VIROLOGY

188, 502-509

(1992)

Rapid in viva Induction of HIV-Specific CD8+ Cytotoxic T Lymphocytes by a 15-Amino Unmodified Free Peptide from the lmmunodominant V3-Loop of GPI 20

Acid

K. J. SASTRY,* P. N. NEHETE,* S. VENKATNARAYANAN,* J. MORKOWSKl,t C. D. PLATSOUCAS,t AND R. B. ARLINGHAUS**’ *Department

of Molecular Pathology and tDepartment of immunology, the University M. D. Anderson Cancer Center, Houston, Texas 77030 Received December

5, 199 1; accepted

February

of Texas

13, 1992

Efforts to generate a vaccine to prevent infection by human immunodeficiency virus (HIV) have focused on inducing neutralizing antibodies. However, cytotoxic T-lymphocyte (CTL) responses are a major immune defense mechanism required for recovery from many different virus infections. Since CTL epitopes can be defined by short synthetic peptides, we searched for HIV peptides that elicit a viral-specific CTL response in mice. We have developed a new method for screening CTL-inducing peptides involving a single injection into the footpad of mice to prime CTLs in the draining popliteal lymph node of mice within IO days. Our results demonstrate that a 15amino acid peptide (aa 315-329) derived from the V3 loop of HIV gp120 caused a rapid induction of peptide-specific and gpl60-specific CD8-positive CTLs. Lysis of targets is specific since cells preincubated with unrelated peptides are resistant to lysis as are cells of a different MHC haplotype pretreated with the cognate peptide. Pretreatment of restimulated node cells with complement plus anti-CD8 but not anti-CD4 removed the lytic activity. We also successfully induced in viva CTL activity with unmodified synthetic peptides from the influenza and Sendai virus nucleoproteins, indicating general applicability of our method for rapid screening of CTL epitopes. Because HIV replication has been reported by several labs to occur mainly in lymph nodes of infected patients, the rapid induction of HIV-specific CTLs in proximal lymph nodes by unmodified peptides emphasizes the physiological significance of our findings toward vaccine and therapeu0 1992 Academic press. Inc. tic approaches.

INTRODUCTION

thetic peptides for in viva generation of virus-specific CTLs (Deres et al., 1989; Aichele et a/., 1990; Kast et al., 1991). Several reports have detected CTLs specific for various human immunodeficiency virus (HIV) gene products in HIV-infected patients or humans or mice immunized with HIV proteins (Walker eta/., 1987; Plata et a/., 1987; Walker et al., 1988; Nixon et a/., 1988; Michel et a/., 1988; Chenciner et a/., 1989; Tsubota et al., 1989). Although specific peptide sequences recognized by these in viva-primed CTLs have been identified, unmodified free synthetic peptides representing these CTL epitopes have not been demonstrated to induce an in viva CTL response directed against cells expressing the corresponding HIV protein or coated with appropriate peptide (Berzofsky, 1991). We describe the induction of CD8+ HIV env-specific CTLs in mice by injecting a synthetic peptide from the immunodominant V3-loop (aa 315-329: RIQRGPGRAFVTIGK, abbreviated hereafter as R15K) of the HIV envelope protein. This peptide sequence was originally identified as a CTL epitope by Berzofsky and collaborators (Takahashi et a/., 1988). In their studies, in viva induction of CTLs was accomplished by infecting Balb/ c mice with a recombinant vaccinia virus expressing HIV env proteins. These CTLs lysed syngeneic target cells preincubated with the V3-loop peptide, R15K.

