Peptides eliciting t-cell cytotoxicity against hiv, pharmaceutical compositions containing the same, and kits for detecting hiv infection containing the same

PEPTIDES ELICITING T-CELL CYTOTOXICITY AGAINST HIV, PHARMACEUTICAL COMPOSITIONS CONTAINING THE SAME, AND KITS FOR DETECTING HIV INFECTION CONTAINING THE SAME

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A SYNTHETIC ANTIGEN EVOKING ANTI-HIV RESPONSE

BACKGROUND OF THE INVENTION

The present invention is related generally to the field of vaccines and diagnostic or prognostic reagents for AIDS. More particularly, the present invention is related to a synthetic oligopeptide which elicits cytotoxicity by T cells against human immunodeficiency virus (HIV) antigens and proliferation of HIV-specific CTL's.

STATE OF THE ART

Various strategies are being developed to combat acquired immunedoficiency syndrome (AIDS). Envelope protein of the human immunodeficiency virus .(.HIV) has been employed as an antigenic agent to generate anti-HlV antibodies. This approach stresses the mechanism of eliciting neutralizing antibodies against the virus.

A different mode of HIV infection, however is the direct cell-to-cell transmission of HIV. Such cellmediated mechanism does not require de novo infection by extracellular virus. Hence, treatment modalities which

89012/2 depend upon the accessibility and neutralization of the virus by the antibodies, would not be effective in the cases of cellmediated infection.

It has been reported that cytotoxic T lymphocytes (CTL) play an important role in resistance to viral infection (Kast, et al.,

J. Exp. Med., 164:723-748 (1986)). However, a synthetic peptide which stimulates increased production of T cells to selectively kill the target (HIV-antigen expressing cells) has not heretofore been known or identified.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide an isolated, synthetic peptide capable of stimulating cytotoxic T cells specifically against HIV antigen-expressing cells. For simplicity, the peptide of the present invention is designated herein as Env-K!.

It is a further object of the present invention to provide a pharmaceutical composition comprising an antigenic amount of the Εην-Κχ peptide to stimulate production of HIV-specific CTL.

It is another object of the present invention to provide a diagnostic or prognostic kit for detecting HIV or predicting the course of HIV infection.

Thus the present invention provides Env-K!, peptide of the amino acid sequence: NH-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-PheVal-Thr-Ile-Glu-Lys or a peptide corresponding to the homologous segment of any HIV isolate which stimulates T cells cytotoxic to cells expressing antigen of that HIV isolate from which the sequence derives.

k 89012/1

The invention also provides a pharmaceutical composition for inducing cytotoxic lymphocytes that specifically recognize cells that express human immunodeficiency virus antigens, comprising a pharmaceutically acceptable carrier and an Env-K! peptide.

Also provided according to the present invention is a diagnostic or prognostic kit for performing an assay for detecting cell-mediated human immunodeficiency virus infection, comprising an Env-K! peptide; a mitogen that is effective for stimulating peripheral blood lymphocytes; a label for detecting the lysis of peripheral blood lymphocytes, and instruction material for performing said prognostic or diagnostic assay.

In FR 2,580,177, corresponding to AU 56126/86, there are described AIDS virus envelope glycoproteins and precursors and their use in immunogenic compositions.

In WO 87/02775 there are described synthetic peptides homologous to the gp41 and gpl20 subunits of the gpl60 envelope glycoprotein of human T cell lymphotropic virus type III (HTLV-III).

None of these references discloses or suggests a peptide having the amino acid sequence defined herein, which stimulates T cells cytotoxic to cells expressing antigen of the HIV isolate from which the sequence derives. Although WO 87/02775 discloses a peptide which partially overlaps with the sequence of the presently claimed peptide, the overlap is only with respect to less than 50% of the claimed peptide. In addition, the claimed peptide includes amino acids which are critical to the activity thereof and yet are not included in the overlap portion with WO 87/02775. Finally, the peptide described in WO 87/02775 is disclosed as being useful for stimulating antibody production. In contrast, the peptide of the present invention stimulates cytotoxic T cells. One skilled in the art simply could not

B 89012/ 1 extrapolate or otherwise derive the presently claimed peptide from that described by WO 87/02775; and one skilled in the art could not predict with any certainty or expectation of success that a peptide for stimulating antibodies would also stimulate cytotoxic T cells.

AU 56126/86 [FR 2,580,177] is directed to antigens related to envelope glycoproteins of the AIDS virus. More specifically, AU 56126/86 describes antigens having molecular weights of 150,000 and 135,000, which possess a polypeptide skeleton comprising a polypeptide sequence in common with the envelope glycoprotein of the LAV virus. There is no teaching or disclosure in AU 56126/86 of a peptide having the amino acid sequence defined herein.

