IDENTIFICATION OF HUMAN T-LYMPHOCYTE EPITOPES IN PATHOGENS FOR VACCINE DEVELOPMENT

Abstract: A method for isolating, from a biological sample derived from a member of a mammalian species, a lymphocyte specific for an antigen of an organism capable of infecting that species; the sample also contains lymphocytes not specific for the antigen, and the method involves contacting the lymphocytes of the sample with the antigen and selecting a lymphocyte which proliferates in response to the antigen, the selected lymphocyte being the desired antigen-specific lymphocyte.

Description: IDENTIFICATION OFHUMAN T-LYMPHOCYTE EPITOPES IN PATHOGENS FOR VACCINE DEVELOPMENT Background of the Invention This invention relates to T-lymphocyte(T cell)- specific antigens, in particular those o.f infectious viral, bacterial, fungal, or protozoan organisms and to vaccines employing fragments of such antigens. Summary of the Invention In general, the invention features a method for isolating, from a biological sample obtained from a member of a mammalian species, a lymphocyte specific for an antigen or an organism capable of infecting that species, which sample contains lymphocytes not specific for the antigen; the method involves contacting the lymphocytes of the sample with the antigen and selecting a lymphocyte which proliferates in response to the antigen, that lymphocyte being an antigen-specific lymphocyte. By "specific for an antigen" it is meant that the major histocompatibility (MHC) antigen recognition structure (referred to as the "surface recognition structure" in Reinherz et al. U.S. Patent No. 4,550,086, hereby incorporated by reference), a variable heterodimeric protein composed of a 49 kd and a 43 kd subunit which are non-covalently associated with the CD3 subunits, specifically recognizes and is activated by (initiates proliferation in response to) the antigen. In preferred embodiments, the organism is a viral, bacterial, fungal, or protozoan organism capable of infecting the mammalian species, and the sample is derived from a member of the mammalian species (e.g., humans) not infected with the organism. Generally, the blood of an uninfected individual contains a huge number of circulating lymphocytes, each having a different antigen-MHC receptor structure and therefore a different antigen specificity; thus the number of lymphocytes specific for an antigen of any given infectious organism is exceedingly low. This is not to say, however, that the number of cells capable of reacting with the antigen is small; on the contrary, in the case, for example, of HIV-1, which causes AIDS, its intact envelope glycoprotein, gpl20, is capable of binding to the CD4 receptor on the surfaces of a large sub-population of T-lymphocytes, the T4 cells. This binding, rather than causing cell proliferation, causes eventual cell death when gpl20 is presented as part of the AIDS virus. Because of this phenomenon, i.e., the binding and uptake of some infectious organism antigens by a receptor possessed by large numbers of cells in a given lymphocyte population, it is desirable according to the invention to contact the antigen with the lymphocytes of the sample under conditions which permit stimulation of proliferation through the antigen-MHC receptor complex, but preclude binding to a widely distributed receptor. The preferred method of presenting the antigen to the lymphocytes thus is not as a soluble reagent, but rather in processed form on the surface of an antigen-presenting cell (APC) which has taken up and processed the antigen. In a preferred format of the method of the invention, the lymphocytes of the sample, prior to being contacted with antigen, are dispersed in a growth medium, e.g., agar containing interleukin-2, which is more conducive to the growth of activated lymphocytes than to the growth of resting lymphocytes. Preferably, in the method, prior to dispersing the lymphocytes in the medium, the lymphocytes are contacted with cells presenting a cell-processed form of the antigen, to enrich the sample in antigen-specific lymphocytes. Once antigen-specific lymphocytes have been isolated according to the above method, the antigen-lymphocyte interaction can be studied; this is of importance, e.g., because, where the antigen is a protein, the region of the protein which is responsible for recognition can be identified. This is preferably carried out by fragmenting the protein and identifying a recognized fragment; this is preferably done by testing protein fragments for the ability to stimulate proliferation of the lymphocyte. The stimulatory fragment thus isolated can be used, in substantially pure (greater than 99% pure w/w) form as a vaccine against the infectious organism containing the antigen. For vaccination, a sufficient amount of the fragment is administered to stimulate activation of either cytolytic T-lymphocytes, or helper T-lymphocytes, or both. The protein fragment (used according to standard vaccine protocols, e.g., coupled to a carrier such as albumin) is superior as a vaccine to whole antigen or cells because it includes only the stimulatory portion of the antigen, and excludes regions which can inhibit the immune response. In addition, while the whole antigen might be taken up by a widely distributed receptor (e.g., in the case of gpl20, the CD4 receptor), and thus wasted, the small fragment will not be recognized by such receptors, and will target only the antigen-MHC receptor complex, to which binding can stimulate both a protective cytolytic T-lymphocyte response and a T-lymphocyte-initiated B-lymphocyte (antibody) response. Further, vaccines made using the fragments of the invention are likely to be less toxic than whole antigen. