Human Immunodeficiency Virus Type 1 Nef Epitopes Recognized in HLA-A2 Transgenic Mice in Response to DNA and Peptide Immunization

Virology 273, 112–119 (2000) doi:10.1006/viro.2000.0360, available online at http://www.idealibrary.com on

Human Immunodeficiency Virus Type 1 Nef Epitopes Recognized in HLA-A2 Transgenic Mice in Response to DNA and Peptide Immunization Johan K. Sandberg,* ,1 Ann-Charlotte Leandersson,* ,† Claudia Devito,* ,† Birgit Kohleisen,‡ Volker Erfle,‡ Adnane Achour,* Michael Levi,§ Stefan Schwartz,§ Klas Ka¨rre,* Britta Wahren* ,† and Jorma Hinkula* ,† *Microbiology and Tumor Biology Center, Karolinska Institutet, 171 77 Stockholm, Sweden; †Swedish Institute for Infectious Disease Control, Department of Virology, 171 82 Stockholm, Sweden; ‡Forschungszentrum Neuberg, Institute for Molecular Virology, 85764 Oberschleissheim, Germany; and §Department of Microbiology, Uppsala University, Uppsala, Sweden Received December 21, 1999; returned to author for revision March 15, 2000; accepted April 14, 2000

We investigated the immune response against a human immunodeficiency virus type 1 (HIV-1) nef DNA sequence administered epidermally in mice transgenic for the human major histocompatibility complex (MHC) class I molecule HLA-A201. Ten potential HLA-A2 binding 9-mer Nef peptides were identified by a computer-based search algorithm. By a cell surface MHC class I stabilization assay, four peptides were scored as good binders, whereas two peptides bound weakly to HLA-A2. After DNA immunization, cytotoxic T lymphocyte (CTL) responses were predominantly directed against the Nef 44-52, 81-89, and 85-93 peptides. Interestingly, the 44-52 epitope resides outside the regions of Nef where previously described CTL epitopes are clustered. Dominance among Nef-derived peptides did not strictly correlate with HLA-A2 binding, in that only one of the high-affinity binding peptides was targeted in the CTL response. The 44-52, 85-93, and 139-147 peptides also generated specific CTLs in response to peptide immunization. T helper cell proliferation was detected after stimulation with 20-mer peptides in vitro. Three Nef regions (16-35, 106-125, and 166-185) dominated the T helper cell proliferation. The implications of these results for the development of DNA-based vaccines against HIV is discussed. © 2000 Academic Press

INTRODUCTION

al., 1999). The emergence of viral epitope escape mutants suggests that CTL responses exert a significant selection pressure during the course of infection (Evans et al., 1999; Koenig et al., 1995; Phillips et al., 1991; Soudeyns et al., 1999). Taken together, these data strongly indicate an important role for CTLs in the containment of HIV-1 infection and support the notion that candidate vaccines should induce strong CTL responses to be successful. Accumulating evidence indicates that the regulatory Nef protein plays a key role in the pathogenesis and development of AIDS (Peter, 1998). Nef is expressed early in infection (Kim et al., 1989) and is responsible for high titer virus replication in vivo (Kestler et al., 1991). Long-time survivors of HIV infection show a lack of disease progression, commonly associated with deletions in the nef gene (Deacon et al., 1995; Kirchhoff et al., 1995) or defective nef alleles (Mariani et al., 1996). Moreover, nef was the only gene absolutely necessary to develop an AIDS-like disease in HIV-1 transgenic mice (Hanna et al., 1998). The Nef protein acts together with Env and Vpu proteins to down-regulate cell surface expression of the major HIV receptor CD4 (Aiken et al., 1994; Chen et al., 1996). Nef also acts to reduce the levels of MHC class I at the cell surface (Schwartz et al., 1996), thus preventing CTL recognition of infected cells (Collins et al., 1998).

