Patent Application: US-80093291-A

Abstract:
the present invention discloses a novel method for the rapid screening of candidate cytotoxic t lymphocyte - inducing compounds , such as peptides , by the in vivo priming of ctls with synthetic peptides . the use of compounds so identified for the development of ctl vaccines for the treatment of various infectious disorders , including the treatment of viral diseases such as aids , herpes , influenza , and feline or bovine leukemia , is also disclosed , as is the use of this methodology for the preparation of specifically primed ctls .

Description:
the present invention thus concerns in one embodiment a novel and rapid method for screening candidate compounds , such as peptides or multimers thereof , to identify appropriate ctl - inducing active compounds for use , e . g ., in ctl vaccines . in preferred screening procedures , candidate peptides or peptide multimers are assayed for in vivo ctl - induction by injecting the test substance into an experimental animal , recovering t cells from the lymph nodes and measuring the activity ( priming ) of such ctls . most preferrably immunization would involve a single intradermal injection of the test peptide in complete fruend &# 39 ; s adjuvant ( cfa ) into an appropriate site , such as the hind - foot pad of a mouse , recovering lymph node t cells , preferrably from the draining popliteal lymph nodes ( pln ), and determining their ctl activity by assaying the t cell - mediated lysis of mhc - matched target cells decorated with the candidate peptide or expressing the parent protein . this procedure allows the results to be obtained only 8 - 10 days after immunization . employment of such a screening procedure will result in the identification of t cell active peptides , whether they be peptides that induce substantially only a ctl response , or also induce an antibody response ( i . e ., b cell reactive ). a plurality of strains of mice that vary in their histocompatibility genes are used for these screenings . peptides that have a broad response in the various mhc genotypes are selected for further study in primates , and finally humans . exemplary assay procedures are found hereinafter . the synthesis of the candidate peptides to be tested in the present invention can be conducted using an automated peptide synthesiser . for example , peptides can be synthesised using the merrifield solid phase method ( merrifield , 1963 ) either on a modified vaga 250 automatic peptide synthesizer or by the “ bag ” method , as described by houghton et al . ( 1985 ). following the synthetic reactions , the completed peptides are then recovered . in the above two methods this involves the removal of the t - boc blocking groups and hydrolysis of the peptide from the resin using hydrofluoric acid ( hf ) treatment at 0 ° c . for 1 hour . the various organic by - products can then be removed , for example by extraction with ether , and the peptides extracted from the supporting resin with a reagent such as 25 % acetic acid . the compounds to be tested for potential ctl - inducing properties are then used to immunise an experimental animal , such as a mouse , rat , rabbit , guinea pig , goat , rhesus monkey , chimpanzee , or the like . in the rapid screening method for the identification of appropriate ctl - inducing candidate compounds , the inventors have found that the intradermal immunization of a mouse can be advantageously employed . intradermal immunization is believed to be particularly effective as it serves to efficiently activate the cell mediated limb of the immune system . the inventors have found that even more advantageous aspects of this immunisation protocol are ( i ) that a single injection of the candidate will usually be sufficient to achieve detectable ctl activation , and ( ii ) that the sensitivity of the assay is such that the use of immunisation modifiers will generally not be required in order to achieve ctl priming . accordingly , in preferred screening embodiments , mice are immunized by intradermal injection into the hind foot - pad with an appropriate composition , such as 100 μg of the test peptide in a 1 : 1 emulsion with complete freund &# 39 ; s adjuvant ( cfa ). after a period of time sufficent to induce a ctl response , such as 10 days , the draining popliteal lymph nodes ( pln ) are surgically removed and the cells were separated by mild homogenization . the pln cells thus obtained , typically between 10 - 50 × 10 6 cells / lymph node / mouse , are maintained in 5 - 10 ml of a solution such as click &# 39 ; s medium , 10 % fcs , 50 μm 2 - mercaptoethanol , and re - stimulated for 5 days , in vitro , with irradiated ( 3300 rads ) syngeneic mouse spleen cells pre - treated with the candidate peptide . a syngeneic cell expressing the parent protein from which the peptide is derived , such as the hiv gp160 protein , would be an appropriate target cell . however , the inventors contemplate that peptide - decorated spleen cells which can be easily generated , for example by pre - treating the cells with the peptide monomer at a concentration of approximately 40 μg / ml for 2 hours at 37 ° c ., may be preferred for use in accordance with the present invention . the re - stimulated effector cells are then washed , for example with rpmi 1640 medium , 10 % fcs , resuspended at an appropriate cell density in the region of 5 × 10 6 / ml , and assayed for ctl activity . a suitable method of assaying for ctl activity is to determine the release of a non - metabolisable radiolabeled substance from specific target cells which have been pre - loaded with the substance . measuring the release of 51 cr from peptide - treated syngeneic cells , as described by platsoucas & amp ; good ( 1981 ), is considered by the inventors to be a particularly suitable method . to generate peptide - expressing target cells for use in the ctl assay , appropriate syngeneic cells , such as p815 cells , are first loaded with radioactive compound such as chromium . this can be achieved by incubating the cells for 2 hours at 37 ° c . with 250 μci of 51 cr . the cells are then washed three times and incubated for an additional 2 hours at 37 ° c . with the monomeric form of the test peptide at a concentration of approximately 40 μg / ml . the cells are then washed twice with a solution such as rpmi medium , 10 % fcs , and then resupended at an appropriate concentration , for example in the region of 5 × 10 4 cells / ml . the target cells thus generated can then be employed in the ctl assay , for example by mixing them with the candidiate effector cells in u - bottom 96 well - microtiter plates such that different effector to target cells ( e : t ) ratios are achieved . alternatively , as discussed above , target cells expressing the parent protein from which the test peptide was derived can be generated and used in the ctl assay . in this method , syngeneic cells are infected for 18 - 20 hours with an appropriate amount , such as 5 × 10 7 plaque forming units , of recombinant vaccinia virus containing the gene for the parent protein which is to be expressed . as a control , syngeneic cells may be infected with recombinant vaccinia virus which carries the gene for an immunologically unrelated protein . at this stage one may wish to confirm the presence of the required protein in the target cells , but not in the control cells , for example by western blotting both infected cell types with specific antibodies . target cells created in this manner can then be labelled with the radioactive compound , in this case 100 μci of 51 cr , and used in the chromium release ctl assay ( platsoucas & amp ; good , 1981 ), as described above . in certain embodiments where the candidate peptides to be screened are derived from the hiv protein gp160 , the recombinant vaccinia virus system can be used to prepare cells which specifically express this protein . for use in this regard , the inventors have found the control ( vsc8 ) and hiv env - expressing ( vpe16 ) recombinant vaccinia viruses obtained through dr . bernard moss via the aids research and reference reagent program , ( division of aids , niaid , nih ) to be of particular use . to confirm the presence of the gp160 protein in such vpe16 - infected p815 target cells , the inventors found it convenient to employ hiv antibody - positive human sera in the western blotting experiments . in using the vpe16 - infected p815 target cells in ctl assays , the inventors determined that it was particularly advantageous to employ 5 × 10 4 target cells / ml and 1 × 10 6 candidate ctl effector cells / ml , mixed so as to obtain an initial e : t ratio of 20 : 1 . in ctl assays conducted using either of the above target cells , the percentage specific 51 cr - release can be calculated as : the maximum release can be determined by measuring the radioactivity released into the supernatant from target cells in which complete lysis has been experimentally induced , such as by using a 5 % solution of the non - ionic detergent triton x - 100 to disrupt the membranes of the cells . spontaneous release can be determined by measuring the radioactivity released into the supernatant from target cells incubated without added effector cells . it is contemplated that valid experiments will have a value for spontaneous target lysis which is between 15 - 20 % of the maximum lysis observed . prior to assaying the pln cells for possible ctl activity , one may desire to first deplete certain cell populations , such as cd4 + or cd8 + , from the mixture of cells . in this regard , re - stimulated pln cells can be treated with either anti - cd4 monoclonal antibody ( clone gk - 1 . 