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiments The drawings are first briefly described. Drawings Figure 1 is a graph showing antigen specificity and MHC restriction of a T-cell clone, designated Een69. Proliferative responses of the CD4 CD8 ~ , gp!20-specific clone Een69 to autologous and allogenic peripheral bloodmonocytes pulsed with the indicated concentrations of gpl20 or tetanus toxoid (TT) were 3 measured in a standard HTdR incorporation assay. Autologous monocytes + gpl20 (o); autologous monocytes + TT ( ) . HLA class II typings of donors were as follows. Autologous donor: DR4,7;DRw53;DQw2,3. Allogeneic donor: DRl,2;DQwl. Figure 2 is a bar graph showing results which permit definitive assignment of the MHC Restriction Specificity of Clone Een69 using transfected murine fibroblasts. Proliferative responses of clone Een69 to various antigen-presenting cells pulsed with media alone or with 10 ug/ml of an acetic acid digest of gpl20 were 3 measured in a HTdR incorporation assay. Antigen-presenting cells included adherent autologous peripheral blood mononuclear cells (aut APC), murine L cells, or transfected L cells wxpressing the indicated human calss II molecules. Een69 is derived from a donor with the class II genotype DR4,7; DRw53; DQw2,3. Figure 3 is a series of graphs permitting identification of human T cell epitopes within HIV-1 gpl20. (A) A region of gpl20 containing predicted and experimentally defined T cell epitopes is shown in detail. Predicted epitopes are underlines. Residues 421-436 represent a putative T cell epitope predicted on the basis of amphipathic o helical character (Berzofsky et al. , 1987; Cease et al . , 1987). Residues 451-455 conform to the sequence pattern (charged or Gly, hydrophobic, hydrophobic, hydrophobic, polar or Gly) found in many T cell epitopes according to the analysis of Rothbard (1986). Structural features in this region of gpl20 include the indicated internal disulfide bond (Lasky et al., 1987) and three N-linked glycosylation sites (*). The acetic acid fragment 413-456 isolated by affinity chromatography on a 5C2E5 monoclonal antibody column and three synthetic peptides used to map T cell epitopes experimentally are indicated. gpl20 fragments and peptides are denoted using an amino acid numbering system that begins with the first Met in the env open reading frame of the H3DCG proviral isolate of HIV-1 (Muesing et al . , 1985). (B) Proliferation of the gpl20-specific clone Een69 to peptide fragments of gpl20. Autologous peripheral blood monocytes were pulsed with the indicated concentrations of various antigen preparations and then used to stimulate Een69 in a standard proliferation assay. Antigens tested included recombinant gpl20, an acetic acid digest of gpl20, purified acetic acid fragment 413-456, an acetic acid digest depleted of fragment 413-456 by passage over 5C2E5 monoclonal antibody column, Herpes Simplex Virus type I glycoprotein D, and tetanus toxoid (TT) . (C) Comparison of the proliferative responses of the DR4-restricted clone Een69 and the DR-2 restricted clone Fenl05 to an acetic acid digest of gpl20 peptides, to the digest after removal of fragment 413-456 on a 5C2E5 affinity column, and to purified fragment 413-456 eluted from the affinity column at pH 3.0 as previously described (Lasky et al., 1987). Autologous monocytes were used as APC. Antigen concentration was 10 μg/ml. Background responses to media alone are subtracted. (D) Mapping of the DR4-restricted epitope with synthetic peptides. Proliferation of clone Een69 to autologous monocytes pulsed with the indicated concentrations of intact gpl20, purified acetic acid fragment 413-456, and synthetic peptides 410-429, 419-444, and 437-456. Figure 4 is a pair of graphs showing delineation of DR4-restricted epitope with truncated peptides. Clone Een69 was tested for proliferative responses to autologous monocytes pulsed with the indicated concentrations of a series f synthetic peptides representing N-terminal (Panel A) and C-terminal (Panel B) truncations of the H3DCG 410-429 epitope. Note that peptide 416-429 could not be tested at concentrations of >3 x 10 ~ M because of its limited aqueous solubility. Figure 5 is a set of graphs showing the effect of naturally-occurring sequence variation in HIV gpl20 on T cell recognition. (A) Proliferation of clone Een69 to autologous monocytes pulsed with the indicated concentrations of various 20 residue synthetic peptides including a peptide representing residues 410-429 of the reference H3DCG sequence and peptides representing the corresponding regions of gpl20 in three other isolates; H3CG, HXB2, and Z3. Sequences of these peptides are given in Table II. Background responses to media alone are subtracted. (B) Proliferative responses of the DR4-restricted clones Een69 (top) and the DR2-restricted clone Fenl05 (bottom) to autologous monocytes pulsed with intact gpl20 molecules derived from the H3DCG and MPF stains. Background responses to media alone are subtracted. Figure 6 iε a set of graphs showing the cytolytic activity of clone Een217 against monocyte, B cell and T cell targets. (A) Autologous peripheral blood monocytes purified by adherence (top) and autologous EBV-transformed B lymphoblastoid cells (bottom) were pulsed with gpl20 or the irrelevant antigen tetanus toxoid (TT) for 16 hr and then used as targets in a standard 51Cr release assay with clone Een217. Antigen concentration was 10 μg/ml . (B) The autologous CD4 + CD8 ~ T cellclone RW17C was pulsed with media or with lOμg/ml of gpl20 for 16 hr and then incubated with clone Een217 in a standard 51Cr release assay. Application of Method to AIDS There follows a description of the application of the above-summarized method to human immunodeficiency virus-1 (HIV-1), the virus which causes acquired immune deficiency syndrome (AIDS) . The method generally involves first isolating, from HIV-uninfected individuals, T-lymphocytes specific for the HIV-1 gpl20 envelope glycoprotein, and then identifying the peptide within gpl20 responsible for binding to those gpl20-specific T-lymphocytes. Before describing those steps in detail, it is useful to present an introductory discussion of AIDS. Introductory Discussion of AIDS Of great importance in AIDS is the nature of the immune response in humans to the externally-disposed envelope glycoproteins of the HIV-1 virion, gpl20 and gp41. These proteins are synthesized in