Other objects and advantages of the present invention will become evident from the detailed description of the invention.

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BRIEF DESCRIPTION OF DRAWINGS .These and other objects, features and many of the attendant advantages of the invention will be better understood ־upon a reading of the following detailed .description when־ considered in connection with the accompanying drawings wherein:

Fig. 1 shows the dose-response of Env-Kj for sensitization of targets for envelope-specific CTL. Effector CTL line cells (5 x 10^/well) were added to the ־assay culture with various concentration of Env-K! in the presence of ^¹CR-labelled neo-gene transfected BALB/c3T3 fibroblast (H-2^) target cells (5 x lO^/well). After 6 hr incubation at 37°C, culture supernatant was harvested ^.and .radioactivity measured in a gamma counter. Specific -51cr-release was calculated as described vide infra. Spontaneous release for the target cells was below 25%.

Fig. 2 shows the phenotype of the H-2^ CTL line specific for Env-Kj-bearing cells. 5 x 10^ of Sl-Cr-labelled BALB/c3T3 neo-gene transfectants were cultured with cells from the long-term anti-gpl60 CTL line at several effector target ratios in the presence of 1 M of Εην-Κχ. The effector cells were pre-treated with monoclonal antibodies anti-L3T4 plus complement (-) anti-Lyt2 plus complement (φ - φ ) or with complement only ( φ - f ). ( □ ) shows no treatment control group.

Fig. 3 shows the CTL specific for peptide Env-K! are restricted by the class I molecule 5 x 10^ effector cells from the long-term line were cultured with 5 x 1051 3־cr-1abelled targets in the presence of various concentrations of Env-K!.

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ΤΙ. 1.1. ( ^ - φ ) and T4.8.3. ( - ) indicate L-cell (H-2^ transfectants expressing and D^, respectively.

Neo-gene transfected 3T3 fibroblasts, 18-Neo, (K^,L^,D^) ( Π ־ D ) were used as a positive control and neo-gene transfected L-cells, L28, (K^,D^) (¢ - ) as a negative target control. Specific lysis in the absence of peptide was -0.2, 0.3, 1.5, and 0.02% for the 4 targets, respectively. Spontaneous release ranged from 14.6 to 16.6%.

Fig. 4. shows .the stimulation of proliferation - of the CTL LINE by Env-K! in the״presence of IL-2. Panel 4A shows 1 x 10^ LINE cells were stimulated with 0.1 μΜ Env-K! (Peptide No. 18) or no peptide with or without 10% IL-2 (the same source as for making and maintaining the LINE) in the presence of 2 x 10^ mitomycin-C treated (50 pg/ml, 37°C, 30 min) 18Neo transfectants (H-2^) in 200 μΐ of medium in 96-well flat-bottom culture plates. After 3-days, wells were pulsed with 1μ Ci of ^H-thymidine (6.7 Ci/mmole) and an additional 16 hr later, activity was measured by β-scintillation counting. Panel 4B shows 1 x 10^ LINE cells were stimulated with 0.1 M Env-Kj (Peptide No. 18) or peptide No.30 or no peptide with or without 10% IL-2 in the presence of 4 x 10^ irradiated (3300 rad) B10.D2 spleen cells (SC) (H-2^ in 200 μΐ of medium in 96-well flat-bottom culture plates. Thymidine incorporation was measured as in panel A.

DETAILED DESCRIPTION OF INVENTION

The above and various other objects and advantages of the present invention are achieved by an isolated, synthetic oligopeptide, Env-Kj, having about 15 amino acid residues in the following sequence:

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Arg - lie - Gin - Arg - Gly - Pro - Gly - Arg Ala - Phe - Vai - Thr - Ila - Gly - Lys, or a functionally equivalent variant thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can ,be used in the practice or testing of - the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, standard procedures or methodology, well known to one of ordinary skill in the are, are followed.

The term variant or functionally equivalent variant as used herein means that the oligopeptide Env-K! of the present invention can be modified by deletion, addition, substitution or derivatization of the amino acid residues described herein, in any suitable manner so long as the resulting peptide acts in a functional manner similar to that of the Env-K! for any HIV isolates. All such variants or derivatives of the Env-K! peptide of the present invention are included herein as being functional equivalents thereof, it being understood that with the modern techniques of chemical synthesis and manipulations several variations of the amino acid sequence of Env-Κχ, while maintaining the essential biological properties thereof, are within the skill of an ordinary artisan in the field to which this invention belongs.

Given the amino acid sequence of Env-K!, the peptide is easily and conveniently synthesized employing the commercially available peptide synthesizers (such as

Applied Biosystems Model 430A) well known to one of ordinary skill in the art.