5 ) plus complement ( c ) (+ anti cd4 + c ), or anti - cd8 monoclonal antibody ( clone 53 - 6 . 72 ) plus c (+ anti cd4 + c ), as described by platsoucas & amp ; good ( 1981 ). it may also be desirable to treat certain pln cells with complement alone (+ c ), to act as a comparison . cells treated in any of these ways can then tested for their capacity to lyse mhc - matched target cells that have been pre - treated with the monomeric form of the peptide or infected with recombinant vaccinia virus expressing the parent protein from which the test peptide was derived . where one desires to identify ctl reactive compounds that do not exhibit an antibody generating capability , peptides identified as being t cell active may be screened to identify those that lack b cell stimulatory activity . such peptides are proposed to be particularly useful in the preparation of vaccines for the treatment or prevention of viral diseases such as aids where an antibody response may in fact enhance infectivity of the causative agent . identification of such peptides is accomplished by injecting a candidate into an immunocompetent animal ( e . g ., mice ) to identify those peptides that fail to generate a significant antibody response to the native protein to whose sequence the peptides correspond in part . for example , in the case of hiv , one would desire to test for the production of antibodies having reactivity against gp120 , gp41 or core proteins , etc . of course , as mentioned above , the invention also concerns the identification of compounds capable of eliciting both a t cell response and a b cell response , in that this latter group will serve to induce protective antibody - based immunity in the immunized host . ctl active compositions of the present invention prime t cells in a way that , when the infecting virus appears at a later date , memory t cells are activated to result in a cell - mediated immune response that destroys target cells that have the corresponding target protein or a portion thereof on their cell surfaces , and thereby the virus . thus , the present invention ultimately involves the preparation of ctl vaccines . peptide multimers may find particular advantages in the preparation of ctl vaccines in accordance herewith . preferred multimers are formed of surfactant - like micelles and polymers . in addition to the amino - terminal lysyl residue , the spacer peptide can contain one to about five additional residues . substantially any amino acid residue can be utilized so long as it does not interfere with the t cell immunizing capacity of an aqueous composition containing the multimer or with the capacity of the di - amide reaction product to form surfactant - like micelles in an aqueous composition . one to about three glycyl residues per spacer peptide are preferred . the before - described peptide and the amino - terminal lysyl residue - containing peptide spacer are peptide - bonded together , and can thus be viewed as a composite polypeptide . the useful diamide is thus a reaction product of the alpha - and epsilon - amino groups of the amino - terminal lysyl residue and two moles per composite polypeptide of the c 12 - c 18 fatty acid . the composite polypeptide can thus be prepared as a single sequence and amidified before or after removal from the resin , where solid phase synthesis is used , by conventional techniques . the phrase “ surfactant - like micelle ” is used herein to emphasize that , in an aqueous composition , the di - amidolysyl composite polypeptide appears to form micelles similar to those formed by surfactants and to distinguish such multimers from submicroscopic structural units of protoplasm built up from polymeric molecules that are also sometimes referred to as micelles . the word “ micelle ” is also sometimes used herein , and when so used has the same meaning as surfactant - like micelle . another multimer form of a previously described peptide is a polymer having a plurality of peptide repeating units . in this case , a peptide containing two terminal cysteines as part of its natural sequence can be selected and synthesized . a peptide lacking such cysteines can be modified by the addition of one or two extra cysteines to the n - and c - terminal ends , respectively . the presence of two cysteines per peptide permits