JOURNAL OF VIROLOGY, Feb. 1992, p. 750-756

Vol. 66, No. 2

0022-538X/92/020750-07$02.00/0 Copyright © 1992, American Society for Microbiology

Three Epitopic Peptides of the Simian Immunodeficiency Virus Nef Protein Recognized by Macaque Cytolytic T Lymphocytes ISABELLE BOURGAULT,* ALAIN VENET, AND JEAN PAUL LEVY Institut Cochin de Genetique Moleculaire, Laboratoire d'Immunologie et Oncologie des Maladies Retrovirales, Institut de la Sante et de la Recherche Medicale Unite 152, H6pital Cochin, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France Received 25 July 1991/Accepted 17 October 1991 In 8 of 12 experimentally infected macaques, the Nef SIVmac 251 protein was recognized by cytolytic T lymphocytes (CTL) and appeared strongly immunogenic. Here, we report experiments which have been performed by using synthetic peptides to precisely determine the epitopes recognized by macaque CTL. Three epitopes of the Nef protein have been defined as CTL targets in three macaques. The epitopic peptides are located in the central region of the protein, and all of them show high homology with peptides of the human immunodeficiency virus type 1 Nef protein recognized by human CTL in association with several human leukocyte antigen molecules. These results suggest that (i) the Nef protein is a good candidate for vaccination not only because of its early expression but also because of its high immunogenicity for CTL, (ii) long peptides covering the central region of this protein could be used as vaccines and could cross the major histocompatibility complex barrier in a large variety of individuals, and (iii) the rhesus macaque is a good animal model in which to test for protection by CTL.

Cytolytic T lymphocytes (CTL) are produced in response to viral infections, and it is now established that they recognize viral peptides in association with major histocompatibility complex (MHC) class I molecules (17, 40). CTL have been found in both human immunodeficiency virus (HIV)-infected individuals (6, 20, 25, 27, 31, 33-35, 42) and simian immunodeficiency virus (SIV)-infected or vaccinated macaques (16, 29, 36, 41, 45, 46). Several epitopes of the structural proteins Env (1, 44), Gag (7, 14, 32), and Pol (21, 43, 44) and the nonstructural protein Nef (9, 10, 19, 26) have been described as human CTL targets. However, epitopic peptides are poorly defined in the SIV model, only one epitope in a SIV Gag protein having been identified (45). It is clear that a vaccine against HIV must induce the synthesis of neutralizing antibodies, but we may assume that this would not be sufficient for full protection against AIDS since the virus can propagate from cell to cell without coming into contact with antibodies. Therefore, induction of an associated CTL response might be essential to kill infected cells, before any release of infectious viral particles can occur. With the aim of producing CTL, synthetic peptides could be useful tools for vaccination. However, it would be necessary to precisely determine the major CTL target peptides. We have therefore studied the CTL response against the Nef protein, for two reasons: (i) the early expression of Nef mRNA in infected cells and (ii) the very high immunogenicity of Nef for CTL in humans (9, 10, 26) as well as in macaques (40a). The CTL responses against HIV-1 Nef protein have been well documented in humans, and several CTL epitopes have been located in the central region of this protein (9, 10, 19, 26). In this study we demonstrate that three peptides of the SIV Nef protein, also located in the central region, are the targets of CTL in three SIV-infected macaques. The sequences of these peptides correspond to previously identified epitopic peptides of the human Nef

