Development of HIV-1 Nef vaccine components: immunogenicity study of Nef mutants lacking myristoylation and dileucine motif in mice

Development of HIV-1 Nef vaccine components: immunogenicity study of Nef mutants lacking myristoylation and dileucine motif in mice

Vaccine 20 (2002) 3413–3421 Development of HIV-1 Nef vaccine components: immunogenicity study of Nef mutants lacking myristoylation and dileucine mot...

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Vaccine 20 (2002) 3413–3421

Development of HIV-1 Nef vaccine components: immunogenicity study of Nef mutants lacking myristoylation and dileucine motif in mice Xaioping Liang∗ , Tong-Ming Fu, Hong Xie, Emilio A. Emini, John W. Shiver Merck Research Laboratories, Department of Virus & Cell Biology, Merck and Co. Inc., Sumneytown Pike, P.O. Box 4, West Point, PA 19486, USA Received 24 September 2001; received in revised form 2 April 2002; accepted 8 April 2002

Abstract In an effort to develop a safe Nef component for use in Cytotoxic T-lymphocyte (CTL)-based HIV-1 vaccines, several versions of Nef constructs lacking myristoylation and dileucine motif were engineered and their abilities to elicit T cell responses were evaluated in mice. Nef-specific murine T cell epitopes were first mapped in three strains of mice (Balb/c, C3H/HeN and C57BL/6), and a pair of dominant Nef-specific CD4+ and CD8+ T cell epitopes were identified in C57BL/6 mice. C57BL/6 mice were subsequently immunized with engineered Nef DNA constructs, and Nef-specific CD4+ and CD8+ T cell responses were determined. A Nef mutant with simple alanine substitutions at the myristoylation and dileucine sites was impaired in its ability to elicit Nef-specific CD4+ and CD8+ T cell responses. Addition of human tissue plasminogen activator (TPA) leader sequence to the N terminus of Nef, which concomitantly inactivates the myristoylation site, significantly enhanced the Nef-specific T cell responses. These findings may have practical implications for developing HIV-1 Nef vaccine component. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: HIV-1; Nef; CTL

1. Introduction Several lines of evidence suggest that HIV-1-specific CTL responses can be efficacious in control of HIV-1 infection and prevention of diseases progression. In primary HIV-1 infection, the appearance of the HIV-1-specific CTL responses has been shown to be temporally associated with the decline of viremia during primary HIV-1 infection [1,2]. In HIV infected long-term nonprogressors, levels of HIV-1-specific CTLs appear to be inversely correlated with plasma viral loads [3]. In SIV infection monkey models, depletion of CD8+ T cells during either acute or chronic stages of SIV infection resulted in uncontrolled viral replication, and the monkeys lacking the CD8+ T cells exhibited accelerated progression to AIDS and death [4,5]. These observations collectively established the role of CTL in the containment of viral replication. This role was further emphasized in several recent SHIV monkey challenge studies showing that CTL responses elicited by vaccination were able to contain viral replication and prevent disease progression [6–8]. The present study concerns HIV-1 Nef as an important component for CTL-based HIV-1 vaccines. Nef is a 27 to 30 kDa N-myristoylated, membrane-associated cytoplasmic ∗

Corresponding author. Tel.: +1-215-652-8215; fax: +1-215-652-7320. E-mail address: xiaoping [email protected] (X. Liang).