Induction of virus-specific cytotoxic T lymphocytes (CTLs) is typically achieved by infection with the virus itself or by infection with a recombinant virus that expresses one or more of the viral gene products. Peptides derived by intracellular processing of viral proteins are presented on the surface of infected cells in association with major histocompatibility complex (MHC) class I molecules for recognition by CTLs (Braciale et a/., 1987; Unanue and Cerottini, 1989). CTL responses constitute a major defense mechanism against viral infections. In some cases full protection can be achieved with virus-specific CTLs even in the absence of an antibody response (Lukacher et al., 1984; Bevan, 1989). The concept of identifying T-cell epitopes in proteins for inclusion in potential vaccine candidates has gained importance as a result of the demonstration by Townsend et al. (1986) that CTL epitopes of influenza nucleoprotein can be defined by short synthetic peptides. Three recent reports in the literature, one each from influenza, lymphocytic choriomenengitis, and Sendai viruses, respectively, described the use of syn’ To whom reprint requests should be addressed.

0042.6822/92

$5.00

Copynght 0 1992 by Academic Press. Inc All rights of reproductron I” any form reserved.

502

l/V V/V0 INDUCTION OF CTLs WITH PEPTIDES

However, to date, in viva induction of CTLs by immunizing mice with this or any other CTL active HIV peptide, in its free form without attaching to either specific lipid tails, carrier molecules, helper T-cell epitopes, or other peptide sequences, has not been demonstrated.

MATERIALS AND METHODS Mice C57Bl/6 and Balb/c mice between 6 and 8 weeks of age were purchased from Charles River Laboratories (Wilmington, MA). Peptide synthesis The peptides were made using the Merrifield solid phase method (Merriefield, 1963) either on a modified Vega 250 automatic peptide synthesizer or by the “Bag” method as described by Houghten eta/. (1985). In either case, removal of T-BOC blocking groups and hydrolysis of the peptide from the resin were accomplished by hydrofluoric acid (HF) treatment at 0” for 1 hr. After HF treatment, ether extraction was used to remove various organic compounds and peptides were extracted from the resin with 25% acetic acid.

Peptide polymers Two types of polymers were prepared: (1) double cysteine polymers linked end to end by disulfide bonds, and (2) lipid micelle polymers formed by attaching an amino terminal lysine to the peptide sequence in question and then coupling a fatty acid to both the (Y and c amino groups (Hopp, 1984). In the first method, cysteines were added to the termini during synthesis. Completed peptides were dissolved at 10 mg/ml at room temperature in 0.1 h/l ammonium bicarbonate. The solution was stirred overnight to allow oxidation of the sulfhydryl groups. The peptide solution was freezedried and analyzed by HPLC to confirm the presence of polymer forms of the peptide. For the lipid micelles, lysine was added as the last amino acid and palmitic acid was attached to the amino groups of lysine as described (Hopp, 1984). Such molecules were insoluble in the usual acetic acid solutions used to extract peptides from the resin. As suggested by Hopp (1984) it was necessary to use 95% acetic acid to dissolve peptides with palmitic acid residues attached.

Generation of CTLs Mice were immunized by intradermal injection into the hind footpad with 100 pug of the peptide in a 1: 1 emulsion with complete Freund’s adjuvant (CFA). After 10 days the draining popliteal lymph nodes (PLN) were

503

surgically removed and the cells were separated by mild homogenization. These PLN cells (typically the cell yield ranged between 10 and 50 X 1O6cells/lymph node/mouse) were restimulated for 5 days, in vitro, with irradiated (3300 rads) syngeneic mouse spleen cells that were pretreated with the monomeric form of the peptide (40 pg/ml) for 2 hr at 37”. The cell mixtures were maintained in volumes of 5-l 0 ml of Click’s medium (Click et a/., 1972) supplemented with 10% fetal calf serum (FCS) and 50 PALM 2-mercaptoethanol.

CTL Assay After 5 days of restimulation, the effector cells were washed three times with RPMI 1640 medium supplemented with 10% FCS and resuspended at a final cell density of 5 X 1O’?ml. The CTL activity against peptidetreated target cells or cells expressing HIV env protein gpl60 was determined in a standard 4-hr “Cr-release assay (Platsoucas and Good, 1981). The percentagespecific 51Cr-release was calculated as 100X (experimental release - spontaneous release)/(maximum release - sponteneous release). Maximum release was determined from supernatants of cells that are lysed by adding 5% Triton X-l 00. Spontaneous release was determined from target cells incubated without added effector cells. All the results presented have a standard deviation value of less than 109/o. Spontaneous lysis of target cells was between 15 and 20% of the maximum lysis observed in all experiments.