the rough endoplasmic reticulum of the infected cell initially in the form of a glycosylated precursor protein (gpl60). This precursor is subsequently cleaved to generate a larger N-terminal glycoprotein (gpl20) which remains non-covalently associated with the smaller membrane-spanning C-terminal fragment (gp4l). The endoproteolytic cleavage of gpl60 into gpl20 and gp41 is essential for viral infectivity. The larger envelope subunit gpl20 exhibits several unique features which are important in the pathophysiology of the disease. First, by viture of its high affinity binding to the T cell surface protein CD4, gpl20 dictates the tissue tropism of HIV and permits viral attachment to and infection of CD4 T lymphocytes. Human mononuclear phagocytes, which express low levels of CD4, can also be infected by HIV and may represent a major reservior of virus in infected individuals. Second, a high degree of sequence heterogeneity is observed within gpl20 when different HIV-l isolates are compared. Molecular analysis of human B and T cell responses to gpl20 is important not only for understanding the complex nature of the host-virus interaction, but also for the development of vaccines. Immunization of experimental animals with gpl20 gives rise to antisera which can neutralize viral infectivity in vitro, presumably by blocking gpl20-CD4 interactions or by blocking post-attachment events. In infected individuals, however the titer of neutralizing antibodies is low. This fact, together with the general observation that T cell immunity plays an important role in the elimination of many viral infections, has led to a great deal of interest in the nature of the human T cell response to gpl20. Cytolytic T lymphocytes (CTL) specific for gpl20 have been detected in infected individuals. However, little is known about the response to gpl20 in non-immuno, seronegative individuals. The nature of this primary T cell response to gpl20 is of particular importance for understanding host-virus interactions during the critical early phases of infection. Knowledge about T cell responses to gpl20 in naive subjects is also crucial for the rational design of gpl20-based vaccines. Isolation of gpl20-specific T-cell clones from Normal Human Subjects The analysis of antigen-specific human T cell responses in normal, non-immune subjects presents a challenging experimental problem because of the extremely low frequency of T cells reactive with a given antigen in non-immune individuals. To overcome this problem, we developed a soft agar cloning method which facilitates the isolation of rare clones from the peripheral blood T cells of normal donors. The technique was carried out as follows. Recombinant gpl20 was produced in Chinese hamster ovary (CHO) cells transfected with a truncated form of the gene encoding the gpl60 envelope precursor protein. The construct used to generate these transfectants was derived from the proviral form of the H3DCG isolate of HIV-1 (Muesing et al . (1985) Nature 313, 450-458) and has been described in detail elsewhere (Lasky et al, (1986) Science 233, 209-212; Lasky et al . , (1987) Cell 5_p_, 975-985). Truncation of the envelope gene was carried out by insertion of a stop codon after the codon for amino acid 531 in order to remove the sequence encoding the transmembrane gp41 subunit and produce a secreted form of gpl20. In addition, to enhance levels of expression, the gpl20 signal sequence and N-terminal 30 amino acids in this construct were replaced by the corresponding region of Herpes Simples Virus type I glycoprotein D (gpD) . As a result of this modification, the mature processed form of recombinant gpl20 contained 25 amino acids from gpD at its N terminus and lacked the first 30 amino acids of the mature processed form of natural gpl20 (Lasky et al., 1986) . This recombinant gpl20 binds to CD4 with high affinity (Lasky et al., 1987) and induced neutralizing antibodies (Lasky et al., 1986). Recombinant gpl20 was purified from CHO cell supernatants by affinity chromatography on gpl20-specific monoclonal antibody columns as previously described (Lasky et al. , 1986, 1987) and was greater than 99% pure by SDS-polyacrylamide gel electrophoresis. MPF gpl20 was expressed and purified in a similar fashion and showed a single 120Kd band on SDS-polyacrylamide gel electrophoresis. Peripheral blood mononuclear cells (100 x 10 ) were isolated by centrifugation through Ficoll-Hypaque discontinuous density gradients and plated in glass Petri dishes for 2 hr. at 37°C in culture medium (CM) consisting of RPMI 1640 supplemented with 10% heat-inactivated pooled AB human serum, glutamine (4mM) , penicillin (50 U/ml) and streptomycin (so μg/ml). Non-adherent cells were removed by gently rinsing, and the adherent cells consisting of >90% Mo-l positive cells by flow cytometry were harvested by scraping in cold Ca-Mg free Hanks' BSS—supplemented with 2% newborn calf serum. The monocyte preparation was 7 then adjusted to 10 /ml in the CM and pulsed with gpl20 (50 μg/ml) for 4-8 hr at 37°C. The non-adherent fraction containing T cells was then added back and g cells were cultured at 2 x 10 /ml in 24-well plates for 1 week. Responding T cells were harvested and incubated with irradiated (4000 R) , gpl20-pulsed autologous adherent cells at 5 x 10 /ml in CM for 18 hr at 37°C and then cloned in a two layer solf agar system (Srendi et al . , 1983) in 6-well tissue culture plates. The bottom layer consisted of 2.5 ml CM supplemented with 10% (v/v) IL2 containing supernatant (Meuer et al . , 1982) and 4.86 mg/ml Noble agar. The 5 upper layer consisted of 6 x 10 responding T cells/well in 0.8 ml of the above medium but with 20% serum and 3.67 mg/ml agar. The isolation of rare clones was facilitated by the fact that large numbers of responding T cells (30 x 10 ) were plated in agar. In addition, these conditions appeared to favor the growth of recently activated T cells, resulting in an enormous enrichment in antigen-specific cells. After 7-10 days, colonies were picked by micromanipulation and transferred to 96 