protein that is abundantly expressed in the early phase of HIV-1 replication [9,10]. Nef is an essential viral protein for efficient HIV-1 replication and plays an important role in viral pathogenesis [11]. Studies have shown that deletion of Nef could result in significant attenuation in HIV-1 and SIV [12,13]. It is also known that Nef constitutes an important viral target for host immune system in HIV-1-infected individuals [3,14]. Nef possesses diverse biological activities, which include altering T cell signal transduction pathway [15], activating T cells and tissue macrophages [16], and down regulating cell surface expression of CD4 and MHC class I [17,18]. Because of these intrinsic, potentially pathogenic properties, wild type Nef may not be suitable for use as a vaccine component. This concern is further underscored by the observation that in transgenic mice expression of Nef alone is sufficient to cause AIDS-like disease [19]. In an attempt to address the safety issue, we constructed Nef mutants lacking myristoylation site and dileucine motif, the two well defined functional structures [20,21], and conducted immunogenicity studies in mice. The results showed that a Nef mutant with alanine substitutions in myristoylation site and dileucine motif elicited lower Nef-specific T lymphocyte responses than did the wild type Nef. This defect, however, could be overcome by fusing Nef with a human tissue plasminogen activator (TPA) leader sequence.

0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 2 ) 0 0 3 0 8 - 0

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2. Materials and methods 2.1. Plasmids The codon optimized nef gene coding for wild type Nef protein of HIV-1 JRFL was assembled from complementary, overlapping synthetic oligonucleotides by polymerase chain reaction (PCR). The nucleotide sequence of the codon optimized gene, along with translated amino acid sequence, is shown in Fig. 1. The PCR primers used were designed in such that a BglII site was included in the extension of 5 primer and an SrfI site and a BglII site in the extension of 3 primer. The PCR product was digested with BglII and cloned into BglII site of a human cytomegalovirus early promoter-based expression vector, V1Jns [22]. The resultant plasmid was named V1Jns/Nef. The gene coding for the Nef mutant with alanine substitutions at residues Gly 2, Leu 174 and 175 was also made from synthetic oligonucleotides by PCR. In this case, a PstI site and a SrfI site were included in the extensions of 5 and 3 PCR primers, respectively. The PCR product was digested with PstI and SrfI, and cloned into the PstI and SrfI sites of V1Jns/Nef, replacing the wild type nef gene fragment with the mutant gene fragment. This resulted in V1Jns/Nef(G2A,LLAA). To construct the expression vector containing TPA leader sequence and Nef fusion gene, a nef gene fragment lacking the first five codons was amplified from V1Jns/Nef by

PCR. Both 5 and 3 PCR primers used in this reaction contained a BglII extension. The PCR amplified fragment was then digested with BglII and cloned into BglII site of the expression vector, V1Jns/TPA [22]. The ligation of the 3 end of TPA leader sequence to the 5 end of the Nef PCR product restored the BglII site and yielded an in-frame fusion of the two fragments. The resultant plasmid is called V1Jns/TPAnef. Construction of V1Jns/TPAnef(LLAA) was carried out by replacing the SacII-Bsu36 fragment of V1Jns/Nef(G2A,LLAA), which encompasses part of the promoter/intron A sequence and the 5 half of the nef gene, with the corresponding SacII-Bsu36 fragment from V1Jns/TPAnef. All the Nef constructs were verified by sequencing. 2.2. Transfection and protein expression Adenovirus transformed human embryonic kidney cell line 293 grown at approximately 30% confluence in minimum essential medium (MEM; GIBCO, Grand Island, MD) supplemented with 10% fetal bovine serum (FBS; GIBCO) in a 100 mm culture dish were transfected with 8 ␮g of plasmids by Lipofectin (GIBCO) following manufacture’s protocol. Twelve hour post-transfection, cells were washed once with serum-free medium, Opti-MEM I (GIBCO), and replenished with 5 ml of Opti-MEM I. Following an additional 60 h incubation, culture supernatants and cells

Fig. 1. Codon optimized nef gene and amino acid sequence. The codon optimized gene was assembled by using overlapping synthetic oligonucleotides by PCR as described in Section 2. The amino acid residues Glycine 2 and Leucine 174 and 175, corresponding to myristoylation site and dileucine motif, respectively, were highlighted in bold.