Cytotoxic T lymphocytes (CTLs) recognize viral peptides presented by major histocompatibility complex (MHC) class I (Pamer and Cresswell, 1998) and have the capacity to lyse infected cells before the production of progeny virions (Yang et al., 1996; Zinkernagel and Althage, 1977). Several lines of evidence support a key role for CTLs in the immune control of human immunodeficiency virus type 1 (HIV-1) infection (reviewed in Brander and Walker, 1999). CTLs are temporally associated with the containment of primary viremia and remain at high frequency for prolonged periods of time (Borrow et al., 1994; Koup et al., 1994). Strong evidence comes from the observation that there is an inverse correlation between the frequency of specific CTLs and viral RNA load in chronic HIV infection (Ogg et al., 1998). Furthermore, antibodymediated depletion of CD8 ⫹ T cells in vivo leads to a dramatic increase in simian immunodeficiency virus (SIV) viral load in macaques (Jin et al., 1999; Schmitz et

1 To whom reprint requests should be addressed at Aaron Diamond AIDS Research Center, The Rockefeller University, 455 1st Avenue, 7th Floor, New York, NY 10016. Fax: (212) 725-1126. E-mail: jsandber@ adarc.adarc.org.

0042-6822/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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RESULTS

TABLE 1 Search for HLA-A2 Binding Peptides in the HIV-1 Nef Protein Rank

a

1 2 3 4 5 6 7 8 9 10 Control

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a

Position

Sequence

Score

85–93 183–191 174–182 108–116 111–119 139–147 44–52 68–76 52–60 81–89 Gag 77–85

AALDLSHFL LEWRFDSRL GMDDPEREV RQDILDLWV ILDLWVYHT LTFGWCFKL ALTSSNTAA VGFPVKPQV ATNADCAWL MTYKAALDL SLYNTVATL

24.7 22.2 16.5 15.5 11.7 10.8 5.0 2.9 1.6 1.6 157.2

a

Scan of the Nef protein sequence for putative HLA-A2 binding peptides was performed using a search engine at the web site: http:// www-bimas.dcrt.nih.gov/molbio/hla_bind/index.html. Analysis is based on coefficient tables deduced from the published literature by Dr. Kenneth Parker. Score indicates estimated half-time of dissociation.

Furthermore, high frequencies of CTLs specific for Nef have been detected in HIV-exposed noninfected individuals (Langlade-Demoyen et al., 1994; Rowland-Jones et al., 1995). These characteristics of the nef gene product are interesting from an immunological perspective and make it a primary target for HIV vaccine development (Montagnier, 1995). A number of approaches to prophylactic and therapeutic vaccination against HIV are currently under evaluation, including the use of viral vectors such as vaccinia and canary pox, live attenuated viruses, recombinant proteins, synthetic peptides, naked DNA, and combinations of different antigen delivery systems (Letvin, 1998). A novel approach to boost immunity in infected persons depends on controlled interruptions of highly active antiretroviral therapy (Ortiz et al., 1999). In this report, we used the HLA-A2 transgenic mouse model to study the Nef-specific HLA-A2-restricted CTL response to an experimental HIV-1 Nef-expressing DNA construct. Ten 9-mer peptide fragments from the Nef protein were selected as possible epitopes based on predicted binding to the HLA-A2 molecule by a computer-driven algorithm. Binding was confirmed using an HLA-A2 stabilization assay. We detected specific and HLA-A2-restricted CTL responses to three of the peptides 2 weeks after immunization with the Nef-expressing DNA-vaccine and demonstrated that three peptides generate CTL responses after peptide immunization. One epitope maps in a region of the protein where no epitopes have been described previously. In addition, we observed and quantified T cell proliferative responses in immunized mice. Our data have implications for the development of a therapeutic DNA vaccine against HIV (Calarota et al., 1999).