well U-bottom plates and restimulated 4 with 5 x 10 gpl20-pulsed irradiated, autologous PBMC in CM with 10% (v/v) IL2 containing supernatants. Peripheral blood monocytes isolated as described above were pulsed with various concentrations of antigen 4 to 8 hr at 37°C in U-bottom plates with 2 x 10 monocytes/well. T cell clones were then added (5 x 10 /well) in a final volume of 0.2 ml CM. After 24 hr, plates were pulsed with 1 μCi/well HTdR. After 48 hr, plates were harvested, and 3HTdR incorporation was determined by scintillation counting. Proliferation assay with murine fibroblasts were performed as follows. Murine fibroblast lines were transfected with full-length class II o and β chain cDNAs as described in Klohe et al (1988). Briefly, the DaP.3 subclone of murine L cells was co-transfected with 20 μg each of class II o and β cDNAs in the Okayama and Berg expression vector and 1 μg of the pSV2-neo plasmid using the calcium phosphate co-precipitation method. Transfectants were selected for neomycin resistance in medium containing the neomycin analogue G418, and transfectants expressing high levels of human class II molecules were isolated by flow cytometric sorting and cloning. Fibroblasts were grown in suspension cultures in bacteriologic petri dishes for 24 hr. in the absence of G418. They were then irradiated (4000R) and plated m flat-bottom wells at 2 x 10 /ml in the presence of media alone or 10 μg/ml of an acetic acid digest of gpl20. After pulsing with antigen for 24 hr at 37°C, 4 clone Een69 (5x10 /well) was added and HTdR was measured as described above. All determinations were performed in triplicate, and standard deviations were almost always <10%. In general, approximately 1-2% of the isolated colonies were found to be antigen- specific. Positive clones were maintained in CM with 10% (v/v) IL2 containing supernatants and were restimulated weekly with irradiated, gpl20-pulsed autologous monocytes. The proliferative responses of a representative gpl20-specific clone designated Een69 are shown in Figure 1. This clone has the cell surface phenotype CD4 CD8 ~ . It showns a dose-dependent response to gpl20 in the presnece of autologous monocytes as a source of antigen-presenting cells (APC) . It does not proliferate in response to autologous APC pulsed with an irrelevant protein antigen (tetanus toxoid) or to gpl20-pulsed APC from a donor with a different class II HLA genotype. Thus the clone is both gpl20-specific and MHC-restricted. The observed proliferation is not simply the result of a non-specific mitogenic effect of gpl20 on CD4 + lymphocytes since other CD4+ T cell clones specific for irrelevant antigens do not proliferate in the presence of gpl20. Table I summarizes the phenotypic and functional properties of the four gpl20-specific clones we analyzed in detail with respect to MHC restriction specificity and gpl20 epitope localization. All four clones had the phenotype CD4 CD8 ~ . Two of the clones showed significant cytolytic activity for autologous gpl20-pulsed B lymphoblastoid targets as is discussed below. Two approaches were used to define the precise MHC restriction specificity of these CD4 + clones. In the first, class II allele-specific typing alloantisera were used to block proliferative responses to gpl20. In this manner, DR2-restricted and DR4-restricted clones were identified (Table I). Definitive confirmation of the restriction specificity of DR4-restricted clones was carried out by using as antigen presenting cells a panel of transfected murine L cells expressing particular human class II molecules (Klohe et al. , 1988). This panel included lines expressing each of the potential class II restriction elements for clones Een69 and Een217. In order to overcome possible defects in antigen processing pathways in fibroblasts, the L cells were pulsed with a set of peptides derived by cleavage of gpl20 with acetic acid (Lasky et al. , 1987). As illustrated in Figure 2, clone Een69 exhibits antigen-specific proliferative responses only in the presence of autologous monocytes and fibroblasts transfected with DR4, confirming the DR4 restriction of this clone assigned in alloantisera blocking studies. Similar results were obtained for clone Een217. Identification of epitopes recognized by qpl2Q-specific T cell clones The next step was the identification of regions within gpl20 to which the isolated gpl20-specific T cell clones respond. Studies in the murine system have demonstrated that the antigenic sites recognized by individual T cells specific for particular protein antigens consist of restricted regions of linear amino acid sequence. This finding reflects the fact that the association of peptide fragments of protein antigens with MHC restriction element is necessary for T cell recognition. To determine whether linear T cell epitopes within gpl20 could be identified for each gpl20-specific clone, we focused initially on a region near the C-terminus of the protein that contained two putative T cell epitopes, one predicted by the amphipathic o-helix algorithm and a second one predicted by the general sequence pattern alogorithm. Acetic acid digestion of gpl20 and isolation of fragment 413-456 on a 5C2E5 affinity column were carried out as described in Lasky et al., 1987, . Briefly, recombinant gpl20 (200 μg) was digested with 0.25 M acetic acid for 18 hr at 100°C under argon (Fig. 3A) . Fragment 413-456 was purified from the digest by adsorption to a 5C2E5 monoclonal antibody column and subsequent elution with 0.1 M sodium acetate, pH 3.0. Synthetic peptides were produced using standard solid phase chemistry (Merrifield, 1983) by one of two methods. One set of peptides was synthesized on a Dupont Coupler 2100 automated peptide synthesizer. Another set of peptides was synthesized manually using chloromethylstyrene-divinyl benzene resin (0.74mMol Cl/g) . The C-terminal residues were esterified to the resin using a twofold excess of Boc protected amino acid and 1.8 fold excess of triethylamine. After refluxing at 80°C for 24 hrs., the remainder of the amino acids