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were collected separately and stored in aliquots at −70 ◦ C till use. 2.3. Western blot analysis Test samples were separated on a 10% SDS-polyacrylamide gel (SDS-PAGE) under reducing conditions and blotted onto a piece of PVDF membrane (Bio-Rad, Hercules, CA). The membrane was then probed with a mixture of gag mAb (#18; Intracel, Cambridge, MA) and Nef mAbs (aa64–68, aa195–201; Advanced Biotechnologies, Columbia, MD), which was followed by reaction with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA). The membrane was then treated with ECL Western blotting detection reagents following manufacture’s protocol (Amersham, Arlington Heights, IL) and exposed to an X-ray film. To evaluate the relative densities of different protein bands, the film was scanned by a densitometer (Personal Densitometer SI; Amersham Biosciences Corporation, Piscataway, NJ), and the band densities were determined by a ImageQuant software (Version 5.0; Amersham). 2.4. Positive selection of CD8+ cells Fractionation of splenocytes into CD8+ and CD8− T cell populations was carried out with Multenyi Cell Sorting System by following manufacture’s protocol (Miltenyi Biotech, Auburn, CA). Briefly, 1 × 107 splenocytes in 90 ␮l of phosphate-buffered saline (PBS) containing 0.5% BSA (PBS–BSA) were incubated with 10 ␮l of anti-mouse CD8 monoclonal antibody-coated beads at 4 ◦ C for 15 min. Cells were washed once with 5 ml PBS–BSA, and applied to a separation column mounted on the Magnetic separator. The column was washed three times with PBS–BSA. The cells that did not bind to the column were kept as CD8− cell population. After removing the column from the magnetic separator, cells were eluted with 3 ml PBS–BSA and kept as CD8+ cell population. The resultant cells in either fraction consistently showed higher than 95% viability. To control for the cell fractionation procedure, the splenocytes from mice immunized with a HIV-1 gag DNA vaccine were run in parallel, and the fractionated cells were checked for their reactivities to a known gag CTL epitope by ELIspot assay. The results showed that only the CD8+ cell fraction contained the gag CTL epitope (data not shown).

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(FBS–RPMI) at 37 ◦ C in a CO2 incubator for 2–4 h. Splenocytes were suspended at 8×106 cells/ml in RPMI-1640 supplemented with 10% FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml of penicillin, 100 U/ml of streptomycin, and 55 ␮M 2-mercaptoethanol. Fifty microliters of cells was added together with 50 ␮l of a testing peptide to each well. Wells containing cells only were included as negative controls. All the samples were performed in triplicate wells. The plates were incubated at 37 ◦ C for 20 h. After incubation, plates were rinsed briefly with distill water and washed three times with PBST. Fifty microliters of biotinylated rat anti-mouse IFN-gamma detecting mAb (XMG1.2; PharMingen) at a concentration of 2 ␮g/ml in PBST containing 1% BSA was then added to each well. Plates were incubated at 24 ◦ C for 2 h, followed by washes with PBST. Fifty microliters of streptavidin-conjugated alkaline phosphatase (KPL, Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) at a dilution of 1:1000 in FBS–RPMI was added to each well. The plates were incubated at 24 ◦ C for an additional 1 h. Following additional washes with BPST, 100 ␮l of BCIT/NBT substrate (KPL) was added for 15 min, and color reaction was stopped by washing the plate with tap water. Plates were air-dried. Spots were countered using a dissection microscope. 2.6. Cytotoxic T lymphocyte (CTL) assay Splenocytes from immunized mice were stimulated in vitro for 7 days with irradiated, peptide-pulsed syngenic na¨ıve splenocytes in the presence of 10 U/ml of human IL-2 (Sigma, St. Louis, MO). EL-4 cells (H-2b) were incubated at 37 ◦ C for 1 h with or without 20 ␮g/ml of a designated peptide in the presence of 51 Cr and used as target cells. For the assay, 1 × 104 peptide-pulsed target cells and a designated number of effector cells were added together to the wells in a 96-well plate and were incubated at 37 ◦ C for 4 h. After incubation, supernatants were collected and counted in a Wallac gamma-counter. Specific lysis was calculated as [(experimental release−spontaneous release)/(maximum release−spontaneous release)] × 100%. Spontaneous release was determined by incubating target cells in medium alone, and maximum release was determined by incubating target cells in 2.5% Triton X-100. The assay was performed with triplicate samples. 2.7. Animal experiments