Identification of HIV-1 Nef-derived HLA-A2 binding peptides In the search for putative HLA-A2 binding peptide sequences in the HIV-1 Nef HXB3 sequence, we used a search algorithm based on individual amino acid binding coefficients described by Parker et al. (1994). The ten 9-mer peptide sequences predicted by this method to have the longest half-time of dissociation in complex with HLA-A2 are shown in Table 1. The well-characterized HIV-1 Gag 77-85 epitope was included as a positive control (Tsomides et al., 1994). Next, a T2 HLA stabilization assay was performed to confirm the binding properties of these peptides (Fig. 1). This assay is based on the accumulation of “empty” MHC class I molecules at the surface of TAP1/TAP2-deficient T2 cells cultured at 26°C, when they are stabilized by the addition of specific extracellular peptides (Ljunggren et al., 1990; Stuber et al., 1992). Overall, the result of the T2 stabilization assay was in agreement with that of the computer-driven algorithm, in that predicted good binders were generally better at stabilizing HLA-A2 on the cell surface. Four Nef-derived peptides, at positions 85-93, 108-116, 111119, and 139-147, bound as efficiently as the control Gag 77-85 sequence (Fig. 1). Two peptides, the 44-52 and the 81-89, stabilized HLA-A2 weakly (Fig. 1). However, two peptides predicted to bind, the Nef 183-191 and 174-182 peptides, were not able to stabilize HLA-A2 (Fig. 1). These data demonstrate the presence of Nef-derived peptide fragments, which bind and stabilize the HLA-A2 restriction element.

FIG. 1. HLA-A2 binding capacity among Nef-derived peptides. Ninemer peptides were tested for HLA-A2 binding by using the T2 MHC class I stabilization assay. Briefly, T2 cells were incubated with indicated amounts of peptide for 12 h at 26°C, washed, chased at 37°C for 30 min, and analyzed for HLA-A2 expression by FACS using the mAb HB54. Expression induced in the absence of peptide was subtracted from all values. Nef-derived peptides were as indicated in the legend, and the Gag 77-85 epitope was used as control.

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FIG. 2. Epitope-specific CTL derived from HIV-1 nef DNA-immunized HLA-A2 transgenic mice. CTL activity after 6 days of in vitro restimulation with peptide was assayed against Con A blasts from HLA-A2 transgenic mice pulsed (F) or not pulsed (E) with specific peptide. Shown is one of two positive experiments of a total of three experiments.

CTL responses against HLA-A2 binding Nef peptides in DNA-immunized mice

more, the selection of CTL epitopes in Nef does not correlate with HLA-A2 binding capacity.

We next investigated whether HLA-A2 transgenic mice immunized epidermally with a single 1-␮g dose of nef DNA develop CTL responses against any of the HLA-A2 binding Nef peptides. Spleen cells were recovered either 2 or 4 weeks after immunization and restimulated with peptides for 6 days. CTL responses were detected against several peptides 2 weeks after immunization (Fig. 2). The Nef 44-52 and 81-89 peptides were efficiently targeted, and the 85-93 sequence was somewhat less efficiently recognized. The 174-182 peptide was weakly recognized in one experiment only. It is notable that the 44-52 and 81-89 peptides are the primary targets of the CTL response, despite not being among the best HLA-A2 binding peptides. No or very weak responses were detected against the 183-191, 108-116, 111119, 139-147, 68-76, and 52–60 peptides. No CTL activity was detected in two of two experiments 4 weeks after immunization, indicating that CTL responses were shortlived (data not shown). Taken together, these data show that a single DNA immunization elicits CTL responses against three of the HLA-A2 binding Nef peptides. Further-

HLA-A2 restricted CTL responses against Nef peptides in peptide-immunized mice We next examined the potential usefulness of the Nefderived peptides for peptide immunization. Two of the peptides recognized after DNA immunization, the 44-52 and 85-93 peptides, and one that was not recognized despite being a good HLA-A2 binder, the 139-147, were dissolved in adjuvant and individually injected subcutaneously into HLA-A2 transgenic mice. These three peptides all generated peptide-specific CTL responses (Fig. 3). CTL responses were clearly restricted by the transgenic human HLA-A2 molecule, because preincubation with specific peptide sensitized T2 target cells but not RMA-S cells for lysis. Thus the Nef-derived peptides elicit peptide-specific and HLA-A2-restricted CTLs when injected in adjuvant. T-cell proliferation against the whole Nef protein and against 20-mer peptides We next measured T-cell proliferation in splenocytes to whole Nef protein and 20-mer peptides 2 and 4 weeks

