first position (P1Y) can improve peptide/MHC-binding stability (12, 21). Building on these studies, we examined the binding stabilities of 2L9V-M184V, 1Y2L9V-M184V, and 1Y2L9V-M184I. All three peptides exhibited much higher affinities for the HLA-A2 molecule in comparison to RT-M184V (FI0.5 = 0.856, 0.245, and 0.332 µM, respectively, compared to 15.1 µM for RT-M184V) (Fig. 1b). In particular, the P1Y mutations of 1Y2L9V-M184V and 1Y2L9V-M184I displayed binding capacities for the HLA-A2 molecule nearly equal to that of the positive control, the highly antigenic influenza virus matrix peptide (FMP) (7) (FI0.5 = 0.176 to 0.245 for 1Y2L9V-M184V, 0.332 to 0.367 for 1Y2L9V-M184I, and 0.338 µM for FMP) (Fig. 1c). These results suggested that the peptide expressing the M184V mutation in the RT-WT epitope could function as at least as potent an antigenic epitope as the RT-WT epitope. We concluded that the best-fitted anchor- or P1Y-substituted peptide might be able to induce an RT-M184V-specific CTL repertoire that could provide an immune counterselective pressure to prevent the M184V viral escape mutation during HIV therapy involving RT inhibitors such as lamivudine.

Recognition of RT variant peptides by RT-WT-specific CTL lines from HHD-2 transgenic mice. To explore this possibility, we first tested the recognition pattern of RT-WT-specific CTLs developed from HLA-A2 transgenic mice. As shown in Fig. 2, the RT-WT-specific CTLs recognized RT-WT and I-RT-WT but not RT-M184V. This result in transgenic mice mirrored the previous finding of Harrer et al. involving human RT-WT-specific CTLs (8) and, together, these results suggest that the lamivudine resistance mutation abolishes recognition by an established CTL response. Furthermore, this result means that the experiment employing HLA-A2-restricted antigen-specific CTLs from HHD transgenic mice is a good model of a human CTL response.


Figure 2
View larger version (14K):
[in this window]
[in a new window]
  		FIG. 2. Recognition pattern of RT-WT (VIYQYMDDL), RT-M184V (VIYQYVDDL), I-RT-WT (IVIYQYMDL), and I-RT-M184V (IVIYQYMDDL) by RT-WT-specific CTL lines. Recognition of RT-WT, I-RT-WT, RT-M184V, and I-RT-M184V by RT-WT-specific CTL lines from HHD transgenic mice as a function of peptide concentration revealed differences in peptide affinities for HLA-A2 and CTL avidity for the same peptide-MHC complexes (effector/target ratio, 10:1). Similar results were obtained in two independent experiments.

 
Recognition of RT-WT peptide by epitope-enhanced peptide-specific CTL lines. Based on the results of the T2-binding assay (Fig. 1), CTL lines specific for 1Y2L9V-M184V, which possessed the best binding ability of all peptides tested, were developed from HHD transgenic mice. In concert with the RT-WT-, RT-2L9V-, and RT-1Y2L9V-specific CTLs previously developed, recognition abilities were compared among these four CTL types for RT-WT and RT-M184V (Fig. 3). RT-WT-specific CTLs failed to recognize either RT-M184V or RT-1Y2L9V, as seen in Fig. 2 and previously described (12). However, in contrast to the RT-WT-specific CTLs, the anchor-modified RT-2L9V-specific CTL line recognized both RT-WT and RT-M184V most strongly among all four CTL types. Based on the titration curve, RT-2L9V-specific CTLs recognized the RT-M184V mutant epitope more efficiently than did the 1Y2L9V-M184V-specific CTLs; nevertheless, the latter CTLs did recognize the RT-M184V peptide. Furthermore, RT-2L9V-specific CTLs were able to recognize RT-WT efficiently, whereas 1Y2L9V-M184V-specific CTLs were not. These findings suggested that the P1Y mutation diminishes the RT-WT-orientated specificity of the induced specific CTLs, while at the same time leading to much stronger affinity for the HLA-A2 molecule. This observation is consistent with our previous data (12). The principle that epitope-enhanced peptide-specific CTLs should exhibit cross-reactivity to the wild-type peptide suggests that the anchor-enhanced RT-2L9V pair of substitutions may also be a superior choice when applying the epitope enhancement strategy to immunization against the drug-induced M184V mutation.