2.5. Enzyme-linked immune spot (ELIspot) assay Nitrocellulose membrane-backed 96 well plates (MSHA plates; Millipore, Bedford, MA) were coated with 50 ␮l of rat anti-mouse IFN-gamma capture mAb (R4-6A2; PharMingen, San Diego, CA) at a concentration of 5 ␮g/ml in PBS per well at 4 ◦ C overnight. Plates were washed three times with PBS containing 0.05% Tween 20 (PBST) and blocked with 10% fetal bovine serum in RPMI-1640

Female mice of 6–10 weeks old were obtained from Charles River Laboratories, Wilmington, MA, and maintained in animal facilities of Merck Research Laboratories in accordance of institutional guidelines. For immunization, mice were injected in quadriceps with 100 ␮l of DNA in phosphate-buffered saline. At specified time points after immunization, spleens were collected for CTL and ELIspot assays.

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2.8. Data analysis Statistical analysis of the experimental data and controls was performed with the two-tailed Student’s t-test, and statistical significance was defined as P < 0.05.

3. Results 3.1. Construction and in vitro expression of Nef and Nef mutants lacking myristoylation and dileucine motif The HIV-1 JRFL Nef is a 216 aa protein. Its second residue glycine and the leucine residues at positions 174 and 175 are well conserved among different Nef sequences. The Gly 2 is an essential residue for the myristoylation signal site and the Leu 174 and 175 constitutes the dileucine motif responsible for the Nef-mediated down regulation of CD4 and MHC class I molecules. To inactivate these functional structures, we substituted all three residues with alanine. The resultant construct was named Nef(G2A,LLAA). Previously we and others found that fusion of TPA leader sequence to the N terminus of an antigen was able to increase the protein expression and immunogenicity of the respective antigen ([23,24]; Shiver, unpublished data). Based on this, we also constructed two Nef mutants by fusing TPA leader sequence to the N terminus of Nef either with or without additional dileucine motif mutation, which were named TPAnef and TPAnef(LLAA), respectively. With both of the fusion mutants, the TPA leader sequence was fused to the sixth N-terminal residue, Ser, to delete the myristoylation signal. Therefore, all three Nef mutants are defective in myristoylation modification. This was confirmed by immunoprecipitation assay using metabolic labeling of transfected cells

with H3-myristate (date not shown). Fig. 2 shows schematic representation of Nef mutants. To evaluate the expression of the Nef constructs, adenovirus-transformed human kidney 293 cells were co-transfected with one of the Nef constructs and a HIV-1 Gag expression vector. Seventy-two hours post-transfection, transfected cells and their respective culture media were collected separately and analyzed by Western blotting, using both Nef- and Gag-specific mAbs (Fig. 3). Cells transfected with the Gag plasmid alone revealed a single band of approximately 55 kDa, consistent with the estimated size of Gag protein. The cells co-transfected with Gag and Nef plasmids revealed the 55 kDa Gag protein band and a major 30 kDa and several minor bands corresponding to Nef-related products. Among the Nef products, a minor band with slower migration rate than that of the major 30 kDa band was present with TPAnef and TPAnef(LLAA), but not with Nef and Nef(LLAA). This may indicate that some of the TPA fusion products could have entered into the endoplasmic reticulum and become glycosylated. Four Nef constructs showed different levels of expression. On the basis of densitometry measurement of the cellular fractions, the calculated ratios of the 30 kDa Nef band over that of the 55 kDa Gag band for Nef, Nef(G2A,LLAA), TPAnef and TPAnef(LLAA) are 1.8, 1.3, 4.9, and 3.3, respectively, suggesting that the relative expression levels for the different Nef constructs are in following descending order: TPAnef, TPAnef(LLAA), Nef and Nef(G2A,LLAA). 3.2. Mapping of Nef-specific CD8+ and CD4+ T cell epitopes in mice In order to characterize immunogenicity of Nef and Nef mutants, experiments were first carried out to map Nef CD8+