were coupled using standard solid phase methods (Memfield, 1983). For these peptides 3H-Ile was incorporated during the coupling of lie in order to facilitate concentration measurements. Samples of each synthetic peptide were subjected to confirmatory amino acid analysis or microsequencing on an Applied Biosystems sequencer. All peptides were purified by HPLC prior to use in functional assays. In general, conditions were as follows. After HF cleavage, crude peptides in 0.1% trifluoroacetic acid (TFA), pH 2.3 were fractionated by reverse phase HPLC on a Vyadac C18 column equilibrated with 0.1% TFA. Peptides were eluted with a 0-60% gradient of acetonitrile with 0.08% TFA. Eluted fractions were dried and then solubilized in phosphate buffer saline (PBS) pH7.4. Precise concentrations of peptides in PBS stocks were determined by measurement of the absorbance at 278 and at 257.4 nm. For peptides containing a single Trp residue and no Tyr residues, the molar extinction coefficient of Trp at 278nm was used. For peptides containing a single Phe and no Trp or Tyr residues, the molar extinction coefficient of Phe at 257.5 nm was used. For peptides contai .ni.ng a 3H-Ile residue, concentration measurements were confirmed by scintillation counting. Specific activities were determined by scintillation counting and quantitative amino acid analysis. For use in functional assays, peptides were diluted in CM and filtered through 0.22 μm Millex GV4 membranes. Purified fragment 413-456 was tested for stimulatory activity in proliferation assays with gpl20-specific clones and autologous monocytes as a source of APC. Clones were also tested for proliferative responses to Herpes Simplex Virus type 1 glycoprotein D (gpD), as the recombinant gpl20 used in these studies contains the 25 N-terminal amino acids of gpD (to enhance levels of expression; see Lasky et al. , 1986) . As indicated in Figure 3B, the DR4-restricted clone Een69 proliferates in response to autologous APC pulsed with gpl20, with an acetic acid digest of gpl20, or with purified fragment 413-456. However, it does not proliferate in response to equivalent weight amounts of the acetic acid digest after depletion of fragment 413-456 on the 5C2E5 affinity column. it also fails to recognize gpD or an irrelevant antigen, tetanus toxoid. Thus Een69 recognizes a epitope within amino acids 413-456 of gpl20. Panel C contrasts the response of the DR4-restricted clone Een69 with that of the DR2-restricted clone Fenl05. Both clones recognize peptide fragments present within the acetic acid digest of gpl20. However, unlike clone Een69, clone Fenl05 proliferates in response to the acetic acid digest even after removal of fragment 413-456 on a 5C2E5 affinity column. Furthermore, Fenl05 does not respond to purified fragment 413-456. From these results, we conclude that DR2-restricted and DR4-restricted clones recognize different regions of the linear amino acid sequence of gpl20. Therefore, at least two spatially separate T cell epitopes exist within the gpl20 molecule. Other clones isolated from the same two donors duplicated the patterns observed with Een69 and Fenl05 (Table I). To determine which residues within fragment 413-456 are recognized by DR4-restricted clones, we tested synthetic peptides representing overlapping segments of this region of gpl20 including peptides spanning the two putative T cell epitopes within this fragment (Figure 3A) . Results of such an experiment are shown in Figure 3D. Intact gpl20, acetic acid fragment 413-456, and synthetic peptide 410-429, which spans the N-terminal region of this fragment, were active. Thus the epitope recognized by DR4-reεtricted T cell clones is contained within the amino acids 410-429. This epitope partially overlaps a regions that has previously been predicted to be a T cell epitope (residues 421-436) on the basis of amphipathic o-helical propensity. However, the synthetic peptide 419-444 which encompasses this amphipathic o-helical region is completely inactive. Peptide 437-456, which contains a sequence pattern commonly found in T cell epitopes according to the analysis of Rothbard (1986), is also inactive. These results thus illustrate the importance of direct experimental determination of T cell epitopes at the clonal level. Precise delineation of essential residues within peptide 410-429 was carried out using a series of truncated peptides. As indicated in Figure 4A, the absence of two N-terminal residues of peptide 410-429 results in a reduction in activity of about ten fold. Peptide 414-429, which lacks the four N-terminal residues, shows a further ten fold decrease in activity. However, peptide 416-429, which lacks the six N-terminal residues, is actually more active than peptide 414-429. Peptide 418-428, which lacks the eight N-terminal residues, shows a >1000-fold reduction in activity relative to peptide 410-429 and is recognized only at very high concentrations (3 x 10 —5M) . Taken together, these results indicate that the six N-terminal residues of peptide 410-429 contribute to but are not essential for recognition, whereas Pro 417 and possibly Leu 416 are more critical, since the absence of both results in a dramatic decrease in activity. A very different pattern is observed with the C-terminal truncated peptides (Figure 4B) . In this case, absence of two, four, or six residues from the C-terminus causes complete loss in activity. In some experiments, weak recognition of peptide 410-427 has been detected at concentrations above 10 ~ M. Based on this analysis, the essential residues of the DR-4 restricted gpl20 epitope are located at the C-terminal end of peptide 410-429, probably beginning at Leu 416 or Pro 417 and extending to Gin 428 or Glu 429. Cytolytic activity of qpl20-specific T cell clones T cell clones specific for gpl20 wer also characterized with respect to cytolytic activity. Cytolytic activity was measured in standard Cr release assays. Target cells at 5 x 10 /ml in CM were pulsed with the indicated concentrations of antigen for the indicated times at 37°C. They were