Figure 3
View larger version (21K):
[in this window]
[in a new window]
  		FIG. 3. Comparison of antigenic potency by CTL lines specific for RT-WT, RT-2L9V (VLYQYMDDV), RT-1Y2L9V (YLYQYMDDV), and 1Y2L9V-M184V. Recognition of RT-WT, RT-M184V, and each cognate peptide by RT-WT-, RT-2L9V-, RT-1Y2L9V-, and 1Y2L9V-M184V-specific CTL lines from HHD-2 transgenic mice as a function of peptide concentration revealed differences in peptide affinities for HLA-A2 and CTL avidity for the same peptide-MHC complexes (effector/target ratio, 10:1). Similar results were obtained in two similar experiments.

 
Recognition of RT-WT by 2L9V-M184V-specific CTLs. Based on findings that 2L9V enhancement is able to induce CTL repertoires able to recognize both wild-type and M184V-substituted epitopes (Fig. 3), a 2L9V-M184V-specific CTL line was developed from HHD transgenic mice to test whether the 2L9V-M184V-induced CTL line could recognize both RT-WT and the RT-M184V mutation (Fig. 4). 1Y2L9V-M184V-specific CTL lines recognized three kinds of M184V-substituted RT peptides, but RT-WT was not recognized. This result was consistent with the data of Fig. 3. On the other hand, the 2L9V-M184V-specific CTL line recognized both the RT-WT epitope to some extent and the RT-M184V epitope even better. Indeed, the activity against RT-M184V was at least as potent as and possibly more potent than that by the 1Y2L9V-M184V-specific CTL line.


Figure 4
View larger version (20K):
[in this window]
[in a new window]
  		FIG. 4. Comparison of antigenic potency by 1Y2L9V-M184V- and 2L9V-M184V-specific CTL lines. Recognition of RT-WT (VIYQYMDDL), RT-M184V (VIYQYVDDL), 2L9V-M184V (VLYQYVDDV), and 1Y2L9V-M184V by 2L9V-M184V-specific and 1Y2L9V-M184V (YLYQYVDDV)-specific CTL lines from HHD-2 transgenic mice as a function of peptide concentration revealed differences in peptide affinities for HLA-A2 and CTL avidity for the same peptide-MHC complexes (effector/target ratio, 10:1). Similar results were obtained in two similar experiments.

 
Based on these results, that RT-2L9V- and 2L9V-M184V-specific CTLs recognize the RT-M184V epitope, we compared RT-M184V-specific IFN-{gamma} production among RT-WT-, RT-2L9V-, and 2L9V-M184V-specific CTLs (Fig. 5). When stimulated with an optimal concentration of peptide, RT-WT-specific CTLs could not produce IFN-{gamma} in response to RT-M184V presented by Jurkat-A2Kb cells. Moreover, RT-2L9V-specific CTLs showed epitope-specific production of IFN-{gamma} against both the RT-WT and RT-M184V epitopes, while 2L9V-M184V-specific CTLs responded only to RT-M184V. Thus, the 2L9V-M184V peptide appears to be the best candidate for inducing CTLs to suppress the drug-induced mutant virus when inducing one-way immune counterpressure.