Fig. 2. Schematic presentation of Nef and Nef mutants. Amino acid residues involved in mutations are presented. Glycine 2 and Leucine 174 and 175 are the sites involved in myristoylation and dileucine motif, respectively. For both versions of TPAnef fusion genes, the putative leader peptide cleavage sites are indicated with “star”, and a exogenous serine residue introduced during the construction of the mutants is underlined. Dashed line: omitted sequence.

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Fig. 3. Western blot analysis of Nef and Nef mutants expressed in transfected 293 cells. 293 cells grown in 100 mm culture dish were transfected with designated plasmids. Seventy-two hours post-transfection, supernatant and cells were collected separately and separated on 10% SDS-PAGE under reducing conditions. The proteins were transferred into PVDF membrane and probed with a mixture of Gag mAb and Nef mAbs, both at 1:2000 dilution. The protein signals were detected with ECL: (A) cells transfected with V1Jns/gag only; (B) cells transfected with V1Jns/gag and V1Jns/nef; (C) cells transfected with V1Jns/gag and V1Jns/nef(G2A,LLAA); (D) cells transfected with V1Jns/gag and V1Jns/TPAnef; and (E) cells transfected with V1Jns/gag and V1Jns/TPAnef(LLAA). The lower case letter ‘c’ and ‘m’ represent medium and cellular fractions, respectively. M.W.: molecular weight marker.

and CD4+ T cell epitopes in mice with common H-2 genetic background. Three strains of mice, Balb/c, C3H/HeN and C57BL/6, were immunized with plasmid V1Jns/Nef. Splenocytes were fractionated into CD8+ and CD8− (hereinafter referred as CD4+ ) T cell populations, and determined for Nef-specific IFN-gamma secreting cells (SFC) by ELIspot assay using a set of overlapping 20aa Nef peptides that encompasses the entire 216-amino acids Nef open reading frame (Fig. 4). No T cell epitope was detected in C3H/HeN mice. Three discrete CD4+ T cell epitopes were found in the Balb/c mice, namely, aa11–30, aa61–80, and aa191–216. A CD8+ T cell epitope, aa51–70, and a CD4+ T cell epitope, aa81–100, were detected in the C57BL/6 mice. The CD8+ T cell epitope was further evaluated in the standard CTL killing assay. Results from several initial experiments showed the peptide aa51–70 induced, albeit specific, only low levels of killings (data not shown). Since CTL epitopes typically consist of 8–11 residues [25], we speculated that the low levels of specific killings observed with peptide aa51–70 was due to the presence of the excessive flanking residues. Two shortened peptides, aa60–68 and aa58–70, were synthesized and tested in CTL assays (Fig. 5). While no killing was observed with Nef aa60–68, significantly enhanced levels of killings were observed with the Nef aa58–70. The data, therefore, confirmed that the CD8+ T cell epitope identified by the ELIspot assay is also

a CTL epitope. Based the predicted binding motifs for H-2 molecules [26], we mapped the minimum sequence for the Nef CD8+ CTL epitope by testing additional five peptides in ELIspot assay, and the result indicated that minimum sequence the epitope is aa58–66 (Table 1). 3.3. Immunogenicity of Nef mutants in mice Having identified Nef-specific CD8+ and CD4+ T cell epitopes, we next examined the immunogenicity of the different Nef constructs in C57BL/6 mice by DNA immunization. Two different immunization regimens were Table 1 Identification of minimum CTL epitope amino acid sequence Nef peptides

Sequence

IFN-gamma SFC/106 splenocytes

Nef58–69 Nef59–79 Nef58–68 Nef58–67 Nef58–66 Medium

TAATNADCAWLE AATNADCAWLE TAATNADCAWL TAATNADCAW TAATNADCA

85 1 69 66 92 1

Nef immune splenocytes were tested for IFN-gamma secreting cells in the presence of designated peptides by ELIspot assay. Results represent average of duplicate samples.