Preparation of peptide-treated target cells Syngeneic target cells (e.g., P815) were labeled with 250 &i of 5’Cr for 2 hr at 37” and washed three times before incubating with the monomeric form of the peptide (40 pg/ml) for an additional 2 hr at 37”. Subsequently the cells were washed twice with RPMI medium containing 10% FCS and resuspended at 5 x 1O4 cells/ml. These cells were then mixed with effector cells in U-bottom 96-well microtiter plates to achieve different effector to target cells (E:T) ratios.

Preparation of target ceils expressing HIV env gpl60 The 5 X 1O6 P815 target cells were infected with 5 X 1O7 plaque forming units of control (VSCS) or recombinant vaccinia virus containing HIV envelope gene (VPEl6) for 18-20 hr prior to labeling with 100 &Ii of 5’Cr (ICN Radiochemicals, Irvine, CA). Cytotoxic activity was determined by the standard 4-hr 5’Cr-release assay as described (Platsoucas and Good, 1981). Western blotting with HIV antibody-positive human sera confirmed the presence of gp160 protein in VPEl6-infected, but not VSC8-infected P815, target

SASTRY ET AL.

504 Immunization

loo

50

25

12

loo

50

with

25

12

loo

50

25

12

Effector:Target Ratio Targets -Ic P515 -

P815+peptlda

FIG. 1. Induction of specific CTLs in mice by immunization with the VB-loop R15K peptide. CTL activity of popliteal lymph node cells from Balb/c mice was measured after immunization with HIV env V3-loop R15K peptide (aa 315-329) in three forms: linear monomer, lipid-tailed micelles formed by conjugation to a dipalmityl-lysine-glytine-glycine at the N-terminus, and disulphide-linked polymers formed by oxidation of cysteine residues added at both the N- and C-termini.

cells. The control and recombinant vaccinia viruses were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, supplied by Dr. Bernard Moss. In these experiments 1 X 1O6cells/ml of CTL effecters and 5 X 1O4cells/ml of targets were mixed to obtain an initial E:T ratio of 20: 1. Depletion of CD4+ or CD8+ cells Restimulated PLN cells were treated with complement alone (+C) or with either anti-CD4 monoclonal antibody (clone GK-1.5) plus C (+anti-CD4 + C) or antiCD8 monoclonal antibody (clone 53-6.72) plus C (+anti-CD8 + C) as described previously (Platsoucas and Good, 1981). Resulting cells were then tested for their capacity to lyse MHC-matched target cells that were either pretreated with the monomeric form of the peptide (P815 + peptide) or infected with recombinant vaccinia virus that expresses the HIV IIIB envelope gene for gpl60 (P815 + VPE16). In these experiments 1 X 10” cells/ml of CTL effecters and 5 X 1O4cells/ml of targets were mixed to obtain an initial E:T ratio of 20: 1.