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FIG. 3. HIV-1 Nef peptide-specific CTL generated by peptide immunization. CTL activity from peptide-immunized HLA-A2 transgenic mice was measured after 6 days of in vitro restimulation with peptide. The HLA-A2-positive cell line T2 was used as target cell. The mouse RMA-S target cell was included as an HLA-A2 negative control. Shown is one representative experiment of three experiments.

after nef DNA immunization. Strong proliferation was observed against recombinant Nef protein after 2 weeks, whereas proliferation was relatively weak 4 weeks after immunization (Fig. 4). Responses primarily focused on the 106-125, 166-185, and 181-205 peptides (Fig. 4). Some proliferation was also observed to the 16-35 peptide and to the peptides spanning positions 121–170 of the protein. Similar to the response detected to whole Nef protein, these peptide-specific proliferative responses quickly decreased, and only weak proliferation was observed 4 weeks postimmunization (Fig. 4). These data

demonstrate that nef DNA immunization elicits helper T-cell responses to several epitopes in HLA-A2 transgenic mice. DISCUSSION HLA-A2 transgenic mice have been used to evaluate CTL responses against human pathogens, including hepatitis B and C virus (Sarobe et al., 1998; Wentworth et al., 1996), influenza virus (Vitiello et al., 1991), human papilloma virus (Wentworth et al., 1996), and recombinant

FIG. 4. T cell proliferation after HIV-1 nef DNA immunization. Proliferative responses in immune splenocytes in vitro to whole recombinant Nef protein and 20-mer synthetic peptides 2 weeks (filled columns) and 4 weeks (open columns) after immunization. Shown is mean and standard error of data from four experiments.

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vaccinia constructs (Woodberry et al., 1999). Immunogenicity in this mouse model has been shown to correlate well, although not completely, with antigenicity for human CTL (Man et al., 1995; Wentworth et al., 1996). Here, we describe the use of HLA-A2 transgenic mice in the evaluation of the T-cell response to an HIV-1 nef DNA construct. CTL responses were observed against several epitopes, identified as putative HLA-A2-presented peptides by a computer-driven algorithm followed by assessment by an HLA-A2 stabilization assay. Helper T-cell proliferation was observed to whole Nef protein and several 20-mer peptides. The Nef peptides exhibiting the best binding to HLA-A2 were not the ones preferentially recognized in the CTL response to nef DNA vaccination. On the contrary, the strongest CTL activity was directed against the 44-52 and 81-89 peptides, both of which were comparably poor at stabilizing HLA-A2. Most interestingly, the 44-52 epitope resides outside the regions of Nef where previously described CTL epitopes are clustered. This is important because it is highly desirable to target several independent epitopes in a vaccine-induced CTL response. A previous study indicated that a 15-mer peptide of the Nef LAI sequence, which included the corresponding amino acids 44–52, could bind HLA-A2 (Choppin et al., 1991). Among the strongly binding peptides, only 85-93 was targeted. Peptides including or overlapping with the 85-93 peptide have previously been described as CTL epitopes restricted by HLA-A3 (McMichael and Walker, 1994) and HLA-A11 (Culmann et al., 1991). It is tempting to speculate that the evolutionary selection pressure exerted by CTL responses has selected against good binding of immunogenic Nef peptide fragments to common MHC class I alleles, as was observed recently in macaques (Evans et al., 1999). In some support of this, we observed that the replacement of amino acid positions 3 and 7 in the 85-93 for phenylalanines, which commonly occur naturally, leads to significantly increased predicted binding to HLA-A2 (data not shown). Also, the strongly binding peptides not targeted by CTLs (e.g., 108-116) may not be efficiently processed and thus not presented at the surface of antigen-presenting cells. No or only very weak CTLs were detected against the 108-116, 111-119, and 139-147 peptides after DNA immunization despite their capacity to efficiently stabilize A2. The absence of CTL responses against these peptides may be due to low intrinsic immunogenicity. Our observation that the 139-147 peptide induced a strong CTL response on immunization in adjuvant speaks against this possibility. Also, CTL recognition of a peptide corresponding to a 1-amino-acid residue longer variant of the 139-147 peptide (i.e., the 138-147) has been described in the human CTL response against HIV (Durali et al., 1998; Haas et al., 1996; Wilson et al., 1999). This discrepancy between the human and mouse HLA-A2-restricted CTL responses may be due to differences in antigen process-