MATERIALS AND METHODS Animals and lymphocyte preparation. Twelve rhesus macaques (Macaca mulatta) were experimentally infected with the SIVmac 251 strain. Nine uninfected macaques served as controls. Of the 12 SIVmac-inoculated rhesus macaques used in this study, 11 became seropositive between 2 and 4 weeks after virus exposure as assessed by Western immunoblotting. Only one rhesus macaque never exhibited antibodies against SIV proteins; it died of AIDS 4 months after infection. The other macaques had no clinical manifestation at the time of the study. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation on lymphocyte separation medium (Flow Laboratories, Glasgow, United Kingdom) and were either used immediately or stored at -180°C in liquid nitrogen. Recombinant vaccinia viruses. Sequences encoding the Nef protein were inserted into vaccinia virus, which served to infect target cells. VAC-Nef (VVTG 3117) (18) produces native nonmodified SIVmac 251 Nef protein. The virus encoding only the wild-type vaccinia virus, strain Copenhagen, was used as a control (Transgene, Strasbourg, France). Peptides. Twenty-five overlapping peptides spanning the Nef amino acid sequence of the SIVmac 251 strain were synthesized by Neosystem, Strasbourg, France, and supplied by the Agence Nationale de Recherche sur le SIDA. Each peptide contained 14 to 16 amino acids. Generation of anti-SIV effector cells. PBMC from rhesus macaques were cultured at 106 cells per ml in culture medium consisting of RPMI 1640 supplemented with 100 U of penicillin per ml, 100 jig of streptomycin per ml, 2 mM L-glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES (N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid) buffer, 2 x 10-5 M 2-mercaptoethanol, and 10% heat-inactivated human AB serum. To induce the expression of SIV antigens on infected CD4+ cells, PBMC were stimulated with 10 p.g of concanavalin A (ConA; Sigma, St. Louis, Mo.) per ml for 3 days. Then, 5% interleukin-2 (IL-2)-containing medium obtained as previ-

protein. *

Corresponding author. 750

VOL. 66, 1992

ously described (15) and corresponding to 5 IU of IL-2 per ml was added, and the cells were diluted at 5 x 105 cells per ml twice a week. Phenotypic analysis of T-celi lines. The T-cell lines were phenotyped by incubating cells with either fluorescein isothiocyanate-conjugated anti-CD4 (OKT4; Ortho Diagnostic Systems, Raritan, N.J.) or phycoerythrin-conjugated antiCD8 (Leu-2a; Becton-Dickinson, Mountain View, Calif.) monoclonal antibodies for 10 min at room temperature, washing once with phosphate-buffered saline. All cells were examined for percent positive-staining cells on an EPIC CS flow cytometer (Coulter). Cell fractionation. PBMC were incubated at 107 cells per ml with either OKT4 (2 ,ug) or Leu-2a (1 ,ug) for 30 min at 4°C. After incubation with OKT4, CD4+ cells were eliminated by addition of rabbit complement (Hoechst Behring) to a final dilution of 1/7 for 30 min at 37°C. After incubation with Leu-2a, CD8+ cells were eliminated by magnetic separation with immunomagnetic beads (Dynabeads; Dynal, Oslo, Norway). The cells were then washed with culture medium. Culture for limniting dilution analysis. Frequencies of antiNef-specific CTL precursors (CTLp) in PBMC were determined by limiting-dilution analysis. PBMC were seeded in round-bottomed microwells at different concentrations (5 x 103 to 4 x 104 per well) in 0.2 ml of culture medium containing 10 jig of Con A per ml. Twenty-four replicates were set up for each concentration. Subsequently, 5% IL-2containing medium was added to each microwell at days 3, 7, and 10. In vitro transformation of B-cell lines. B-lymphoblastoid cell lines (B-LCL) were generated by incubating serial dilutions of PBMC with supernatant of S 594. S 594, kindly provided by N. Letvin, is a cell line producing the immortalizing baboon herpesvirus (herpesvirus papio). After immortalization, the B-LCL were cultured in culture medium supplemented with 10% fetal calf serum. CRT. To obtain target cells presenting SIVmac gene products, we incubated B-LCL at a concentration of 106 cells per ml with recombinant vaccinia virus (20 PFU per cell) for 18 h at 37°C in a 5% CO2 humidified atmosphere. To sensitize target cells with peptides, we incubated the peptides overnight at a concentration of 20 p.M with 106 B-LCL under the same conditions. B cells were then washed and labeled with 100 ,uCi of Na251CrO4 (Amersham) for 1 h, washed twice, and used as target cells. The 51Cr release test (CRT) was performed by using V-bottomed 96-well microtiter plates. The cytolytic activity of anti-SIV cell lines was measured by mixing 5 x 103 51Cr labeled target cells with effector cells at various effector-to-target-cell ratios in a final volume of 0.2 ml per well. LDA cultures were tested after 14 days of culture, when proliferation was judged satisfactory as determined by the optical microscope. The cells were resuspended with a micropipet, divided in two 0.1-ml aliquots, and transferred to plates, where 5 x 103 labeled target cells were added. Plates were incubated for 4 h at 37°C, after which 0.1 ml of supernatant was harvested from each well and analyzed by a gamma counter. Spontaneous release was determined after incubating target cells with medium alone and never exceeded 20% of the total 51Cr incorporation. Results were expressed as specific chromium release: 100 x (experimental cpm - spontaneous cpm) / (maximum cpm -