jetted (liposome formation of the peptide, syngeneic cells pretreated with the peptide, and monomeror polymer or micelle forms of the peptide), the tissue origin of the effector cells (spleen and lymph node cells), and number of immunizations (from a single injection of the peptide to up to five additional boosts). Finally, we observed that a single immunization of Balb/c mice in the hindfoot pad with the R15K peptide in CFA consistently resulted in generation of CTLs in the draining PLN within just 10 days. These PLN cells, when restimulated for 5 days by incubating with irradiated spleen cells pretreated with the R15K peptide, specifically lysed MHC-matched target cells (P815, H-2d) preincubated with the peptide (see Materials and Methods). To determine the optimal form of the peptide for induction of peptide-specific CTL responses in viva, we prepared the R15K V3-loop peptide in three different configurations: a linear monomer, micelles formed by conjugating the peptide to a dipalmityl-lysine-glycineglycine at the N-terminus (Hopp, 1984; Sastry and Arlinghaus, 1991), and a disulphide-linked polymer formed by oxidation of cysteine residues added at both the N- and C-termini. Representative results obtained with each of three forms of the R15K peptide are shown in Fig. 1. ln vivo induction of CTL responses directed to target cells pretreated with the R15K peptide was consistently observed in 8 of 12 mice immunized with the monomeric form, in 13 of 13 mice immunized with the micelle form, and in 6 of 8 mice immunized with the disulfide polymer form of the peptide. Most important, CTLs induced by all three forms of the peptide also specifically lysed P815 cells infected with

100

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80

Monomer

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2.5

Rapid induction of HIV env-specific CTLs in mice with the V3-loop R15K peptide In order to develop a method for rapid and consistent induction of specific CTLs in mice, we tried several different protocols that involved variations in the route of immunization (intravenous, subcutaneous, intraperitoneal, and intradermal), the form of the peptide in-

20

10

5

Effector:Target

RESULTS

Polymer

L-,z

otc”-* 20

with

Lipid Tailed Micelle

2.5

20

IO

6

2.5

Ratio

Targets -

P515+vsc8

-

P515+vPE16

FIG. 2. CTLs induced by immunization with the R15K peptide specifically lyse MHC-matched cells expressing gpl60. CTLs from Balb/ c mice immunized in viva with any one of the three forms of the R15K peptide lysed MHC-matched target cells infected with recombinant vaccinia virus expressing the HIV IIIB envelope protein, gpl60 (P815 + VPE16). These immune cells did not lyse MHC-matched target cells infected with a control vaccinia virus (P815 + VSC8).

IN V/V0 INDUCTION TABLE1 PEPTIDEAND TARGETCELL SPECIFICW OF CTLs INDUCEDIN BALE/C MICE BYTHE HIV env V3-LOOP R15K PEPTIDE %Specific

lysis at various E:T” ratios

Target cells

1OO:l

5O:l

25:l

P81 5b P815+A84C P815 + B106d P815 + 8105"

7.2 76.7 4.2 6.7

0 67.2 0.6 0

0 65.9 0 0

3A9’ 3A9 + A84g

7.0 10.0

0.6 8.0

0 0

a Effector to target cell ratio. ’ MHC-matched target cells (H-2”). c H-2d cells pretreated with HIV env V3-loop peptide. d H-2d cells pretreated with a influenza virus peptide (Deres et a/., 1989). e H-2d cells pretreated with a Sendai virus peptide (Kast et al., 1991). ’ MHC-mismatched target cells (H-2k). 9 H-2’ target cells pretreated with HIV env V3-loop peptlde.

505

OF CTLs WITH PEPTIDES

experiments, treatment of the CTL effecters from mice immunized with the monomeric form of the peptide with a monoclonal antibody against the CD8 antigen plus rabbit complement abolished the cytotoxicity against peptide-treated targets and HIV env-expressing targets (Fig. 3A). In contrast, pretreatment of effector cells with a monoclonal antibody to CD4 antigen plus complement or complement alone had no significant effect. Similar results were also obtained with CTLs generated in mice immunized with the peptide in micelle form (Fig. 3B) as well as the polymeric form (data not shown). Our finding that CD8+ CTLs are induced by the R15K peptide is consistent with the fact that P815 target cells used in all these experiments are known to express MHC class I but not class II gene products (Maryanski et a/., 1985). Our results demonstrate that CTLs