ing (Braud et al., 1998). Similarly, we observed no CTLs against the 183-191 peptide, despite the finding that the corresponding 10-mer can be targeted by the human response (Haas et al., 1996). However, this may be due to the fact that the 9-mer variant apparently does not bind to the HLA-A2 molecule (Fig. 1). Controversy exists concerning the HLA-A2 restriction of the Nef 190-198 CTL epitope (Brander et al., 1998; Connan et al., 1994; Hadida et al., 1995; Hunziker et al., 1998). Because the corresponding peptide in the HXB3 Nef sequence scored very low in the computer-based predicted binding and no binding was observed in the T2 stabilization assay (Hunziker et al., 1998), we did not include this peptide in our experiments. In summary, we have identified several HIV-1 Nef epitopes presented by HLA-A2 in mice transgenic for this human MHC class I molecule. This approach of identifying human CTL epitopes seems promising, although the relevance of these epitopes for the human immune response must be confirmed. Our results further confirm the usefulness of computerized predictions of the strength of MHC–peptide interactions in combination with MHC stabilization assays to identify candidate viral epitopes. MATERIALS AND METHODS Cell lines T2 is a hybrid between the two human cell lines 0.174 and CEM, and it has an antigen processing deficiency due to a deletion in the MHC class II region that includes the TAP1 and TAP2 genes. RMA-S is a TAP2-deficient variant of the Rauscher virus-induced B6 lymphoma RMA (Ljunggren and Karre, 1985). Cell lines were maintained at 37°C and 5% CO 2 in RPMI 1640 tissue culture medium supplemented with 5% FCS, 50 ␮g/ml streptomycin, 100 ␮g/ml penicillin, and 2 mM L-glutamine. Concanavalin A (Con A)-activated blasts were generated by culturing erythrocyte-depleted splenocytes in 5 ␮g/ml Con A for 2 days in tissue culture medium as described above with 10% FCS. Synthetic peptides Synthetic peptides corresponding to HIV-1 Nef (HXB3) protein sequence positions 44-52 (ALTSSNTAA), 52-60 (ATNADCAWL), 68-76 (VGFPVKPQV), 81-89 (MTYKAALDL), 85-93 (AALDLSHFL), 108-116 (RQDILDLWV), 111-119 (ILDLWVYHT), 139-147 (LTFGWCFKL), 174-182 (GMDDPEREV), and 183-191 (LEWRFDSRL) and the HIV-1 Gag protein position 77-85 (SLYNTVATL) (Tsomides et al., 1994) were used for CTL experiments. For the stimulation of T helper cell activity in proliferation assays, 20 mers with 5-amino-acid overlap covering the complete Nef protein were used. Peptides were synthesized using solid phase F-moc chemistry.