polymerization of the subunit peptide by air oxidation to form oxidized cysteine ( cystine )- linked polymers and / or cyclic peptides . such multimers enhance immune recognition of the peptide without the need of a carrier . the lysine - terminated spacer peptide can contain one to about five amino acid residues in addition to the lysyl residue , and the one or two added terminal cysteine residues are not included in counting the length of a peptide of the present invention . a peptide containing terminal cysteine residues is referred to as a di - cysteine - terminated peptide or more simply , a di - cys peptide . details for preparing polymers containing di - cys peptide repeating units are provided hereinafter . an aqueous composition ( inoculum ) of the present invention comprises an immunologically effective amount of a peptide or peptides , whether in multimeric form or not , dissolved or dispersed in a pharmaceutically acceptable aqueous medium . such compositions are also referred to as inocula . the phrase “ pharmaceutically acceptable ” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human . the preparation of an aqueous composition that contains an immunizing molecule such as a peptide or multimer as an active ingredient is well understood in the art . typically , such compositions are prepared as injectables , either as liquid solutions or suspensions ; solid forms suitable for solution in , or suspension in , liquid prior to injection can also be prepared . the preparation can also be emulsified . a peptide , e . g ., a peptide multimer , can be formulated into a composition in a neutral or salt form . pharmaceutically acceptable salts , include the acid addition salts ( formed with the free amino groups of the peptide ) and which are formed with inorganic acids such as , for example , hydrochloric or phosphoric acids , or such organic acids as acetic , oxalic , tartaric , mandelic , and the like . salts formed with the free carboxyl groups can also be derived from inorganic bases such as , for example , sodium , potassium , ammonium , calcium , or ferric hydroxides , and such organic bases as isopropylamine , trimethylamine , histidine , procaine , and the like . upon vaccine formulation , it is administered in a manner compatible with the dosage formulation , and in such amount as is immunologically effective . by “ immunologically effective amount ” is meant an amount of composition is used that contains an amount of a peptide multimer sufficient to induce an effective ctl response in the host animal ( mammal ) such as by the induction of specifically targeted cytotoxic t cells . the presence of such cytotoxic t cells is assayed as discussed hereinafter . in the context of a vaccine , the quantity of peptide and volume of composition to be administered depends on the host animal to be immunized , the capacity of the host animal &# 39 ; s immune system to activate t cells , and the degree of protection desired . precise amounts of active peptide multimer required to be administered depend on the judgment of the practitioner and are peculiar to each individual . however , suitable dosage ranges are of the order of about 10 micrograms ( μg ) to about 500 milligrams ( mg ), preferably about 50 μg to about 1 mg , and more preferably about 100 μg of active ingredient peptide multimer per individual . a minimal volume of a composition required to disperse the immunizing amount of peptide multimer is typically utilized . suitable regimes for initial administration and booster shots are also variable , but are typified by an initial administration followed in one or two week intervals by a subsequent injection or other administration . a composition can also include an adjuvant as part of the excipient . adjuvants such as complete freund &# 39 ; s adjuvant ( cfa ), incomplete freund &# 39 ; s adjuvant ( ifa ) for use in laboratory host mammals are well known in the art , and are used illustratively herein . pharmaceutically acceptable adjuvants such as alum can also be used . synthetic peptides of 7 to about 30 amino acid residues in length were prepared corresponding to the selected conserved domains of the core and gp160 ( gp120 and gp41 ) molecules using the solid - phase technique of merrifield ( 1963 ) using a modified vega 250 automated peptide synthesizer or by the “ bag ” method described in houghten ( 1985 ). in either case , removal of the t - butyloxycarbonyl ( t - boc ) amino acid blocking groups and the hydrolysis of the peptide from the resin were carried accomplished by hydrofluoric acid ( hf ) treatment at 0 ° c . for one hour . the peptide - containing mixture was then extracted with diethyl ether to remove non - peptide organic compounds and the synthesized peptides were extracted from the resin with 25 % acetic acid ( w / v ). nineteen ( 19 ) synthetic peptides have been prepared that correspond to conserved domains of the gp120 molecule and the gp41 molecule by this procedure , and are listed in table 1 . the synthesized peptides correspond to designated conserved domains ( regions ) of gp160 in hiv as shown . 1 the n - and c - terminal amino acid residues of each peptide are numbered as to their position in the gp160 amino acid residue sequence according to modrow et al . virol ., 61 : 570 ( 1987 ). a dash (-) indicates that the sequence continues on the next line . two types of high molecular weight ( multimeric ) forms of the peptides listed in table 1 were prepared . the principal form of multimer was a di - cysteine ( di - cys terminated ) polymer in which a plurality of peptides were linked end - to - end by disulfide bonds . these di - cysteine polymers were produced by adding cysteine residues to the termini of each peptide during synthesis . the di - cysteine - terminated ( di - cys ) peptides were then dissolved ( 10 mg / ml ) in ammonium bicarbonate ( 0 . 1m ) at room temperature (− 25 ° c .) and stirred for about 16 hours to effect oxidation of the sulfhydryl groups to produce polymer forms of the peptides . the peptide solution was freeze - dried and analysed by hplc to confirm the presence of polymer forms of the peptide . the second type of high molecular weight form produced was a surfactant - like micelle formed by linkage of an amino - terminal lysine - containing spacer peptide ( lys - gly - gly -) to the peptide sequence to form a composite polypeptide , and then coupling a c 12 - c 18 fatty acid , such as palmitic acid , to both the alpha and epsilon amino groups , as in hopp ( 1984 ). the c 12 - c 18 fatty acid - containing peptides produced are then extracted in 95 % acetic acid and utilized to form large micelles in the aqueous composition that exhibit increased immunogenicity relative to the peptides . di - cys polymer multimers of all of the peptides listed in table 1 were prepared . aqueous peptide micelle multimers have been prepared of peptides designated 61 , 63 , 65 and 67 , and are designated as peptides 62 , 64 , 66 and 68 , respectively . peptides designated 103 through 117 were utilized only in their di - cys polymer multimeric forms . the high molecular weight , multimeric forms produced correspond to multiple copies of specific regions of gp120 and gp41 in hiv . for ease of designation , the multimer forms will be designated by the peptide number from which it is composed — that is , peptide 61 refers to the di - cys multimeric ( polymeric ) form of peptide 61 and peptide 66 refers to the aqueous micelle form of peptide 65 , whereas peptide 103 - 117 refers to a polymeric multimer . peptides 65 and 66 correspond to the region of gp120 that binds to the cell surface t4 receptor . peptides 61 and 62 correspond to a region near the amino - terminal portion of gp14 that represents a major immunodominant epitope seen by aids patients &# 39 ; sera . aqueous compositions of the multimers ; i . e ., the di - cys peptide polymers and micelles produced in example 1 were assayed for their ability , or lack of ability to elicit an anti - peptide antibody response in balb / c mice , an immunocompetent mouse strain . groups of balb / c mice ( 6 - 8 - week - old females , 3 to 5 mice / group , charles river laboratories ) were immunized by subcutaneous ( s . c .) or intraperitoneal ( i . p .) injection of a peptide multimer ( 100 μg / injection ) in complete freund &# 39 ; s adjuvant ( cfa ) ( 1 : 1 ratio ). booster injections ( 50 μg of peptide multimer ) in incomplete freund &# 39 ; s adjuvant ( ifa ) ( 1 : 1 ) were given at 6 and 10 weeks after the initial immunization . each mouse was bled from its retro - orbital plexus at two - week intervals and the serum was pooled for individual mice in each group . an elisa assay was performed on each serum to detect the presence of anti - peptide antibodies utilizing peroxidase - conjugated goat anti - mouse igg ( boehringer mannheim biochemicals , indianapolis , ind .) as the second antibody . preliminary results for peptides 61 - 68 are shown in table 2 , whereas further refined results for peptides 61 , 63 , 65 , 67 and 103 - 117 are shown in table 3 . it was found that the high molecular weight forms of peptides 65 , 66 , 67 , 68 , 105 to 110 , 112 , 114 , 115 and 117 elicited high antibody titers , whereas peptides 61 , 62 , 63 , 64 , 103 , 104 , 111 , 113 and 116 produced very low to negligible amounts of anti - peptide antibodies . similar results were obtained for antibody responses in b6c3 f1 mice ( charles river laboratories ), another immunocompetent strain . some of the sera were further assayed for antibody response ( reactivity ) with native gp160 , and the results , shown in table 4 , demonstrate that these peptides do not represent b cell epitopes since there was no immunoreaction with native gp160 . the high molecular weight , multimeric di - cys peptide polymeric forms of the peptides described in example 1 were assayed for their elicitation of a t cell proliferative response as in millich et al . ( 1985 ). mice ( 3 or 5 mice / group ) were injected in the right hind footpad with a 1 : 1 mixture of peptide polymer ( 100 μg / injection ) and cfa . peptides 61 , 63 , 65 and 67 were injected into b6c3 f1 mice ( h - 2 kxb , charles river laboratories ) and a . swxbalb / c f1 mice ( h - 2 sxd , jackson labs , bar harbor , me .). draining popliteal lymph node ( pln ) cells were harvested after ten ( 10 ) days , and cultured ( 2 × 10 5 cells / well ) in 96 - well microtiter plates in 0 . 2 ml of click &# 39 ; s medium ( click et al ., 1972 ) containing various concentrations of synthetic peptide , gp160 , an unrelated proteinaceous material or medium alone , for 96 hours at 37 ° c . in a humidified atmosphere of 5 % co 2 . during the final 16 - 18 hours of culturing , 3 h - thymidine ( 3 h - tdr ) ( 1 μci / well , 6 - 7 ci / mmole , icn radiochemicals ) was added . the cells were harvested onto filter strips and 3 h - tdr incorporation was monitored . the data are presented in fig1 , 3 , 4 and 5 . fig1 illustrates the results for peptides 61 , 63 , 65 , 67 in balb / c mice , and those results are expressed as a stimulation index ( si ) representing the fold increase in radioactivity counts in the presence of antigen compared to background values where no antigen was added . the si values with the different peptides were compared to that obtained with tuberculin purified protein derivative ( ppd ) as a positive control antigen . fig2 - 5 illustrate the peptide - specific 3 h - tdr incorporation for t cell responses ( delta cpm ) in mice with differing major histocompatibility ( mhc ) haplotypes , b6c3 f1 ( c57b1 / 6xc 3 h / hcj ) mice ( fig2 and 4 ) and ( a . swxbalb / c ) f1 mice ( fig3 and 5 ), for all of the synthetic peptides . the 3 h - tdr incorporation values represent the difference between the radioactivity values obtained in wells containing antigen and in control wells without added antigen . the non - specific proliferation of pln cells was determined by including an unrelated peptide in the assays , shown as a horizontal bar for each peptide . all of the assayed peptides exhibited good t cell proliferative responses in b6c3 f1 mice , whereas all of the assayed peptides , except peptides 105 , 107 , 109 and 111 , exhibited good t cell proliferative responses in a - swxbalb / c f1 mice . it was demonstrated by the results above and those described in example 2 that peptides 61 , 63 , 103 , 104 and 113 do not stimulate anti - peptide antibody production but are very good immunogens , in their disulfide ( di - cys ) polymeric form , for eliciting a strong t cell response directed against both the corresponding peptide and the native hiv envelope protein gp160 . t cell proliferation measured by 3 h - tdr incorporation , was also similarly assayed as a function of the t cell antigen concentration , using various amounts of native gp120 or gp160 as one control , and ppd as another control . pln from b6c3 f1 mice were used in these studies . the results for peptides 104 and 105 versus gp120 are shown in fig6 a and 6b , respectively ; those for peptides 61 and 63 versus gp160 are shown in fig7 a and 7b , respectively ; and those for peptides 65 and 111 versus gp120 are shown in fig8 a and 8b , respectively . groups of 3 to 5 syngeneic female mice ( 6 to 8 weeks of age ) are immunized by intradermal injection in an appropriate site with an aqueous composition containing an immunizing ( ctl - stimulating ) amount of peptide either as monomer or as the before - discussed multimers in cfa ( 1 : 1 ). ten ( 10 ) days after immunization , draining pln cells and spleen lymphocytes are obtained and restimulated in vitro by culturing for six ( 6 ) days with irradiated syngeneic normal spleen cells that were pre - treated with the same synthetic peptide as immunogen . the presence of cytotoxic t lymphocytes ( ctl ) is determined by a 4 - hour 51 cr release assay as follows . the pln cells are maintained for five days at 37 ° c . in clicks medium containing 10 % fetal calf serum ( fcs ) together with irradiated syngeneic normal spleen cells that were pre - treated with the approriate test peptide . these cells are designated as the effector cells , and express h - 2 d mhc class i antigen . target cells ( phytohemagglutinin - stimulated ( pha ) blasts of syngeneic mouse spleen cells or p815 mouse cells expressing a corresponding hiv protein ) are washed three times with serum - free rpmi 1640 medium and then admixed , contacted and maintained ( incubated ) at 37 ° c . for about 1 . 5 to about 2 hours together with 250 μci of sodium chromate ( specific activity 200 - 400 ci / g of 51 cr , new england nuclear , boston , mass .). the target cells are subsequently washed with rpmi 1640 medium containing 10 % fcs , and resuspended in rpmi 1640 with 10 % fcs and different concentrations of peptide . these cells are then washed 3 times with rpmi containing 10 % fcs and resuspended at 5 × 10 4 cells / ml . a 100 μl aliquot of each cell suspension is added to a well of a 96 - well - u - bottom microtiter plate . a 100 μl aliquot of the appropriate effector cell suspension ( 5 × 10 6 cells / ml ) is added to each well and a twofold serial dilution made to obtain different effector - to - target cell ( e : t ) ratios . control wells receive 0 . 1 ml of rpmi medium with 10 % fcs alone in the absence of effector cells to obtain a value for spontaneous 51 cr release , and receive 0 . 1 ml of 5 % triton x - 100 detergent to obtain a value for maximum 51 cr release . the plates are incubated at 37 ° c . for about 4 hours , following which 100 μl of supernatant from each well is monitored in a gamma counter to determine 51 cr release . the percent cytotoxicity is calculated as ( effector   cell - stimulated   release ) - ( spontaneous   release ) maximum   release - spontaneous   release × 100 example 5 — rapid assay of hiv - specific ctls induced by an immunodominant peptide . a peptide with the sequence riqrgpgrafvtigk ( herein referred to as r15k ), from the hiv gp160 immunodominant v3 - loop has previously been identified as a ctl immunodominant epitope in h - 2 d mice ( takahashi et al ., 1988 ). in the original studies ctls , induced in vivo by infecting balb / c mice with recombinant vaccinia virus expressing hiv env proteins , were shown to lyse syngeneic target cells pre - incubated with r15k . unfortunately , to date , immunizing mice with free r15k peptide has been unsuccessful in inducing ctls ( berzofsky , 1991 ). accordingly , the present inventors sought to examine the induction of cd8 + hiv r15k - specific ctls in 6 - 8 week old balb / c mice by employing differing sites of inoculation , differing forms of the peptide , and recovering the effector cells from different tissue origins . it was observed that draining popliteal lymph nodes ( pln ) of mice immunized in the hind - foot pad with 100 μg of the r15k peptide ( in a 1 : 1 emulsion with cfa ) were the best source of ctl effectors against irradiated ( 3300 rads ) syngeneic target cells preincubated with monomeric r15k ( 40 μg / ml for 2 h at 37 ° c .). this has particular importance and physiological significance because human lymph nodes have been described as the primary site of hiv replication ( kaneshima et al ., 1991 ; fauci 1991 ). an important aspect of this immunization protocol is that ctls could be recovered by mild homogenization from pln surgically removed after only 10 days . to determine the optimal form of the peptide for consistent induction of peptide - specific ctls in vivo , the r15k peptide was prepared in three different configurations : a ) linear monomer , b ) disulphide - linked polymer formed by oxidation of cysteine residues added at both the n - and c - termini and c ) micelles formed by conjugating the peptide to a dipalmityl - lysine - glycine - glycine at the n - terminus ( hopp , 1984 ; sastry and arlinghaus , 1991 ). it was found that a single immunization of balb / c mice in the hind - foot pad with any of the above r15k forms in cfa consistently resulted in generation of ctls which specifically lysed mhc - matched target cells ( p815 , h - 2 d ) pre - incubated with the peptide . such responses were observed in 8 of 12 mice immunized with the monomeric form , in 13 of 13 mice immunized with the micelle form and 6 of 8 mice immunized with the disulfide polymer form of the peptide . lysis of mhc - matched target cells without peptide pre - treatment ( p815 ) was not observed . representative results are shown in fig9 . the ctls induced by all three forms of the peptide also specifically lysed p815 cells infected with a recombinant vaccinia virus expressing hiv gp160 ( vpe16 ), but not cells infected with a control vaccinia virus ( p815 + vsc8 ) ( fig1 ). western blotting with hiv antibody - positive human sera confirmed the presence of gp160 protein in vpe16 - infected , but not in vsc8 - infected p815 target cells . the peptide - induced ctls in balb / c mice were h - 2 restricted . they lysed only peptide pre - treated h - 2 d target cells ( p815 ) but not peptide - treated 3a9 target cell , which are expressing the h - 2k haplotype ( table 5 ). representative results with ctls generated in mice immunized with the micelle form of r15k peptide are shown in table 5 . similar results were obtained with ctls induced by injection of the monomeric and polymeric forms of the r15k peptide . experiments were performed to determine whether the virus - specific ctls were cd8 + or cd4 + . treatment of the ctl effectors from mice immunized with monomeric peptide with anti - cd8 monoclonal antibodies ( mabs ) and rabbit complement abolished the cytotoxicity against peptide treated , or env expressing , targets ( fig1 a ). in contrast , pre - treatment of effector cells with anti - cd4 mabs 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 ( fig1 b ) and polymeric forms . the induction of cd8 + ctls by r15k is consistent with the use of p815 target cells which express mhc class i , but not class ii , gene products ( maryanski et al ., 1985 ). mhc class i - restriction is commonly observed with cd8 + effector ctls . since the ctl epitope studied in the present investigation is in the middle of an immunodominant b - cell active region of the hiv gp120 , the b - cell activity of the r15k peptide was investigated . either as monomer or lipid tailed micelle or after conjugation to klh , this peptide failed to induce in mice a measurable titer of anti - peptide antibody . however , the mice immunized with peptide conjugated to klh did make antibodies against klh , showing that the mice are immunocompetent . sera from mice immunized in the foot pad were also tested , when it was observed that no anti - peptide antibodies were formed . despite the results presented immediately above , the location of r15k in a variable region ( v3 - loop ) of hiv gp160 could be viewed as a reason for not selecting this peptide as a potential vaccine candidate . however , a comparison of gp160 amino acid sequences from 245 different hiv isolates has shown that as little as five different consensus sequences can be defined on a serological basis among all the viral isolates ( larosa et al ., 1990 ). therefore , the inventors propose that a cocktail of ctl - inducing peptides from v3 - loop regions encompassing all the principal hiv groups ( which may be five or less ) may be sufficient for generating ctls specific for cells expressing gp120 from most if not all hiv strains . such a mixture would then serve as a prototype vaccine for evaluation for prevention of hiv infection of humans . the protocol developed for the induction of hiv - specific ctls was believed to be generally applicable to the identification , selection and assay of any peptide with unknown epitope specificity , for its ability to prime ctls in vivo . accordingly , the in vivo peptide - induction of ctls specific for influenza virus was examined . deres et al ., ( 1989 ) had previously shown that a synthetic peptide r − , tyqrtralvtg ( aa 147 - 158 ), corresponding to a portion of the nucleoprotein of influenza virus , could prime influenza virus - specific ctls in mice in vivo only when covalently linked through the n - terminus to tripalmitoyl - s - glycerylcysteinyl - seryl - serine ( p 3 css ). however , using the protocol described above , specific cd8 + ctls , that lysed target cells pre - treated with this peptide , could be induced in vivo by immunisation with the free synthetic peptide ( table 6 ). thus , this method was indeed found to be useful in systems other than those related to the hiv virus . furthermore , it is believed that this rapid screening method will have medical utility for developing candidate vaccines and therapeutics for various infectious diseases . changes may be made in the construction , operation and arrangement of the various parts , elements , steps and procedures described herein without departing from the concept and scope of the invention as defined in the claims . the following references are hereby incorporated by reference for the subject matter as specified in the specification and to the extent that they disclose , teach , enable or provide a basis for various aspects of the present invention . ahearne et al ., iii international conference on aids , held in washington , d . c ., jun . 1 - 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