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a recombinant vaccinia virus expressing HIV envelope protein gpl60 (Fig. 2). A gross comparison of the efficiency with which HIV envelope expressing target cells are lysed by CTLs from the three forms of the peptide indicated that CTLs induced by the micelle form are more efficient than those from the polymeric form which in turn are more efficient than the CTLs from mice immunized with the monomeric form. These results clearly demonstrate that the CTLs generated in Balb/c mice with Rl5K peptide from the V3-loop can specifically lyse appropriate target cells expressing the HIV envelope protein. We also observed that CTLs generated in mice immunized with the mice/e form of R15K peptide did not lyse P815 cells preincubated with unrelated peptides (Table 1). Similar results were obtained with CTLs induced by injecting Balb/c mice with the monomeric or polymeric forms of the R15K peptide (data not shown). These results demonstrate the peptide specificity of the CTLs induced in vivo by the R15K peptide. The peptide-induced CTLs in Balb/c mice were H-2 restricted as evident from the lysis of only peptide-pretreated H-2d target cells (P815) but not peptide-treated 3A9 target cells, which express the H-2k haplotype (Table 1). The RI 5K peptide induces

CD8-positive

CTLs.

Experiments were performed to determine whether the virus-specific CTLs were CD8+ or CD4+. In these

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Effector: Target Ratio Targets -PBlB+peptide

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FIG. 3. CTLs Induced by the Rl5K peptide are CD8-positive. CTLs induced by in vko immunization with either the monomeric form (A) or the lipid-tailed micelle form (B) of the HIV VB-loop R15K peptide were depleted by preincubation with anti-CD8 plus C (+ anti-CD8 + C) but not by antr-CD4 plus C (+ anti-CD4 + C) or C alone (+C). MHC-matched target cells were erther pretreated with the monomeric form of the peptide (P815 + peptrde) or infected with recombinant vacclnla virus expressing HIV IIIB envelope protein gp160 (P815 + VPElG).

506

SASTRY ET AL.

induced by the HIV env peptide, R15K, are in fact MHC class l-restricted, as is commonly observed with CD8+ effector CTLs. However, we have not tested target cells expressing MHC class II antigens and therefore can not rule out the possibility of MHC class II restriction in this present study. The R15K peptide was tested for its capacity to induce CTL response in three other inbred mouse strains representing H2k, H2”, and H2b haplotypes (C3H/HeJ, A.SW, and C57Bl/6 mice, respectively). No CTL responses were detected against syngeneic target cells preincubated with the R15K peptide (data not shown). These results established the H2d specificity of the R15K peptide.

The R15K HIV peptide fails to induce anti-peptide antibody Since the R15K peptide is in the middle of an immunodominant B-cell active region of the HIV gpl20, we investigated the antibody-inducing activity of the R15K peptide. We observed that, either as monomer or lipid tailed micelle or after conjugation to KLH, this peptide failed to induce in Balb/c mice a measurable titer of anti-peptide antibody (less than 1:200). However, mice immunized with R15K peptide, conjugated to KLH did make antibodies against KLH (Elisa titer more than 1: 106), showing that the mice are immunocompetent and that the peptide has no inhibitory effects in mice for antibody response against antigenic proteins. Sera from mice immunized in the footpad were also tested; it was observed that no anti-peptide antibodies were formed (Elisa titer less than 1:40). Past experience in our laboratory with anti-peptide antibodies to some proto-oncogene products (mos and abl) raised in either rabbits or mice showed that antibodies with Elisa titers < 1:5000 (against the immunizing peptide) failed to react with the native protein in Western blot analysis or in vitro protein kinase assays.

Induction of peptide-specific CTLs in mice by injection of influenza and Sendai virus synthetic peptides Using this protocol we were also able to demonstrate in viva induction of CTLs in Balb/c mice with a synthetic peptide (B106) (aa 147-158 R-, TYQRTRALVTG) from the nucleoprotein of influenza virus. Deres et al. (1989) have previously shown that this peptide can prime influenza virus-specific CTLs in mice only when covalently linked at its N-terminus to P,CSS group. However, using the free monomer peptide with our immunization protocol, we demonstrated in viva priming of MHC class 1 restricted CD8+ CTLs that