spontaneous cpm). Determination of the frequency of CTL precursors. Estimates of precursor frequency were obtained by the weighted-mean method from the Poisson distribution relationship

CTL EPITOPIC PEPTIDES OF SIV Nef PROTEIN

751

between the responding cell number and the logarithm of the percentage of nonresponding (negative) culture as described by Taswell (39). Cultures in which 51Cr release exceeded the mean spontaneous release by 8 to 10% (i.e., always above 3 standard deviations) were considered positive for cytolytic activity. The 95% confidence limit for each frequency was calculated. We verified, by using the x2 test, that the points were aligned and that the kinetics were in line with the single-hit model. The straight lines were traced only when the calculated x2 was inferior to the theoretical x2 value for each degree of freedom. RESULTS The Nef protein is highly immunogenic for CD8+ CTL in SIV-infected macaques. It has been demonstrated previously

that the Gag (16, 29, 36, 45, 46), Pol (46), and Env (46) proteins of SIV might be the targets of simian CTL. More recently, specific cytolytic activities directed against eight different SIV proteins have been determined in 12 SIVinfected macaques (40a). Most animals had CTL directed against structural proteins, and, of the five nonstructural SIV proteins also recognized, Nef was particularly antigenic for CTL since an unequivocal activity was found in eight macaques (Table 1). Of the four remaining macaques, only one produced CTL against Rev, Vif, and Vpx and against structural proteins. The other three animals did not produce any SIV-specific CTL (data not shown). A very low activity was detected in most cases against targets infected with the wild-type vaccinia virus except in macaque RH 483 (Table 1). Under the same experimental conditions, no CTL has been generated against any protein after stimulation of PBMC from 9 seronegative macaques or from 7 of our 12 experimental macaques when tested prior to infection (data not shown). After 14 days of culture, the phenotype of cultured cells was mainly CD8+ (65% + 6%; range, 57 to 74) as shown in Table 1. The cytolytic activity was clearly mediated by CD8+ cells since it disappeared after elimination of these cells, whereas CD4 depletion had no effect on the lysis, as illustrated in Fig. 1 for macaques RH 483 and RH 1238. Three peptides of the SIV Nef protein can be recognized by CTL from three different macaques. To determine the region(s) of the Nef protein recognized by Nef-specific CTL, we tested 25 overlapping peptides for their capacity to sensitize target cells. Three peptides were recognized by three of the four macaques that we were able to test regularly (Table 2). The target peptide of the CTL of the last macaque could not be identified. The three identified peptides, 108 to 123, 155 to 169, and 164 to 178, were all located in the central region of the protein. No activity was seen either against autologous target cells without peptide or against heterologous target cells incubated with the peptide (data not shown). In addition, we have verified that peptides were not toxic for target cells. Furthermore, effector cells were CD8+ cells since depletion of CD8+ cells at the time of the CRT Fig. completely abrogated the cytolytic activity, as shown toin 123. 2 with macaque RH 499 recognizing the peptide 108 Frequency of anti-Nef protein and anti-Nef peptide CTLp among PBMC. The frequency of anti-Nef CTLp in PBMC was determined by limiting-dilution analysis. After 14 days of culture, specific cytolytic activity was tested against target cells infected with SIV-Nef vaccinia virus. As illustrated for macaque RH 505 in Fig. 3, the frequency of anti-Nef CTLp was very high, in contrast to the frequency detected against wild-type vaccinia virus. The nature of

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BOURGAULT ET AL.