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Mice and immunizations

Proliferation assay

Mice transgenic for the chimeric MHC class I molecule composed of ␣1/␣2 from HLA-A2 and ␣3 from H-2K b on C57Bl/6 background (Vitiello et al., 1991) were bred at the Microbiology and Tumor Biology Center of the Karolinska Institutet. Animal care was in accordance with institutional guidelines, and mice were used at an age of 6–12 weeks. The plasmid used for DNA vaccination contained the HIV-1 nef gene from the HXB3 strain under the control of the human cytomegalovirus (CMV) immediate-early promoter, as described by Hinkula et al. (1997). Plasmid (1 ␮g) was delivered in the abdominal skin by the helium pulse Accell device (courtesy of J. Haynes, Auragen, Madison, WI) coated onto 0.95-mm gold particles (Aldrich Chemical, Milwaukee, WI) (Haynes et al., 1994). Control animals were given empty plasmid vector and/or PBS. To elicit CTLs by peptide immunization (Sandberg et al., 1998), 100 ␮g peptide was dissolved in distilled water and mixed with incomplete Freund’s adjuvant in a 1:1 ratio by sonication and injected subcutaneously in the base of the tail. At 12 days after peptide immunization and 2 or 4 weeks after DNA immunization, 25 ⫻ 10 6 immune splenocytes were cocultured with 25 ⫻ 10 6 2000-rad-irradiated splenocytes in the presence of 0.1 ␮M peptide in 12 ml complete medium (RPMI 1640 supplemented with 10% FCS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 5 ⫻ 10 ⫺5 M 2-mercaptoethanol, 2 mM Lglutamine, and 50 ␮g/ml streptomycin/100 ␮g/ml penicillin) at 37°C and 5% CO 2 . Splenocytes from four DNA-immunized mice were pooled and then split into separate cultures during in vitro culture with peptide. Six days later, these cells were used as effector cells in a 51 Cr-release assay.

Spleen cells (2 ⫻ 10 5 cells/well) were cultured for 5–6 days in RPMI 1640 supplemented with 2 mM L-glutamine, 100 ␮g/ml penicillin/50 ␮g/ml streptomycin, and 10% fetal calf serum in the presence of 1 ␮g/ml whole antigen or 5 ␮g/ml peptide. Then 50 ␮l/well 3H ⫹-labeled thymidine (1 ␮Ci) was added, and cells were incubated for 16 h before cells were harvested and thymidine incorporation was measured using a ␤-counter. Stimulation index (SI) was calculated by dividing the mean cpm values in triplicate antigen-stimulated wells with the mean cpm of medium control wells. CTL assay CTL activity was measured in a standard 51Cr-release assay. Briefly, peptide coated T2, RMA-S, or Con A blast target cells were prepared by incubating cells with 10 ␮M peptide for 1 h at 37°C. Coated cells were labeled with 10 ␮l of 10 mCi/ml 51Cr for 1 h at 37°C. Titrated numbers of effector cells were incubated with 3 ⫻ 10 3 of 51 Cr-labeled target cells for 4 h at 37°C and 5% CO 2. After incubation, released radioactivity was measured and specific lysis was calculated according to the formula: specific release (%) ⫽ [(experimental release ⫺ spontaneous release)/(maximum release ⫺ spontaneous release)] ⫻ 100. ACKNOWLEDGMENTS This work was supported by The Swedish Cancer Foundation, The Swedish Society for Medical Research, The Swedish Medical Research Council, and Karolinska Institutet. We thank Dr. Douglas F. Nixon for valuable discussions and Dr. Cristina Cerboni for kind gift of the HB54 mAb supernatant. J.K.S. was supported by a fellowship from the Helmuth Hertz Foundation.

REFERENCES HLA-A2 peptide binding assays Initial scan of the HXB3 Nef protein sequence for putative HLA-A2 binding peptides was performed using a search engine at the Web site http://www-bimas. dcrt.nih.gov/molbio/hla_bind/index.html. The analysis is based on coefficient tables deduced from the published literature by Dr. Kenneth Parker (Parker et al., 1994). The peptide stabilization assay was used to test candidate binders for real HLA-A2 binding (Ljunggren et al., 1990; Stuber et al., 1992). T2 cells were incubated overnight at 26°C in complete medium with titrated concentrations of peptide, followed by washing and a 40-min chase at 37°C. Cells were then incubated with HB54 mouse mAb specific for HLA-A2 for 30 min on ice, washed, and stained with goat anti-mouse FITC conjugate (PharMingen, San Diego, CA) on ice for 30 min. After washing, analysis was performed using an FACScan (Becton Dickinson, Sunnyvale, CA) using Cellquest software.

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