TABLE 2 IN Wo PRIMINGOF PEPTIDESPECIFICCTLs IN BALE/C MICE WITHA FREE SYNTHETICPEPTIDE(5106) FROM INFLUENZAVIRUS NUCLEOPROTEIN

Treatment to effector cells

% Specific lysis at various E:T’ ratios Target cells

16O:l

8O:l

4O:l

2O:l

No treatment No treatment No treatment

P815b P815 + B106” P815 + B105d

5.7 83.9 11.0

0 76.6 26.0

0 57.1 19.3

2.5 47.3 16.0

+ Complement + anti-CD4 +C + anti-CD8 +C

P815 + B106 P815 + B106 P815 + B106

83.3 67.2 2.5

73.6 59.3 0

58.2 40.7 0

39.7 25.8 0.4

0 0

0 0

ND 0

ND 0

No treatment No treatment

3Age 3A9 + B106’

Note. ND, Not done. ’ E:T, Effector to target cell ratio. ’ MHC, matched target cells (H-2”). ’ H-2d target cells pretreated with the influenza virus peptide (B106) (Deres et al., 1989). d H-2” target cells pretreated with the Sendai virus peptide (B105) (Kast et al., 1991). e MHC-mismatched target cells (H-2’). ‘H-2’ target cells pretreated with the influenza virus peptide (Deres et a/., 1989).

lysed target cells pretreated with this peptide (Table 2). Kast et al. (1991) recently described protection of C57Bl/6 mice against lethal Sendai virus infection by in viva priming of virus-specific CTLs with a free synthetic peptide. Employing our immunization protocol we have also been successful in inducing an in viva CTL response in C57Bl/6 mice (H2b) with this synthetic peptide (B105: NP 321-336, HGEFAPGNYPALWSYA) (data not shown), which represents an immunodominant CTL epitope from the nucleoprotein of Sendai virus. Thus, our results demonstrate that the immunization protocol we have described here has general applicability to rapidly screen (within 10 days) unmodified free synthetic peptides representing potential vaccine candidates from a wide variety of viral antigens to induce antigen specific in viva CTL response in mice expressing appropriate MHC haplotypes.

DISCUSSION The data presented here demonstrated for the first time that an unmodified free synthetic peptide, free of carrier molecules, lipid tails, helper T-cell epitopes, or other peptide sequences from the envelope protein of HIV, efficiently induces viral-specific CTLs in mice. This peptide is derived from the immunodominant V3-loop of the HIV envelope protein gpl20. Another important aspect of these studies is that it describes a new

IN V/V0 INDUCTION

method for rapid screening of synthetic peptides for their ability to generate antigen-specific CTLs in lymph nodes of experimental animals. More importantly, this immunization protocol has general applicability because we have been able to induce CD8+, MHC-restricted antigen-specific CTLs in lymph nodes of mice by injection of two peptides from different viruses. These include peptides from the nucleoproteins of influenza and Sendai viruses. It was recently reported that the human lymph nodes are the primary site of HIV replication (Kaneshima eta/., 1991; Pantaleo et a/., 1991). It was therefore suggested that appropriate strategies to inhibit HIV multiplication should be directed to these tissue sites. Thus, we believe that our findings in this regard are extremely important and physiologically relevant because the immunization protocol we have developed enables rapid induction of viral-specific CTL responses in lymph nodes, the principal sites of HIV replication in vivo. However, at this stage we do not have any evidence for inducing CTL responses in lymph nodes of humans. While this paper was being prepared for publication, Hart et al. (199 1) reported that HIV-specific CD8+ CTLs can be induced by multiple injections over a number of weeks at several sites in mice by HIV peptides composed of a T-cell determinant fused to the amino terminus of an HIV CTL epitope. However, these results raise crucial questions by implicating that unmodified free synthetic peptides representing CTL epitopes cannot be used for in vivo priming of CTL responses. With our method, a single intradermal injection of the HIV env peptide, in its unmodified free form and without the need to attach either T-helper cell epitopes or other peptide sequences, into the footpad of Balb/c mice was sufficient to generate HIV-specific CTLs within the lymph node proximal to the site of inoculation after just 10 days. The resulting CTLs efficiently lysed syngeneic mouse cells either pretreated with cognate peptide or expressing the gp160 precursor protein. Furthermore, our experiments show that such CTLs are CD8+ and that the response is restricted to cells that express the appropriate major histocompatibility complex (MHC) haplotype. We have used this new protocol to screen a number of other synthetic peptides representing T-cell epitopes from the HIV envelope protein that we recently identified (Sastry and Arlinghaus, 1991). However, none of these peptides induced CTLs in Balb/c mice. It is possible that some of these peptides will be functional in generating antigen-specific CTLs in other inbred strains of mice that express appropriate H2 haplotypes or in humans who are outbred populations and therefore express a combination of different MHC