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TABLE 1. PBMC of SIV-infected rhesus monkeys lyse autologous target cells expressing SIV Nef protein % Specific lysisc at E/T ratiod of:

Effector cellsa Macaque no.

CD4/CD8 ratioe

RH 471

24/60

RH 473

21/65

RH 475

40/57

RH 477

39/53

RH 483

11/74

RH 485

45/38

RH 493

28/62

RH 495

22/69

RH 499

23/2

RH 501

28/61

RH 505

29/59

RH 1238

17/69

Target cellsb

WT Nef WT Nef WT Nef WT Nef WT Nef WT Nef WT Nef WT Nef WT Nef WT Nef WT Nef WT Nef

200:1

100:1

50:1

25:1

10 46 0 4 1 2 0 30 34 56 1 3 0 2 8 21 5 22

6 22 2 2 1 4 0 30 36 53 1 4 0 1 4 16 5 17

5 7 0 3 1 4 0 13 33 46 1 3 0 0 2 10 3 10

9

16 35 1 13 3 17

5 21 3 11 0 10

3 1 0 0 0 2 0 12 29 4 1 1 0 0 3 6 2 5 11 19 1 3 2 6

28 6 23 0 22

PBMC were obtained from 12 SIV-infected monkeys, cultured in the presence of ConA for 3 days, and then maintained in 5% IL-2-containing medium. b Target cells were autologous B-LCL immortalized by herpesvirus papio and infected with a wild-type (WT) or Nef recombinant vaccinia virus. Target cells (5 x 103) were labeled with 51Cr and incubated for 4 h with various numbers of effector cells. d E/T ratio, effector-to-target-cell ratio. Positive results are in bold type. ePhenotypes were performed on cell cultures at day 14.

a

anti-Nef CTLp tested in these experiments was confirmed by the depletion of CD4+ or CD8+ effector cells at the onset of the cultures for limiting-dilution analysis. Treatment with anti-CD4 plus complement did not change the frequency of CTLp with regard to PBMC. In contrast, the elimination of

a)

CD8+ cells greatly reduced the number of CTLp, indicating that the majority of these CTLp were CD8+ cells (Fig. 3). For the seven macaques tested, the frequencies of the anti-Nef CTLp were between 1/5,000 and 1/35,000 (Table 3). Knowing the global anti-Nef CTLp frequencies, we mea-

b) CD4-depleted ,A Unfractionated PBMC Unfractionated PBMC

,o CD4-depleted

2 0 ,

10

u

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10

*1

CD8-depleted $I 101

i01

10.1

I

6

1001 &I iLl

iti

50-1

n CD8-depleted

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EFFECTOR/TARCET RATIO

monkeys RH 483 (a) and RH 1238 (b). Effector cells were unfractionated PBMC monoclonal antibody plus complement (0) or PBMC depleted of CD8+ magnetic beads (O) at the time of CRT. Target cells were autologous antibody B-LCL infected with Nef recombinant vaccinia virus. The percent lysis observed on the target infected with the wild-type vaccinia virus has

FIG. 1. Specific cytolytic of anti-Nef CTL from cells (A) or PBMC depleted of treatment with an anti-CD4 cells by treatment with an anti-CD8 monoclonal bound to

activity by CD4+

been subtracted from all

the

experimental

values.

VOL. 66, 1992

CTL EPITOPIC PEPTIDES OF SIV Nef PROTEIN

TABLE 2. Cytolytic activities in the presence of different Nef peptides Cytolytic activity for monkey and E/T ratiob:

Peptidea (amino acid position)

1-105 101-115 108-123 116-130 125-147 146-160 155-169 164-178 176-190 184-247

H49 RH 499 200:1 100:1