then washed and labelled with 51 Cr (100μCi/10 6 cells) for 1.5 hr. at 37°C. Targets were then washed three times and added to V-bottom microtiter plates at 3000/well in CM. Effector cells were added at the indicated ratios in a final volume of 0.2 ml. Plates were centrifuged (300 x G, 5 min) and then incubated at 37°C for 4-6.5 hr. After recentrifuging in the cold, aliquots were removed from each well for assay of gamma radioactivity. Percent specific lysis was calculated according to the formula ((E-C)/(M-C)) x 100 where E is the experimental value in cpm, C is the control release vlaue, and M is the maximal release value. C was determined as the average release in control wells from which effector cells were omitted. M was determined as the average release in wells to which 1% NP40 was added in place of effector cells. C was 15-25% of M. All determinations were performed in triplicate. As shown in Fig. 6A the DR4-restricted CD4 + CD8~ clone Een217 shows cytolytic activity towards autologous peripheral blood monocytes that have been prepulsed with gpl20. Monocytes are not lysed when prepulsed with an irrelevant protein antigen, tetanus toxoid, indicating that the observed cytolysis is antigen-specific. Clone Een69 is also cytolytic, but is a less efficient killer on a per cell basis. Thus the presentation of gpl20 by autologous monocytes can lead not only to the proliferation of CD4 + CD8~, gpl20-specific clones as shown above, but also to direct lysis of the APC. Similar antigen-specific cytolysis is observed when the autologous EBV-transformed B lymphoblastoid cell line Laz 509 is used to present gpl20 to clone Een217 (Fig. 6A) . These data indicate that certain gpl20-specific clones belonging to the CD4 CD8 ~ subset possess significant antigen-specific cytolytic activity towards la monocytes and B lymphoblastoid cells. Other CD4 + CD8 ~ gpl20-specific clones such as Fenl05 and Fenl09 show little if any cytolytic activity (Table I), consistent with the notion of functional heterogeneity among CD4 CD8 ~ T cells. Given the above results, we considered the possibilty that activated autologous T cells might also be able to present gpl20 to gpl20-specific class-II restricted CTL. In this regard, the activation of human T cells is known to result in the synthesis and cell surface expression of class II MHC gene products. Therefore, activated T cells exposed to gpl20 might also be targets for lysis by class II-reεtricted, gpl20-specific CTL. To investigate this possibility, we used the previously described CD4 + CD8 ~ clone RW17C which was derived from the same donor as Een69 and Een2l7 and which was specific for an irrelevant antigen, namely ragweed antigen E. R 17C cells were pulsed with gpl20 and then labelled with Cr and incubated with clone Een217 in a standard Cr release assay. As shown in Fig. 6B, Een217 shows significant lytic activity against gpl20-pulsed autologous RW17C cells. These targets are not lysed in the absence of gpl20. Thus, gpl20-specific CTL can lyse activated autologous CD4 + CD8 ~ cells that have been exposed to gpl20. Allogeneic (DR4-) CD4 + T cells clones pulsed with gpl20 are not lysed by Een217, indicating that the lysis is MHC-restricted (data not shown) . The gpl20-specific lysis of autologous T cells provides a potential mechanism both for the destruction of HIV-infected T cells and for the lysis of non-infected T cells that have taken up gpl20 released from infected cells of HIV virions. Because of the potential importance of this gpl20-specific cytolytic reaction in the pathophysiology of AIDS, we carried out a detailed analysis of the types of T cells that were subject to destruction through such a mechanism. We tested clone Een217 for gpl20-specific lytic activity against a variety of T cell targets including resting and activated peripheral T cells as well as CD4 CD8 ~ and CD4 ~ CD8 T cell clones all derived from the same donor as clone Een217. These target cell populations were pulsed with either medium alone, with intact gpl20 or with synthetic peptide 410-429 which encompasses the epitope recognized by clone Een217. Data from several experiments are summarized in Table III. Clone Een217 fails to lyse a T cell enriched population of resting peripheral blood lymphocytes. This is not unexpected εince these resting T cells do not express class II MHC gene products (Table III). However, there is significant lysis of class II-positive, activated autologous T cell blasts pulsed with gpl20. Substantially higher lysis is observed if the T cell blasts are pulsed with peptide 410-429. When T cell blasts are separated into CD4 + CD8 ~ and CD4 ~ CD8 + subsets using antibody and complement mediated lysis, then only the CD4 CD8 ~ subset is able to present intact gpl20 to clone Een2l7. However, both subsets are lysed following exposure to peptide 410-429. Taken together, these results demonstrate that both CD4 + CD8 ~ and CD4 ~ CD8 + T cell blasts can present peptide antigen in a manner which allows for recognition by class-II restricted CTL. Nevertheless, it appears that only CD4 + CD8 ~ activated T cells are able to take up and process intact gpl20 in a manner which allows for recognition of the 410-429 epitope. This notion was confirmed using a series of autologous T cell clones. CD4 CD8 ~ clones such as RW17C and even Een217 itself are lysed by Een217 effectors after exposure to either gpl20 or peptide 410-429. In contrast, the CD4 ~ CD8 + clone Ephll is lysed by Een217 when pulsed with peptide 410-429 but not when pulsed with intact gpl20. These results most likely reflect the fact that although activated CD4 ~ CD8 + T cells express appropriate class II MHC gene products for presentation of peptide antigens to class II-restricted CTL, they lack a high affinity uptake mechanism for gpl20. CD4 CD8 ~~ clones on the other hand can readily take up gpl20 via its high affinity interaction with CD4 (McDougal et al . , 1986). Effect of HIV qenomic heterogeneity on T Cell Recognition of HIV. Little is currently known about how the marked sequence variability in