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Fig. 4. Mapping of Nef-specific CD8+ and CD4+ T cell epitopes. Three strains of mice, Balb/c, C57BL/6 and C3H/HeN, were immunized with 50 ␮g of V1Jns/Nef and boosted twice in a 4-week interval. Two weeks following last immunization, splenocytes were isolated and fractionated into CD4+ and CD8+ cells using Miltenyi’s magnetic cell separator (Miltenyi). The resultant CD4+ and CD8+ cells were then tested in an ELIspot assay against individual Nef peptides. SFC: IFN-gamma secreting spot-forming cells.

performed. In the first regimen, mice were immunized twice in a 2-week interval, and immune responses were measured 2 weeks post each injection (Fig. 6A). In the second regimen, mice were immunized twice in a 5-week interval, and the immune responses were measured 4 weeks after each

injection (Fig. 6B). With both immunization regimens, two TPA fusion mutants, TPAnef and TPAnef(LLAA), elicited higher CD8+ T cell responses than did the wild type Nef. In contrast, the simple alanine substitution mutant, Nef(G2A,LLAA), elicited lower immune responses than did

Fig. 5. Cytotoxic T cell assay with Nef CTL epitope. Splenocytes of Nef-immunized C57BL/6 mice were stimulated in vitro with peptide-pulsed, irradiated na¨ıve splenocytes for 7 days. Following the in vitro stimulation, cells were harvested and tested in a standard 51 Cr-releasing assay using peptide-pulsed EL-4 cells as targets. Open symbols: killings of EL-4 cells without peptide; solid symbols: killings of EL-4 cells pulsed with peptide.

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Fig. 6. Comparison of immunogenicity of Nef and Nef mutants. (A) C57BL/6 mice were immunized with 100 mcg of the indicated Nef constructs and boosted once 14 days later. Fourteen days after each immunization, five mice were sacrificed and splenocytes from individual mice were collected and tested against the Nef-specific CD8+ T cell epitope (aa58–66) and CD4+ T cell epitope (aa81–100) peptides. (B) C57BL/6 mice, 10 per group, were immunized with 100 mcg of the indicated Nef constructs and boosted once 5 weeks later. Four weeks after each immunization, five mice were sacrificed and splenocytes from each mouse were collected and tested individually against the Nef-specific CD8+ T cell epitope (aa58–66) and CD4+ T cell epitope (aa81–100) peptides. The data represent means plus standard deviation of 5 mice. Asterisk (∗) denotes that the values of these groups are significantly different from those of respective wild type Nef groups (P < 0.05).

the wild type Nef. The most pronounced difference among these constructs was observed at 2 weeks post-priming immunization in the first regimen. For example, only did one of five mice that received Nef(G2A,LLAA) show above background Nef-specific CD8+ IFN-gamma SFCs, while all five mice that received wild type Nef developed positive Nef-specific CD8+ IFN-gamma SFCs. The mice that received either TPA fusion mutant developed over 100-fold higher Nef-specific CD8+ IFN-gamma SFCs than did the mice that received wild type Nef. Although more variable, the Nef-specific CD4+ T cell responses showed similar profiles as those of CD8+ T cell responses.