Figure 5
View larger version (23K):
[in this window]
[in a new window]
  		FIG. 5. Comparison of antigen-specific IFN-{gamma} production by RT-WT-, RT-2L9V-, and 2L9V-M184V-specific CTLs. Each CTL line was cultured with Jurkat-A2Kb cells and with 1 µM peptide or no peptide. The culture supernatants at 48 h were assayed with an IFN-{gamma} enzyme-linked immunosorbent assay kit according to the company's instructions. ND, not detected. Similar results were obtained in two similar experiments.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Combination drug therapies for the treatment of AIDS have dramatically decreased the number of AIDS-related deaths. RT inhibitors, such as the nucleoside analog 3TC, are important components of these multidrug treatment regimens (4, 14, 16). However, the selective pressure for escape from RT inhibitors often leads to escape mutations in RT. In the case of 3TC, such pressure usually leads to the selection of highly resistant HIV with the substitution of valine for methionine at position 184 of HIV-1 RT. Similar mutations at the equivalent position in hepatitis B virus polymerase and in simian immunodeficiency virus and feline immunodeficiency virus RTs confer resistance to 3TC (17). Compounding this problem, this M184V mutation enables HIV to escape immune pressure of CTLs specific for the wild-type RT sequence in patients with HLA-A2, which is the most common HLA class I molecule worldwide. This mechanism implies that a single mutation can lead to the escape of HIV not only from the drug-induced pressure of RT inhibitors but also from CTL-induced immune selective pressure in patients displaying the HLA-A2 haplotype, resulting in the appearance of drug-resistant and CTL-resistant HIV strains. These facts support our hypothesis that a potential novel approach to prevent the appearance of drug-resistant strains might be the induction of CTLs specific for drug-induced mutant epitopes through the use of a vaccine designed using an epitope enhancement strategy (Fig. 6).


Figure 6
View larger version (17K):
[in this window]
[in a new window]
  		FIG. 6. Schematic representation of a therapeutic strategy to produce immune back-pressure to prevent outgrowth of escape mutations that confer drug resistance on HIV, based on the pressure balance hypothesis. Either 3TC or CTLs specific for the wild-type reverse transcriptase with methionine (M) at position 184 can select for escape mutants expressing valine (V) at that position (the M184V mutation), the most common drug resistance mutation in lamivudine-treated HIV-infected individuals. However, CTLs specific for the M184V mutant sequence could provide immune selective back-pressure to prevent outgrowth of the escape mutant sequence and prolong the usefulness of the drug lamivudine. Analogous strategies could be used for other drug resistance mutations.

 
To address this issue, we first examined whether the RT-M184V drug-resistant mutant possessed the ability to bind the HLA-A2 molecule. As shown in Fig. 1, the RT-M184V peptide displayed binding to HLA-A2 at least as good as or better than RT-WT, indicating that RT-M184V could be a CTL epitope restricted to HLA-A2. In addition, we assessed whether the RT-WT-specific CTL line developed from HHD transgenic mice could recognize RT-M184V. The RT-WT-specific CTLs failed to recognize RT-M184V. Thus, the RT-M184V mutation allows escape not only from the drug 3TC but also from immune selective pressure by CTLs specific for the wild-type epitope, VIYQYMDDL. This result, which is consistent with data derived from a human CTL line (8), suggests that such a CTL study in mice expressing this HLA molecule, as the sole class I MHC molecule, should be directly translatable to human vaccines.

The binding data confirmed the prediction that peptides with position 1 replaced by tyrosine (P1Y), such as 1Y2L9V-M184V, exhibit a superior binding ability to HLA-A2 (12, 21). However, to develop an improved vaccine, not only must the binding affinity to the MHC molecule be improved, but also the CTLs induced by the improved peptide must display cross-reactivity to the wild-type epitope of a pathogen. For this reason, the 1Y2L9V-M184V-specific CTL line was developed from HHD mice to assess cross-reactivity of the 1Y2L9V-M184V-specific CTLs to the M184V mutation and RT-WT (Fig. 3). 1Y2L9V-M184V-specific CTLs recognized RT-M184V but not RT-WT. This finding indicated that the P1Y mutation induced a CTL repertoire skewed away from clones recognizing the wild-type sequence. This result is consistent with our previous data with RT-1Y2L9V (12). In contrast, the RT-2L9V-specific CTL line recognized both RT-M184V and RT-WT more efficiently than did 1Y2L9V-M184V-specific CTLs. RT-2L9V is an enhanced epitope of RT-WT containing the optimum anchor residues for peptide binding to HLA-A2, which are leucine and valine at the second and ninth positions of the peptide, respectively (15). Its binding ability with HLA-A2 is approximately eightfold greater than that of RT-WT (12). Generally, amino acids in the anchor positions of a CTL epitope are thought not to participate in alteration of T-cell receptor recognition. Nevertheless, the fact that the 2L9V substitution produces reactivity to the M184V epitope by specific CTLs might suggest that any adverse interaction at peptide position 6 (RT184) between antigenic peptide and the T-cell receptor might be sufficiently weak that it is more than compensated by the increased affinity for the MHC molecule afforded by the 2L9V substitutions. Alternatively, the optimized anchor residues may stabilize the appropriate conformation. Based on these findings, the 2L9V-M184V substitution may be the best candidate for a protective vaccine epitope against the RT-M184V mutant.