OF CTLs WITH PEPTIDES

507

antigens. Indeed, the HIV V3-loop R15K peptide (aa 315-329) demonstrated in the present investigation to be capable of inducing HIV env-specific CTLs in vivo in Balb/c mice (H2d), has been previously shown to be a major immunodominant epitope recognized by CTLs from patients infected with HIV in the context of HLAA2, -Al, and -B8 (Clerici et al., 1991). Therefore, this peptide or a related peptide may be medically useful for the induction of HIV-specific CTLs in humans. Virus-specific CTLs appear to be best suited for protection against HIV infection because the virus spreads primarily by cell-cell contact (Lewis el al., 1988; Hosmalin et a/., 1990). The value of an antibody response in preventing infection by HIV has been brought into question by the discovery that anti-HIV antibodies can enhance HIV infection of cells in vitro (Takeda et a/., 1988; Robinson et al., 1989; Homsy et al., 1990). Moreover, enhancing antibodies may play a role in the disease process as reported by Homsy et al. (1990). We have proposed a novel HIV vaccine strategy to overcome the possible detrimental effects of anti-HIV antibodies by use of peptides that elicit a strong CTL response but lack the ability to prime B cells in a manner that produce antibodies with high affinity for the virus (Sastry and Arlinghaus, 1990). Of interest, the HIV R15K peptide failed to induce a significant titer of anti-peptide antibody in Balb/c mice, either as free or KLH crosslinked peptide. It remains to be determined whether this peptide can induce anti-peptide or even anti-gpl20 antibodies in primates and man. One constraint for selecting this peptide as a potential vaccine candidate is that it is in a region of the HIV envelope protein that has high sequence variability among various strains of HIV. Recently, however, LaRosa et al. (1990) compared amino acid sequences of the envelope protein from 245 different HIV isolates and concluded that the sequence variability in the V3loop is minimal and that as little as five different consensus sequences can be defined on a serological basis among all the viral isolates. Based on this information, we propose that a cocktail of CTL-inducing peptides from the appropriate V3-loop regions encompassing all the principal HIV groups (which may be five or less) would be sufficient for generating CTLs specific for cells expressing gpl20 from most if not all HIV strains. Such a mixture would then serve as a prototype vaccine for the prevention of HIV infection in humans.

ACKNOWLEDGMENTS This work was supported in part by funds from the National Cancer Institute (Al29308) and University Cancer Foundation from the University of Texas M. D. Anderson Cancer Center. R.B.A. holds the Hubert L. Stringer Chair in Cancer Research. All the synthetic

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peptides were prepared in the synthetic antigen core facility supported by funds from Grant CA16672 and our thanks to Mr. Ron Shonk, the peptide chemist, for preparing polymer and micelle forms of peptides. We thank Tammy Trllcek and Linda Jackson for manuscript preparation. Animals are maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care, and in accordance with current United States Department of Agriculture, Department of Health and Human Services, and National Institutes of Health regulations and standards (supported in part by Grant CA1 6672).

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