different HIV isolates affects the recognition of viral proteins by T lymphocytes. Using the gpl20-specific clones described above, we have analysed this problem using both synthetic peptides and intact gpl20 preparations (Figure 5). Having delineated the epitope recognized by DR4-restricted gpl20-specific clones, we prepared a series of synthetic peptides representing the sequences of different isolates of HIV-l in the region of gpl20 recognized by these clones (residues 410-429). Within this 20 amino acid segment of gpl20, the HIV-1 isolates studied showed from one to ten amino acid changes relative to the reference H3DCG sequence (Table II). The ability of these variant peptides to stimulate the proliferation of clone Een69 in the presence of autologous monocytes was aεsesεed in a standard proliferation assay. As illustrated in Figure 5A, three types of responses to peptides representing the naturally occurring variants of HIV gpl20 were observed. One variant peptide HXB2, actually stimulated a greater proliferative response than equivalent amounts of the reference H3DCG peptide. The majority of variants, represented by the H3CG sequence, showed a significant (3-60 fold) reduction in stimulatory capacity relative to H3DCG. A third group of variant peptides, represented by the Z3 sequence, were completely ineffective in stimulating proliferation of clone Een69 even at 3 x 10 ~ M. In order to determine whether the dramatic effects observed with synthetic peptides were representative of cross-reactivity at the intact protein level, we prepared recombinant gpl20 from one particular HIV strain, known as the MPF isolate, and tested it for the capacity to stimulate DR2-restricted and DR4-restricted gpl20-specific clones. As shown in Fig. 5B, the intact MPF gpl20 was recognized only weakly by the DR4-restricted clone Een69 and not at all by the DR2-restricted clone Fenl05. Thus at both the peptide and protein level, the naturally occurring sequence variation in HIV can strongly affect recognition by T cell clones, usually resulting in a marked decrease in responsiveness to the variant. Inhibition experiments with non-stimulatory peptides indicate that this sequence variation can alter the interactions of gpl20 peptides with both the T cell receptor and the restricting MHC gene products. Data from the analysis of a large series of variant peptides are summarized in Table II. Analysis of dose-response curves indicates that the naturally occurring variation in gpl20 generally caused a reduction in activity of at least 70%, and more often greater than 90%. A single conservative amino acid substitution can produce a decrease in activity of almost ten fold, aε shown for the H3CG peptide (Phe to lie at position 423). Only in one case was recognition enhanced (HXB2 isolate) . This may reflect the Glu to Lys substitution which occurs at position 429 in this strain (see Discusson) . It is of note that variant peptides with several changes in the critical residues 416-429 such as ELI and Z3 were not recognized even at the highest concentrations tested (3 x 10 —5 M) . Thus, despite the fact that in comparison to certain highly variable portions of the gpl20 sequence the T cell epitope identified here contains a set of relatively conserved residues, the naturally occurring sequence variation in HIV-1 is often sufficient to prevent completely cross-recognition by T cell clones. Vaccines As is mentioned above, the gpl20-derived peptides which are recognized by the T-cell recognition structures of gpl20-specific cells can be used as vaccines for AIDS. Because of the above-described HIV sequence variation, such vaccines will most likely be strain-specific, i.e., no one vaccine will be effective against all strains. However, such universal pan-strain vaccines are much more likely to be useful for other, 10 more invariable pathogens, e.g., protozoans such as the malaria-causing amoeba Plasmodium falciparum; viruses such as the Herpes simplex viruses, and bacterial pathogens such as the agent which causes Hansen's diεeaεe (leproεy). Vaccines to all of these can be made ■ j t using methods analogous to those described above, i.e., pathogen—specific T-cells are isolated and used to identify specifically recognized epitopes on the pathogenic cell. Other embodiments are within the following 20 claims. Table I Properties of qpl20-sρecific T Cel 1 Clones Clone Donor 3 Phenotype *3 Ag Specificity 0 Cytolytic MHC Activity* 3 Restriction 13 CD2 CD3 CD4 CD8 la gpl20 TT gpD 413- 456 Ken69 E + ++ DR4 Een217 + +++ DR4 Fen105 F + DR2 Fen109 F + DR2 10 (see two pages following for notes) (notes to Table I) a Donors were healthy HIV-seronegative adults. HIV serologic testing was performed by the Blood Bank of the Dana-Farber Cancer Institute. Cell surface phenotype of gpl20-specific clones was determined by indirect immunofluorescence with the relevant monoclonal antibodies on an Epics V cell sorter as previously described (Siliciano et al. , 1986). Monoclonal antibodies: CD2, 3T48B5; CD3, RW28C8; CD4, 19Thy5D7; CD8, 21Thy2D3; la (monomorphic class II), 9-49. Clones were tested for proliferative response to the indicated antigens by incubation with autologouε monocyteε prepulsed with lOug/ml antigen for 6 hr. at 37°C. Proliferation was measured by incorporation of HTdR after 48 hr. Antigens tested include recombinant gpl20, tetanus toxoid (TT) , Herpes Simplex Virus type glycoprotein D (gpD), and an acetic acid fragment of recombinant gpl20 representing residues 413-456 (see text). +, stimulation index >10; -, stimulation index <1.5. Antigen-specific cytolytic activity towards autologous gpl20-pulsed monocyte or B lymphoblast targets as measured m a standard 51Cr release assay at an E:T ratio of 10:1. +++, specific lysis >40%; ++, specific lysis - 20-40%; -, specific lysis <5%. MHC restriction specificity as defined by blocking experiments with allele-specific typing alloantisera. Autologous gpl20-pulsed monocytes were reacted with 1/5 dilutions of a panel of class II allele-specific alloantisera (kindly provided by the Tissue