To test this idea further, we developed the 2L9V-M184V-specific CTL line from HHD mice in order to compare the recognition patterns between 1Y2L9V- and 2L9V-M184V-specific CTLs (Fig. 4). The RT-2L9V-specific CTL line also recognized RT-WT and RT-M184V, whereas RT-M184V-specific human CTLs recognized only RT-M184V and not RT-WT (19). However, the recognition ability of the RT-WT peptide by 2L9V-M184V-specific CTLs was slightly weaker than that of RT-2L9V-specific CTLs. Moreover, 2L9V-M184V-specific CTLs can produce IFN-{gamma} against the RT-M184V epitope but not the wild-type epitope, and the RT-WT-specific CTLs did not respond to the drug-resistant escape mutant M184V (Fig. 5). Thus, the 2L9V-M184V-specific CTLs more selectively recognize the drug-resistant escape mutants and therefore may more effectively exert immunologic counterpressure to balance the selective pressure of lamivudine for resistance mutants (Fig. 6). Therefore, 2L9V-M184V may be a worthy therapeutic vaccine candidate to prevent or delay appearance of the M184V mutation induced by the nucleoside RT inhibitor lamivudine used in antiretroviral therapy in individuals expressing HLA-A2 (Table 2). This strategy may work most effectively if used early in lamivudine therapy, when the resistant variants represent a very minor component of the viral swarm. Based on these considerations, we would suggest that RT-2L9V may function as a prophylactic vaccine to induce more broadly cross-reactive CTLs against HIV, whereas 2L9V-M184V may be more effective as a therapeutic vaccine that may work synergistically with highly active antiretroviral therapy that includes lamivudine as one of its components. Although this epitope contains the major resistance mutation for lamivudine, it may be necessary to include epitopes containing resistance mutations at other sites in reverse transcriptase to completely prevent the outgrowth of resistant strains. Nevertheless, if CTL immunity to this epitope can slow the appearance of resistance mutations at residue 184 relative to other resistance mutations in the protein, it would show proof of principle of the strategy and possibly prolong the duration of efficacy of lamivudine in the immunized subjects.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Comparison of recognizing pattern by CTLs

 
This investigation stresses the novel theoretical possibility of a therapeutic vaccine involving an epitope-enhanced peptide strategy to prevent or delay the appearance of HIV-1 drug-resistant mutants during antiretroviral therapy. Additionally, these studies provide a model for the production of enhanced epitopes that can be applied to the construction of next-generation vaccines, applicable to all forms of vaccines, peptide, DNA, recombinant viral or bacterial vector, or live attenuated virus. Finally, these studies define and demonstrate the efficacy of a prototype conserved enhanced epitope that can be incorporated into many candidate vaccines currently under investigation.


   ACKNOWLEDGMENTS
 
We thank François Lemonnier for a gift of the HHD-2 mice and Ira J. Berkower for critical reading of the manuscript and helpful suggestions. We also thank Samuel Broder, James T. Snyder, Igor Belyakov, Amiran Dzutsev, SangKon Oh, and Jong Myun Park for helpful discussions and Lisa Smith for secretarial assistance.

This research was supported in part by the Intramural Research Program of the NCI, NIH.


   FOOTNOTES
 
* Corresponding author. Mailing address: Vaccine Branch, Center for Cancer Research, National Cancer Institute, Building 10, Room 6B-04 (MSC#1578), NIH, Bethesda, MD 20892-1578. Phone: (301) 496-6874. Fax: (301) 480-0681. E-mail: berzofsk{at}helix.nih.gov. Back

{triangledown} Published ahead of print on 18 August 2006. Back

{dagger} Present address: Division of Rheumatology and Allergy, Department of Internal Medicine, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki 216-8511, Japan. Back