The details of the materials and the methods are now described.

Mice: BALB/c (H-2^) and C3H/HeN (H-2^ mice were obtained from the Jackson Laboratories (Bar Harbor, ME). BALB.K (H-2^ and BALB/c mice used in certain experiments were bred in the colony maintained at NIH. Mice were used at 6-12 weeks of age. '

Recombinant vaccinia viruses: vSC-8 (recombinant vaccinia vector containing the bacterial lacZ gene), and vSC-25 (recombinant vaccina vector expressing the HIV env glycoprotein gp 160 of the HTLV ΙΙΙβ isolate without other HIV structural or regulatory proteins) were the same as described by Chakrabarti, et al. Nature, 320: 535-537 (1986).

Transfectants: An EcoRV fragment containing the region of HIV clone BH.8 encoding gp 160 used to construct vSC-25 was cloned into the SV40-drived expression vector pcEXV-3. This DNA was co-transfected along with pSV2neo into either BALB/c-3T3 (H-2^ or DAP.3 L cell (H-2^k) fibroblasts using a standard calcium phosphate precipitation method. Transfectants were also prepared with PSV2neo alone. Individual clones were picked after selection G418 (Geneticin, GIBCO), expanded, and tested for expression of the gp 160 gene by RNA hybridization using the EcoRB fragment labelled by random hexamer priming as probe. Tl.1.1 and T4.8.3 are L cell transfectants expressing and D^ class I MHC molecules, respectively.

Monoclonal antibodies (inAb): The following inAb were used: Anti-Lyt2 (3.155; rate IgM) and anti-L3T4 (RL172.4; rat IgM)

ו _CTL generation: Mice were .immunized i.v. with

10%⁷.PFU ._of ._recombinant vaccinia viruses. Three to 14 weeks later, immune spleen cells (5xl0^/ml in 24-well culture plates in complete T-cell medium) were jrestimulated for 6 days in vitro with either 2.5 x ΙΟθ/ml

....״of ^recombinant .״vaccinia virus-infected syngeneic spleen .cells (1 hr, 37°, MOI of 10:1) or 2 x 10⁵/ml of gp

160-gene transfected, MHC-identical fibroblasts. A long term CTL line was also generated by repetitive stimulation of immune cells with mitomycin- treated, gp

160 gene-transfected fibroblasts and 10% Con-A supernatant-containing medium.

.CTL assay: After culture for 6 days, cytolytic activity of the restimulated cells was measured by the method of Bennink, et al., Nature, 311:578-579 1984) using a 6 hr assay with various ^^Cr-labelled targets were mixed with various concentrations of peptide at the beginning of the assay. The percent specific ⁵¹Cr release was calculated as 100 (experimental release - spontaneous release)/(maximum release - spontaneous release). Maximum release was determined from supernatants of cells that were lysed by addition of 5%

Triton-X 100. Spontaneous release was determined from target cells incubated without added effector cells.

Peptide synthesis: Synthetic peptides 13 to 23 residues long were prepared using the multiple simultaneous peptide method of solid-phase peptide synthesis, in polyproylene mesh tea-bags as described by Houghten, Proc. Natl. Acad. Sci., U.S.A., 82:5131-35 (1985). Conventional .blocked BQC amino-acids and HF cleavage were used. xPeptides were .desalted by .reverse phase chromatography on C-18 sep-Pak columns (Waters)

ג and analyzed by HPLC. They were prescreened for lack of nonspecific toxicity or mitogenicity in a lymph node T cell proliferation assay.

Alternatively, Env-K! is also synthesized by commercially available peptide synthesizers, such as Applied Biosystems Model 430A.

Demonstration that peptide Env-Kl is an immunodominant epitope for HIV envelope-specific CTL, stimulates proliferation of HIV-specific CTL and sensitizes targets to be killed by these CTL.

Conditions for eliciting murine CTL specific for HIV gp 160. The ability of recombinant vacinia viruses to prime and stimulate CTL specific for the products of inserted viral genes was used to generate CTL specific for HIV gp 160 using BALB/c (H-2^d)mice. Specificity for gp 160 was found at three levels—lymphocyte priming, re-stimulation, and effector function (Table 1). Thus, only the recombinant .vaccinia virus expressing gp 160 (vSC-25), no the recombinant vaccina virus containing the bacterial lacZ gene (vSC-8), could prime mice for development of CTL able to kill the gp 160-expressing fibroblast, 15-12 (.compare groups 2 and 4, and. groups 5 and 6). Likewise, only syngeneic vSC-25 infected cells, or the gp 160-expressing transfectant 15-12, but not cells infected with control vaccinia virus vSC-8, could restimulate immune T cells in vitro to kill the specific target (groups 4 and 6 vs group 3). CTL from gp 160-primed and restimulated spleen cells preferentially killed gp-160 expressing targets vs control H-2 matched targets (groups 4 and 6). The modest but significant nonspecific killing of control targets (18 neo) by vSC-25 restimulated spleen cells was absent when restimulation was done with the transfectant rather than vaccinia-infected cells. Vaccinia (vSC-25)-infected targets served as a positive control. Finally, a long-term line of CTL effectors specific for gp-160 expressing targets was established by repetitive stimulation of spleen cells from vSC-25-inununized mice with 15-12 transfectants and Con A supernatant (group 7).