Typing Laboratory at the Dana-Farber Cancer (notes to Table I, continued) Institute) and then tested for ability to stimulate proliferation of the relevant clones. Restriction specificities could be readily identified based on the capacity of the antisera of the indicated specificity to produce >50% inhibition relative to normal human serum controls. Antisera with irrelevant specificities produced <15% inhibition. In the case of clones Een69 and Een217, DR4 restriction was confirmed using transfected fibroblasts expressing appropriate human class II molecules as described in the text. HLA types of donors are as follows. Donor E: A2,25; B13,38; Bw4; DR4,7; DRw53; DQw2,3. Donor F: Al,24; B7,8; DR2,3. Table II Effect of rimary Sequence Variabilily on gp!20 recognition by T cells Strain 3 Sequence (410-429)-*-* Relative Concentration Potency For 50% Maximal Stimulation (M) HE3DCG GSDTITLPC IKQFINMWQE d 1.00 7.5 x 10-8 MAL STGS 1 K (pending) HAT -N- •IV- 0.27 2.7 x 10-7 10 NY5 NNE--II— I-- 0.14 5.4 x 10-7 Oo H3CG 0.13 5.6 x 10 " ARV2 -N I--- -I 0.075 1.0 x 10 -6 WMJ2 N-TL -I G 0.027 2.8 x 10 -6 CDC42 TG-I -I—R---V <0.0025 >3.0 x 10 -5 15 ELI TNTN Q- -I-K-VAG <0.00075 >1.0 x 10 .-4 Z3 CTGN -VVRT—G <0.00075 >1.0 x 10 ~4 MPFe — - :Ό (see page following for notes) (noteε to Table II) a The sequence of a peptide representing residueε 410-429 of the reference H3DCG gpl20 is show on the first line. Peptides repreεenting the corresponding region of gpl20 in other isolates are listed in order of potency relative to the reference H3DCG sequence. Sequence data are from Ratner et al., 1985, Muesing et al., 1985; Wong-Staal et al. (1985), Benn et al. (1985), Wain-Hobson et al. (1985), Rabεon and Martin (1985), Hahn et al. (1986), Willey et al. (1986), Starcich et al. (1986), and from the compilation by Myers et al. (1988). Reεidues differing from the reference H3DCG sequence in the region 410-429 are indicated. c Relative potency was determined from dose-response curves for the proliferation of clone Een69 to peptide-pulsed autologous monocytes (as in Figure 5A) . The relative potency of a given peptide is calculated as the ratio between the concentration of the reference H3DCG peptide required for 50% maximal proliferation and the concentraton of the variant peptide required for 50% maximal proliferation. The critical residues of the DR4-restricted epitope as defined by experiments with truncated peptides are indicated in bold. e The MPF variant was tested at the intact protein level. See Figure 5B. Table III Lysis of various target populations by clone Een217 Targets 3 Phenotype* 0 % Specific Lysis c CD4 CD8 la None gpl20 410-429 Exp. #1 Resting T cells (+)d (+) T cell blasts (+) (+) T4+ blasts + CoO T8+ blasts 10 Exp. #2 R 17C + Een217 + Ephll + (see page following for notes) (notes to Table III) Target cells were all derived from Donor E and included resting and activated T lymphocytes, T lymphoblasts separated into CD4 + CD8 ~ and CD4 ~ CD8 subsets by antibody and complement mediated lysis (see Experimental Procedures), and a set of T cell clones. Clone Ephll is a mitogen-activated CD4 ~ CD8 + clone with unknown antigen specificity. Cell surface phenotype was determined by indirect immunofluorescence on an Epics V cell sorter as previously described (Siliciano et al. , 1986). c Target cells were pulsed with media alone, intact gpl20 (10 μg/ml, 16 hr.) or peptide 410-429 (10 μg/ml, 4 hr.) and then labeled with 51 CR and incubated with clone Een217 in a standard 4 hr. 51CR release assay. E:T = 20:1. Parentheseε indicate a mixed population of CD4 + CD8 ~ and CDr-CD8 ~ T cells.

Claims: Claims • 1. A method for isolating, from a biological sample derived from a member of a mammalian species, a lymphocyte specific for an antigen of an organism capable of infecting said mammalian species, said sample containing lymphocytes not specific for said antigen, said method comprising contacting said lymphocytes of said sample with said antigen and selecting a lymphocyte which proliferates in response to said antigen, said selected lymphocyte being said antigen-specific lymphocyte. 2. The method of claim 1 wherein said organism is a viral, bacterial, fungal, or protozoan organism, and said member of said mammalian species is not infected with said organism. 3. The method of claim 1 wherein said antigen, prior to being contacted with said sample, is contacted with cells capable of taking up and processing said antigen and presenting a processed form of said antigen to said lymphocytes of said εample. 4. The method of claim 1 wherein the lymphocytes of said sample, prior to being contacted with said antigen, are dispersed in a growth medium which is more conducive to the growth of activated lymphocytes than to the growth of resting lymphocytes. 5. The method of claim 4 wherein, prior to said step of dispersing said lymphocytes in said medium, said lymphocytes are contacted with cells presenting a cell-processed form of said antigen to enrich said sample in antigen-specific lymphocytes. 6. The method of claim 4 wherein said medium contains interleuken-2. 7. The method of claim l wherein said antigen is a protein and said method further comprises identifying a fragment of said protein recognized by said lymphocytes by treating said protein to form protein fragments, and then testing said fragments for the ability to cause proliferation of said antigen-specific lymphocytes. 8. The protein fragment of claim 7, in substantially pure form. 9. A vaccine comprising the protein fragment of claim 8. 10. A method of vaccinating a mammal against an infectious viral, bacterial, or protozoan organism comprising administering to said mammal a cytolytic T-lymphocyte or helper T-lymphocyte activation stimulating amount of the vaccine of claim 9.

Inventors: REINHERZ ELLIS L, SILICIANO ROBERT F, REINHERZ, ELLIS, L., SILICIANO, ROBERT, F.
Applicants: DANA FARBER CANCER INST INC, DANA-FARBER CANCER INSTITUTE, INC.
Application year: 1989
Main classification: A61K45
IPC codes: A61K35, A61K38, A61K39, C07K14, C12N5, C12N15, G01N33