{ddagger} Present address: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Berzofsky, J. A., J. Ahlers, J. Janik, J. Morris, S. Oh, M. Terabe, and I. M. Belyakov. 2004. Progress on new vaccine strategies against chronic viral infections. J. Clin. Investig. 114:450-462.[CrossRef][Medline]
  2. Berzofsky, J. A., J. D. Ahlers, and I. M. Belyakov. 2001. Strategies for designing and optimizing new generation vaccines. Nat. Rev. Immunol. 1:209-219.[CrossRef][Medline]
  3. Boucher, C. A., N. Cammack, P. Schipper, R. Schuurman, P. Rouse, M. A. Wainberg, and J. M. Cameron. 1993. High-level resistance to (–) enantiomeric 2'-deoxy-3'-thiacytidine in vitro is due to one amino acid substitution in the catalytic site of human immunodeficiency virus type 1 reverse transcriptase. Antimicrob. Agents Chemother. 37:2231-2234.[Abstract/Free Full Text]
  4. Carpenter, C. C., M. A. Fischl, S. M. Hammer, M. S. Hirsch, D. M. Jacobsen, D. A. Katzenstein, J. S. Montaner, D. D. Richman, M. S. Saag, R. T. Schooley, M. A. Thompson, S. Vella, P. G. Yeni, and P. A. Volberding. 1998. Antiretroviral therapy for HIV infection in 1998: updated recommendations of the International AIDS Society-USA panel. JAMA 280:78-86.[Abstract/Free Full Text]
  5. Firat, H., S. Tourdot, A. Ureta-Vidal, A. Scardino, A. Suhrbier, F. Buseyne, Y. Riviere, O. Danos, M. L. Michel, K. Kosmatopoulos, and F. A. Lemonnier. 2001. Design of a polyepitope construct for the induction of HLA-A0201-restricted HIV 1-specific CTL responses using HLA-A*0201 transgenic, H-2 class I KO mice. Eur. J. Immunol. 31:3064-3074.[CrossRef][Medline]
  6. Gao, Q., Z. Gu, M. A. Parniak, J. Cameron, N. Cammack, C. Boucher, and M. A. Wainberg. 1993. The same mutation that encodes low-level human immunodeficiency virus type 1 resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine confers high-level resistance to the (–) enantiomer of 2',3'-dideoxy-3'-thiacytidine. Antimicrob. Agents Chemother. 37:1390-1392.[Abstract/Free Full Text]
  7. Gotch, F. M., J. Rothbard, K. Howland, A. R. M. Townsend, and A. J. McMichael. 1987. Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature 326:881-882.[CrossRef][Medline]
  8. Harrer, E., T. Harrer, P. Barbosa, M. Feinberg, R. P. Johnson, S. Buchbinder, and B. D. Walker. 1996. Recognition of the highly conserved YMDD region in the human immunodeficiency virus type 1 reverse transcriptase by HLA-A2-restricted cytotoxic T lymphocytes from an asymptomatic long-term nonprogressor. J. Infect. Dis. 173:476-479.[Medline]
  9. Johnson, V. A., F. Brun-Vezinet, B. Clotet, B. Conway, R. T. D'Aquila, L. M. Demeter, D. R. Kuritzkes, D. Pillay, J. M. Schapiro, A. Telenti, and D. D. Richman. 2003. Drug resistance mutations in HIV-1. Top. HIV Med. 11:215-221.[Medline]
  10. Newberg, M. H., D. H. Smith, S. B. Haertel, D. R. Vining, E. Lacy, and V. H. Engelhard. 1996. Importance of MHC class 1 {alpha}2 and {alpha}3 domains in the recognition of self and non-self MHC molecules. J. Immunol. 156:2473-2480.[Abstract]
  11. Nijman, H. W., J. G. A. Houbiers, M. P. M. Vierboom, S. H. van der Burg, J. W. Drijfhout, J. D'Amaro, P. Kenemans, C. J. M. Melief, and W. M. Kast. 1993. Identification of peptide sequences that potentially trigger HLA-A2.1-restricted cytotoxic T lymphocytes. Eur. J. Immunol. 23:1215-1219.[Medline]
  12. Okazaki, T., D. C. Pendleton, F. Lemonnier, and J. A. Berzofsky. 2003. Epitope-enhanced conserved HIV-1 peptide protects HLA-A2-transgenic mice against virus expressing HIV-1 antigen. J. Immunol. 171:2548-2555.[Abstract/Free Full Text]