Identification of an immunodominant-CTL epitope on gp 160. A series of 41 overlapping peptides in 17 clusters covering 46% of the HIV gp 160 env protein sequence, selected by use of the amphipathicity algorithm for searching for immunodominant helper T-cell epitopes but including some additional regions of the sequence as controls, was synthesized to study helper epitopes and was used to search for CTL epitopes as well. To identify the T-cell epitopes recognized by gp 160-specific BALB/c CTL, various concentrations of peptide were added to the effectors and ^¹Cr-labelled fibroblast tumor- targets at the start of the assay culture. The cytotoxic activity of three types of effector cells was measured: vSC-25-immune spleen cells stimulated once in vitro either with the 15-12 transfectant or with vSC-25 infected-autologous cells (presumably polyclonal effector populations), and the long-term CTL line (possibly an oligoclonal population). The results show that among the various peptides tested, only one (No. 18, henceforth called Env-K!) could sensitize H-2^ target cells for high levels of specific killing by all three types of effectors (Table 2). Therefore, it is concluded that the sequence contained in peptide Env-K! contains an immunodominant epitope of gp 160 for cytotoxic cells of the H-2^d haplotype. Some other peptides (No. 21, No. 41, No. 42, No. 47) appeared to marginally sensitize target cells, but these effects were insignificant compared to the activity of,Env-K!. Env-K! was found to be surprisingly potent in sensitizing targets for being killed by HIV-specific CTL at very low concentrations of the peptide. For instance, at 0.1 μΜ peptide, the killing was still on a plateau, and only at 0,.01 μΜ peptide did the killing fall to half-maximal (Fig. 1). All of the active peptides were identified as capable of forming an amphipathic helix.

Surface phenotype and Class I MHC restriction of the anti-gp 160 CTL. Treatment of the CTL effector cells with anti-Lyt 2 monoclonal antibody plus .rabbit complement, but not anti-L3T4 antibody plus complement or complement alone led to loss of killing activity on either the 15-12 fibroblasts transfected with the gp 160 gene or the control fibroblasts incubated in the presence of Env-K! (Fig. 2). These data show that the effector cells which recognize and kill HIV envelope gp 160 protein-expressing cells in our system, including .those specific for Env-K!, are conventional Lyt2+L3T4(CD8+CD4-) CTL.

The fibroblasts used as targets in these experiments express class I but not class II MHC gene products. Therefore, the gp 160-specific CTL capable of lysing the 15-12 transfectants or the peptide-bearing fibroblasts were likely to be class I MHC molecule-restricted, as is usual for Lyt2+ effected T cells. In the H-2^d haplotype there are three major class I molecules known to be used as restriction elements, K^d,D^d, and L^. To determine which of these was involved in recognition of Env-Kj, we used two L-cell (H-2^ transfectants, Tl.1.1 and T4.8.3, expressing L^ or

D^, respectively Figure 3 demonstrates that recognition ή

of Env-Kj is restricted by the class I D^d molecule, not the molecule.

H-2k mice appear to be low responders for cytotoxic T cells to gp 160. We investigated a second

- haplotype, H-2^k, using the same protocol as in the case of H-2^. L cells (H-2^k) were similarly transfected with and shown to express the gp 160 gene, but no cytotoxic activity could be detected against these targets using spleen cells from vSC-25-immune BALB.K (H-2^k) mice stimulated in vitro with syngeneic vSC-25-infected spleen cells or with the L-cell transfectant itself (Table 3).

Similarly, none of the peptides tested produced targets that could be killed by such effector cells (Table 2).

Despite the lack of gp 160-specific CTL activity, the

BALB.K mice were effectively primed and restimulated as they produced potent vaccinia-specific CTL (Table 3, from the same experiment as Table 2). Similar results were obtained with C3H (H-2״) mice (Table 3 and data not shown). Because BALB.K mice are congenic to BALB/c responder mice but bear the H-2^k haplotype, the difference in responsiveness is MHC-linked. These results suggest that H-2^ is a CTL low responder to gp

160 as assayed under the present conditions, in contrast to H-2^, which is a high responder haplotype.

Demonstration that Env-Kl stimulates proliferation of CTL specific for the envelope of HIV. The peptide Env-K!;was tested for its ability to stimulate proliferation in vitro of the CTL line demonstrated above to be specific for the envelope of HIV. The data in Figure -4'demonstrate that Env-K! stimulates proliferation of these HIV-specific CTL cells, as measured by incorporation of tritiated thymidine into DNA, and that this stimulation is enhanced in the presence of the lymphokine interleukin-2(IL-2).

Env-Kl as a diagnostic reagent. rf a person suspected of being exposed to AIDS is tested for antibodies to HIV and is determined to be seronegative, . ״it .is -Still possible that that person is carrying the virus but has not made antibodies, because a certain .percentage of exposed individuals do not -develop antibodies for a significant period of time after exposure, and some may never develop antibodies.

Nevertheless, that person may have developed a cell-mediated immune response in the form of CTL specific for the envelope of the virus. Because that person’s CTL are specific not only for the virus but also for that .individual's major histocompatibility complex (MHC) .antigens, it is not possible to test for these on ׳transfected tumor targets as was done in the case of the mice, .unless one is lucky enough to have transfected tumor that shares MHC (HLA) molecules with the individual to be tested. Because there are so many human HLA types, iot is not feasible to have transfected cells of every type available. Although it is possible to produce a transformed tumor line from the individual and transfect it with HIV genes, this is a very difficult, time-consuming, laborious process and would not be feasible to do for large numbers of people. Infecting the individual’s cells with HIV requires the appropriate cell type that can be infected, and would be hazardous for laboratory workers to handle the concentrated virus.

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Use of the vaccinia recombinant.described above may give many false positive results since־most ־individuals in the U.S. born before 1972 were immunized with vaccinia as a smallpox vaccination, and so would .have -vaccinia-specific CTL. Purified proteins are generally not taken up by cells in such a way as to .make .them targets for CTL. However, small peptides such as Env-K! are capable of sensitizing targets for CTL, as we have shown herein above. Therefore, it would be relatively simple to use Env-K! as a diagnostic reagent to test for the presence of HIV-specific CTL in the peripheral blood of an individual. This can be achieved by standard procedures; first, produce PHA or ConA_blasts_of_the peripheral blood lymphocytes of the individual to be tested, label these with 51cr as indicated herein above, incubate these with the peptide Env-K! under standard culture conditions similar to those given herein above, but modified for human CTL assays, and add fresh peripheral blood lymphocytes from the same individual. After about 6 hours, one measures the amount of 51cr released into the culture medium, and compares־this-with controls treated identically but without .any־;peptide, or with a control peptide, without any fresh lymphocytes, and with the maximum ^¹Cr release produced by detergent lysis of the target cells. If there is specific release, the individual can be judged to be carrying the HIV virus, even though no antibodies could be detected.

Env-Kl as a prognostic reagent. Although it is not yet known which arms of the immune system, if any, are protective against an HIV infection, CTL is likely to play a significant role for the reasons outlined herein above. Therefore, it is possible that seropositive but still clinically healthy individuals, who have CTL able to kill virus infected-cells, would be less likely to .develop clinical AIDS than those without :such _CTL. Therefore, Env-K! could be used to test ^seropositive individuals at various stages of disease by the methods indicated in the preceding section on diagnostic use, and if there exists a significant correlation between healthy clinical status and presence of CTL specific :for ΣΕην-Κ! this will serve as a prognostic test to predict .which seropositive individuals are at greater .or ^lesser .risk for developing clinical immunodeficiency or ״full-blown AIDS.

Env-Kl as a therapeutic agent: Because the peptide Env-Kj., can stimulate the proliferation and expansion of a population of HIV-specific CTL, Env-K! can be used as a therapeutic agent to enhance the immune response of a patient already infected with the virus. Such therapeutic modality could be better employed in conjunction with other therapeutic or ־preventive strategies for the control of the disease.

An advantage of the present invention is that, for the first time, a pure CTL-stimulatory antigenic product has been identified and synthesized in vitro without any contact with dangerously live infectious virus or product derived from such virus, that it has none of the risks involved in the handling of or exposure to HIV or HIV-derived products.

Another significant aspect of the present invention is that the peptide of the present invention makes it possible to detect and prevent cel!-mediated HIV infection which may not produce any antibodies at all. Hence, those tests which depend on the detection of anti-HIV antibodies in a body sample, would be of no use in those cases of HIV infection where antibodies are not produced. The antigen of the present invention now provides means of detecting cell-transmitted HIV infection in the absence of antibodies, which was not heretofore possible by the currently employed antibody detecting tests. Likewise, when the mode of infection is cell-transmission as described herein above, neutralizing antibodies would not be protective. Hence, a vaccine using the Env-K! as an antigen, for the first time, provides a method of preventing HIV infection, not through antibody-neutralization mechanism, but by killing HIV-infected cells by proliferating CTL’s produced through antigenic stimulation of T cells by Env-Kj.

A pharmaceutical composition including a vaccine in accordance with the present invention comprises an effective antigenic or therapeutic amount of Env-K! to produce CTL’s and a pharmaceutically acceptable carrier such as physiological saline, non-toxic, sterile buffer and the like. Of course, additives such as preservatives, sterilants, adjuvants and the like, well known to one of ordinary skill in the art, could also be included in the pharmaceutical composition to maintain or increase the efficacy of the composition.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

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Table 1. Priming and boosting requirements for cytotoxic T cells induction and long-term CTL line production in H-2^ mice*

<td rowspan="2"> Immuni -zation</td><td rowspan="2"> Restimu -lation</td><td rowspan="2"> E/T Ratio</td><td colspan="3"> % Specific lysis on</td>

<td> 18 Neo</td><td> 15-12</td><td> 15-12/vSC25</td>

<td> vSC-8</td><td> vSC8־</td><td> 40/1</td><td> 6.3</td><td> 2.3</td><td> 70.9</td>

<td></td><td></td><td> 13/1</td><td> 3.7</td><td>.6</td><td> 58.3</td>

<td></td><td></td><td> 4/1</td><td> 1.6</td><td>:2</td><td> 42.3</td>

<td> vSC-8</td><td> vSC-25</td><td> 40/1</td><td> 15.1</td><td> 27.3</td><td> 65.8</td>

<td></td><td></td><td> 13/1</td><td> 6.3</td><td> 8.4</td><td> 59.2</td>

<td></td><td></td><td> 4/1</td><td> 3.3</td><td> 1.0</td><td> 47.1</td>

<td> vSC-25</td><td> vSC-8</td><td> 40/1</td><td> 8.0</td><td> 1.1</td><td> 73.9</td>

<td></td><td></td><td> 13/1</td><td> 3.8</td><td>.9</td><td> 58.3</td>

<td></td><td></td><td> 4/1</td><td> 2.1</td><td> 2.9־</td><td> 41.1</td>

<td> vSC-25</td><td> vSC-25</td><td> 40/1</td><td> 26.1</td><td> 49.3</td><td> 66.2</td>

<td></td><td></td><td> 13/1</td><td> 11.4</td><td> 25.5</td><td> 56.9</td>

<td></td><td></td><td> 4/1</td><td> 4.2</td><td> 12.1</td><td> 49.0</td>

<td> vSC-8</td><td> 12־15</td><td> 40/1</td><td> 0.8</td><td>.8</td><td> 11.4</td>

<td></td><td></td><td> 13/1 .</td><td> 2.6</td><td>.6</td><td> 9.0</td>

<td></td><td></td><td> 4/1</td><td> 1.8</td><td>.3</td><td> 5.2</td>

<td> vSC-25</td><td> 15-12</td><td> 40/1</td><td> 6.2</td><td> 55.9</td><td> 54.4</td>

<td></td><td></td><td> 13/1</td><td> 3.2</td><td> 38.9</td><td> 46.3</td>

<td></td><td></td><td> 4/1</td><td> 2.1</td><td> 24.9</td><td> 3.1</td>

<td> vSC-25</td><td> 15-12+</td><td> 40/1</td><td> 15.0</td><td> 44.0</td><td> NT</td>

<td></td><td></td><td> 20/1</td><td> 4.5</td><td> 39.1</td><td> NT</td>

<td></td><td></td><td> 10/1</td><td> 5.5</td><td> 33.5</td><td> NT</td>

<td></td><td></td><td> 5/1</td><td> 2.1</td><td> 33.2</td><td> NT</td>

Table 1 Continued

BALB/c (Η-2Φ mice were primed i.v. with 10⁷ PFU of recombinant vaccinia virus expressing the HIV env protein gp!60 (vSC-25) or the bacterial lacZ gene (vSC-8). The'spleen cells were restimulated in vitro either with 2.5 x 1/ml of recombinant vaccinia virus (vSC-25 or vSC-8)infected syngeneic spleen cells or with 2 x lO^/ml of gpl60־gene transfected BALB/c 3T3 cells (15-12). CTL activity was measured against neo-gene transfected 3T3 cells (18 Neo), 1512־, and vSC-25 infected 15-12 target cells (15-12/vSC-25). NT: not tested. E/T: effector to target ratio.

¹Long-term CTL line produced by repetitive stimulation of vSC-25immune spleen cells with transfectant 15-12 plus lymphokines.

<td colspan="5"> Table 2 Identification of target epitopes in gpl60 in H-2^ and H-2^ mice</td>

<td rowspan="2"> Peptide (sequence position)</td><td colspan="3"> % Specific 51 Cr Release* BALD/c(H-2d)_________</td><td rowspan="2"> _ BALB.K(H-2k) V25/V25H ____ (40/1)</td>

<td> V25/15-12+ (40/1)</td><td> LINEt (10/1)</td><td> V25/V25S (40/1)</td>

<td> 1 ( 93-107)</td><td> 4.0</td><td> 1.8</td><td> 1.7</td><td> 1.0</td>

<td> 2 ( 98-112)</td><td> 3.3</td><td> 4.5</td><td> 2.6</td><td> 0.9</td>

<td> 3 (102-116)</td><td> 5.7</td><td> 5.0</td><td> 0.6</td><td> 0.8</td>

<td> 4 (103-117)</td><td> 1.8</td><td> 0.0</td><td>.6</td><td> 0.4</td>

<td> 5 (105-117)</td><td> 0.5</td><td> 0.5</td><td> 3.6</td><td>.2</td>

<td> 7 (107-121)</td><td> 1.2</td><td>.1’*</td><td> 2.6</td><td> 0.6</td>

<td> 8 (112-127)</td><td> 5.1</td><td> 3.3</td><td> 7.5</td><td> 0.2 .</td>

<td> (155־141) 9</td><td> 4.7</td><td> 0.4</td><td>.2</td><td> 0.1</td>

<td> 10 (157-171)</td><td>.7</td><td>.5**</td><td> 0.5</td><td> 0.6</td>

<td> 11 (231-245)</td><td> 4.4</td><td> 1.4</td><td> 2.5</td><td> 0.3</td>

<td> 12 (236-250)</td><td> 2.0</td><td> 3.3</td><td> 4.8</td><td> 1.6</td>

<td> 14 (252-273)</td><td> 2.0</td><td> 0.4</td><td>.6</td><td> 1.2</td>

<td> 17 (267-282)</td><td> 5.8</td><td> 4.5</td><td> 4.1</td><td>.4</td>

<td> 18 (308-322)</td><td> 67.6</td><td></td><td> 52.5</td><td> 1.6</td>

<td> 19 (317-331)</td><td>.0</td><td>.6**</td><td>.2</td><td> 0.9</td>

<td> 20 (335-349)</td><td> 6.4</td><td> 0.3</td><td> 5.3</td><td> 0.0</td>

<td> 21 (343-357)</td><td>^! 10.8</td><td> 8.4**</td><td> 14.1</td><td> 0.5</td>

<td> 22 (354-368)</td><td>.1</td><td>.5</td><td>.6</td><td> 0.7</td>

<td> (376־362) 23</td><td> 0.9</td><td> 5.9**</td><td> 0.5</td><td> 0.7</td>

<td> 26 (421-436)</td><td>.8</td><td>.4</td><td> 1.6</td><td> 1.1</td>

<td> 28 (425-439)</td><td> 2.6</td><td>.2**</td><td>.1</td><td> 0.8</td>

Table 2 Continued

<td> 29 (430-444)</td><td> 3.2</td><td> 1.6</td><td> 0.2</td><td> 1.0</td>

<td> 30 (476-490)</td><td> 5.2</td><td> *״3.9</td><td>.0</td><td> 0.8</td>

<td> 33 (485-499)</td><td> 6.0</td><td> 1.9</td><td> 0.4־</td><td> 0.7</td>

<td> 35 (553-574)</td><td>.1</td><td>.3**</td><td>.2</td><td> 0.8</td>

<td> 36 (612-626)</td><td> 2.1</td><td>^; 10.1♦*</td><td> י 3.0־</td><td> 1.0 '</td>

<td> 37 (619-633)</td><td> 1.7</td><td> 8.9</td><td> 0.6</td><td> 2.1</td>

<td> (643־629) 39</td><td> 7.7</td><td> *״4.8</td><td> 0.4</td><td> 0.6</td>

<td> 40 (632-646)</td><td>.5</td><td>.4</td><td> 1.9־</td><td> 0.2</td>

<td> 41 (637-651)</td><td> 21</td><td> *״9.7</td><td> 0.7</td><td> 2.2</td>

<td> 42 (657-671)</td><td> 12.0</td><td> 31</td><td> LQ</td><td> 2.2</td>

<td> 43 (662-676)</td><td> 2.3</td><td>.1</td><td> 0.2־</td><td> 1.7</td>

<td> 45 (723-737)</td><td>.8</td><td>.1</td><td> 0.9</td><td> 1.5</td>

<td> (794־780) 47</td><td> 9.9</td><td> *״10.5</td><td> 1.5־</td><td> 1.1</td>

<td> 50 (794-808)</td><td> 4.7־</td><td> *״1.8</td><td> 0.6־</td><td> 0.4</td>

<td> 51 (799-813)</td><td> 1.2</td><td> 2.6</td><td> 0.1</td><td> 0.9</td>

<td> 52 (821-835)</td><td> 3.9</td><td> 0.8</td><td> 1.9</td><td> 0.6</td>

<td> 53 (827-841)</td><td> 0.5</td><td> *״3.4</td><td> 2.0־</td><td> 0.1־</td>

<td> 55 (834-848)</td><td> 3.1</td><td> *״4.4</td><td> 4.4־</td><td> 0.0</td>

<td> 56 (839-853)</td><td> 2.8</td><td> 1.9</td><td>.6</td><td> 0.8־</td>

<td> 57 (842-856)</td><td> 0.6</td><td> *״1.2-</td><td> 1.4־</td><td> 0-1.</td>

<td> None</td><td> 1.7</td><td> 3.0</td><td> 3.4</td><td> 0.8</td>

- :20 Table 2 Continued

.. 1:

ג * As targets, I8Neo transfectants (H-2^) plus ΙΟμΜ synthetic peptide were used for BALB/c effectors and L28 (H-2^) plus ΙΟμΜ peptide were used for BALB.K effectors. The ratio in parentheses is the effector/target ratio. ^725/15-12 indicates effector cells derived from vSC25־immunized BALB/c spleen cells restimulated in vitro with the 1512־ transfectant (see Table 1). tLINE indicates the CTL line derived from vSC25 immunized BALB/c spleen cells repetitively stimulated with 15-12 and 10% Con־A supernatant. The results marked** were obtained at an effector to target ratio of 20/1 instead of 10/1. No peptide control was 3.0% for both ratios.

§V25/V25 indicates effector cells derived from vSC25־immune BALB/c spleen cells restimulated in vitro with vSC25־infected syngeneic spleen cells. TV25/V25 indicates the effector cells derived from vSC25 immune BALB.K spleen cells restimulated in vitro with vSC25 infected syngeneic spleen cells. Controls for BALB.K cells from the same experiment are in Table 3.

Table 3. H-2^ (BALB.K and C3H) mice fail to respond with CTL specific for gpl60

<td> _H1V env</td><td colspan="5"> transfectants expressing the HIV envelope protein״*_____</td>

<td> Immuni- zation</td><td> Restimu־ E/T lation Ratio</td><td> 25-20</td><td> ' L28</td><td> S^Cr release(%) 25-20/V25</td><td> L28/V25</td>

<td> vSC25־</td><td> vSC-25 inf. 80/1</td><td> 1.2</td><td> 0.5</td><td> 56.2</td><td> 57.7</td>

<td> (BALB.K)</td><td> 40/1</td><td> 1.3</td><td> 0.8</td><td> 47.3</td><td> 44.4</td>

<td> -</td><td> 20/1</td><td> 0.7</td><td> 0.6</td><td> 39.4</td><td> 39.0</td>

<td> vSC25־</td><td> vSC-25 inf. 80/1</td><td> 3.7</td><td> 1.7</td><td> N.T.</td><td> 60.1</td>

<td> (C3H)</td><td> 40/1</td><td> 1.9</td><td> 0.4</td><td> N.T.</td><td> 51.9</td>

<td></td><td> 20/1</td><td> 0.1־</td><td> 0.1</td><td> N.T.</td><td> 41.5</td>

<td> vSC-25</td><td> 60/1 20־25</td><td> 11.8</td><td> 15.7</td><td> N.T.</td><td> 35.7</td>

<td> (C3H)</td><td> 20/1</td><td> 6.8</td><td> 8.5</td><td> N.T.</td><td> 16.7</td>

<td></td><td> 7/1</td><td> 3.9</td><td> 2.3</td><td> N.T.</td><td> 5.6</td>

BALB.K (H-2k) or C3H (H2־^) spleen cells immunized with vSC25־ were boosted in vitro with either the gpl60-expressing L-cell transfectant, 2520־, or vSC-25-infected syngeneic spleen cells. After culture for 6 days, cytolytic activity of the restimulated cells was measured against 2520־, or neo-gene transfected L-cells (H-2^), L28, as a control target or vSC־25־infected 2525-20) 20־/V25) and L28 (L28/V25), as positive control targets.