4. Discussion In this communication, we report construction and characterization of Nef mutants lacking myristoylation site and dileucine motif as a part of our effort to develop a safer

Nef vaccine component. The reason to target the mutations at myrisoylation site and the dileucine motifs is of two-fold. First, the targeted sites do not overlap with any of the human Nef CTL epitopes identified so far [3]. Therefore, the mutations at these sites will have minimum effects on the number of CTL epitopes available in humans. Second, the functional properties of myristoylation and dileucine motif have been relatively well defined. Myristoylation is essential for Nef membrane localization, and the membrane associated Nef has been shown to be responsible for most of the known Nef functions [9,27,28]. In addition, HIV-1 and SIV Nef mutants lacking the myristoylation site have been shown to be defective in in vitro replication or avirulent in vivo [12,20,29,30]. The dileucine motif on the other hand is well characterized for its role in down regulation of surface expression of CD4 molecules in the viral infected cells [17,31]. In view of these properties, Nef constructs with mutations targeted at myristoylation and dileucine motif are likely to be safe for use as a vaccine component.

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We found that a Nef mutant generated by simple alanine substitutions at myristoylation site and dileucine motif had reduced immunogenicity, and in contrast, the TPA fusion mutants showed significantly enhanced immunogenicity. The relative immunogenicity of the different mutants appears to correlate with the levels of protein expression in the transfected cells (Figs. 3 and 6). This assertion is consistent with previous observations reported by Li et al. [23] and Malin et al. [24]. These authors showed that adding TPA leader sequence to various Mycobacterium tuberculosis proteins was able to increase the protein expression in in vitro transfected cells and to enhance antibody as well as CTL responses in mice. Tobery and Siliciano previously showed that by fusing ubiquitin to Nef whereby targeting the Nef antigen to undergo rapid cytoplasmic degradation was able to significantly increase the induction of Nef-specific CTL responses [32,33]. Therefore, with respect to enhancing Nef immunogenicity, two different approaches have now been identified. It will be interesting in future studies to compare the ubiquitin and TPA fusion constructs to elucidate optimal forms of Nef antigen for vaccine uses. The current study identified several Nef-specific T cell epitopes, including an H-2b -restricted CD8+ CTL epitope, Nef58–66. The delineation of Nef-specific CD8+ T cell epitope was instrumental in the current study to evaluate the ability of the Nef mutants to elicit CD8+ T cell responses, and it will prove to be valuable for future studies of Nef immunogenicity in the murine system. Asakura et al. previously reported two murine H-2d -restricted HIV-1 IIIB CTL epitopes [34], which are located at residues 73–81 and 132–150, respectively. However, we found that peptides corresponding to these epitopes failed to detect any CTL responses in Balb/c mice (H-2d ) following immunization with either HIV-1 JRFL Nef or HIV-1 NL4.3 Nef DNA (data not shown). As indicated in the top panel of Fig. 4, the ELIspot assay involving a set of overlapping peptides covering the entire JRFL Nef sequence could not detect any signal in the regions corresponding to these two epitopes. Based on database search, the JRFL Nef CD8+ T cell epitope, Nef58–66, was found highly conserved among a large number of primary HIV-1 isolates. Compared to the commonly used NL4.3 and IIIB sequences, the JRFL epitope differs by a single amino acid residue; the corresponding sequences for the NL4.3 and IIIB Nef are TAANNAACA and TAATNAACA, respectively. It remains to be determined whether the respective sequences of NL4.3 and IIIB Nef can also function as CTL epitopes. With respect to the Nef-specific CD4 T cell epitopes, the JRFL Nef191–216 (H-2d ) is colinear with that of the IIIB Nef CD4+ CTL epitope previously reported by Michel et al. [35]. The remaining three epitopes, namely, Nef11–30 (H-2d ), Nef61–80 (H-2d ), and Nef81–100 (H-2b ), represent newly defined Nef-specific CD4+ T cell epitopes. The present study represents the initial effort to address the safety issue for using Nef as a vaccine component in the context of immunogenicity. Whether the highly immuno-

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