  13. Pascolo, S., N. Bervas, J. M. Ure, A. G. Smith, F. A. Lemonnier, and B. Perarnau. 1997. HLA-A2.1-restricted education and cytolytic activity of CD8+ T lymphocytes from ß2 microglobulin (ß2m) HLA-A2.1 monochain transgenic H-2Db ß2m double knockout mice. J. Exp. Med. 185:2043-2051.[Abstract/Free Full Text]
  14. Pluda, J. M., S. Broder, and R. Yarchoan. 1992. Therapy of AIDS and AIDS-associated neoplasms. Cancer Chemother. Biol. Response Modif. 13:404-439.[Medline]
  15. Rammensee, H. G., T. Friede, and S. Stevanoviic. 1995. MHC ligands and peptide motifs: first listing. Immunogenetics 41:178-228.[Medline]
  16. Reijers, M. H., G. J. Weverling, S. Jurriaans, F. W. Wit, H. M. Weigel, R. W. Ten Kate, J. W. Mulder, P. H. Frissen, R. van Leeuwen, P. Reiss, H. Schuitemaker, F. de Wolf, and J. M. Lange. 1998. Maintenance therapy after quadruple induction therapy in HIV-1 infected individuals: Amsterdam Duration of Antiretroviral Medication (ADAM) study. Lancet 352:185-190.[CrossRef][Medline]
  17. Sarafianos, S. G., K. Das, A. D. Clark, Jr., J. Ding, P. L. Boyer, S. H. Hughes, and E. Arnold. 1999. Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-branched amino acids. Proc. Natl. Acad. Sci. USA 96:10027-10032.[Abstract/Free Full Text]
  18. Sarobe, P., C. D. Pendleton, T. Akatsuka, D. Lau, V. H. Engelhard, S. M. Feinstone, and J. A. Berzofsky. 1998. Enhanced in vitro potency and in vivo immunogenicity of a CTL epitope from hepatitis C virus core protein following amino acid replacement at secondary HLA-A2.1 binding positions. J. Clin. Investig. 102:1239-1248.[Medline]
  19. Schmitt, M., E. Harrer, A. Goldwich, M. Bauerle, I. Graedner, J. R. Kalden, and T. Harrer. 2000. Specific recognition of lamivudine-resistant HIV-1 by cytotoxic T lymphocytes. AIDS 14:653-658.[CrossRef][Medline]
  20. Schuurman, R., M. Nijhuis, R. van Leeuwen, P. Schipper, D. de Jong, P. Collis, S. A. Danner, J. Mulder, C. Loveday, C. Christopherson, S. Kwok, J. Sninsky, and C. A. B. Boucher. 1995. Rapid changes in human immunodeficiency virus type 1 RNA load and appearance of drug-resistant virus populations in persons treated with lamivudine (3TC). J. Infect. Dis. 171:1441-1449.
  21. Tourdot, S., A. Scardino, E. Saloustrou, D. A. Gross, S. Pascolo, P. Cordopatis, F. A. Lemonnier, and K. Kosmatopoulos. 2000. A general strategy to enhance immunogenicity of low-affinity HLA-A2. 1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur. J. Immunol. 30:3411-3421.[CrossRef][Medline]
  22. Wainberg, M. A., H. Salomon, Z. Gu, J. S. G. Montaner, T. P. Cooley, R. McCaffrey, J. Ruedy, M. H. Hilary, N. Cammack, J. Cameron, and W. Nicholson. 1995. Development of HIV-1 resistance to (–)2'-deoxy-3'-thiacytidine in patients with AIDS or advanced AIDS-related complex. AIDS 9:351-357.[Medline]

Journal of Virology, November 2006, p. 10645-10651, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01351-05





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
JVI.01351-05v1
80/21/10645    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Okazaki, T.
Right arrow Articles by Berzofsky, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Okazaki, T.
Right arrow Articles by Berzofsky, J. A.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Mol. Cell. Biol. Microbiol. Mol. Biol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS