Patent Application: US-76346201-A

Abstract:
a method of vaccinating a mammal against a disease state , comprising administrating to said mammal , within an appropriate vector , a nucleotide sequence encoding an antigenic peptide associated with the disease state ; additionally administering to said mammal a compound which enhances both humoral and cellular immune responses initiated by the antigenic peptide , the compound being selected from the list contained herein , wherein the compound is preferably tucaresol or a physiologically acceptable salt or ester thereof , where appropriate .

Description:
throughout this specification and the appended claims , unless the context requires otherwise , the words “ comprise ” and “ include ” or variations such as “ comprising ”, “ comprises ”, “ including ”, “ includes ”, etc ., are to be construed inclusively , that is , use of these words will imply the possible inclusion of integers or elements not specifically recited . as described above , the present invention relates to vaccination methods , and in particular to improvements of methods of vaccination involving the introduction into a mammal of dna which encodes for an antigenic protein or peptide , such that the protein or peptide will be expressed within the mammalian body to thereby induce an immune response within the mammal , against the antigenic protein or peptide . such techniques are well known and are fully described in ( 1 ) as referred to above . it is possible for the vaccination methods according to the present application to be adapted for protection of mammals against a variety of disease states such as , for example , viral , bacterial or parasitic infections , cancer , allergies and autoimmune disorders . some specific examples of disorders or disease states which can be protected against or treated by using the methods or compositions according to the present invention , are as follows : hepatitis viruses a , b , c , d & amp ; e , hiv , herpes viruses 1 , 2 , 6 & amp ; 7 , - cytomegalovirus , varicella zoster , papilloma virus , epstein barr virus , influenza viruses , para - influenza viruses , adenoviruses , coxsakie viruses , picorna viruses , rotaviruses , respiratory syncytial viruses , pox viruses , rhinoviruses , rubella virus , papovirus , mumps virus , measles virus . mycobacteria causing tb and leprosy , pneumocci , aerobic gram negative bacilli , mycoplasma , staphyloccocal infections , streptococcal infections , salmonellae , chlamydiae . breast cancer , colon cancer , rectal cancer , cancer of the head and neck , renal cancer , malignant melanoma , laryngeal cancer , ovarian cancer , cervical cancer , prostate cancer . rhinitis due to house dust mite , pollen and other environmental allergens it is to be recognised that these specific disease states have been referred to by way of example only , and are not intended to be limiting upon the scope of the present invention . the dna sequences referred to in this application , which are to be expressed within a mammalian system , in order to induce an antigenic response , may encode for an entire protein , or merely a shorter peptide sequence which is capable of initiating an antigenic response . throughout this specification and the appended claims , the phrase “ antigenic peptide ” is intended to encompass all peptide or protein sequences which are capable of inducing an immune response within the animal concerned . most preferably , however , the dna sequence will encode for a full protein which is associated with the disease state , as the expression of full proteins within the animal system are more likely to mimic natural antigen presentation , and thereby evoke a full immune response . some non - limiting examples of known antigenic peptides in relation to specific disease states include the following : hiv — gp120 gp40 , gp160 , p24 , gag , pol , env , vif , vpr , vpu , tat , rev , nef hsv — gl , gh , gm , gb , gc , gk , ge , gd , icp47 , icp36 , icp4 tb — mycobacterial super oxide dismutase , 85a , 85b , mpt44 , mpt59 , mpt45 , hsp10 , hsp65 , hsp70 , hsp90 , ppd 19 kda ag , ppd 38 kda ag . in order to obtain expression of the antigenic peptide within mammalian cells , it is necessary for the dna sequence encoding the antigenic peptide to be presented in an appropriate vector system . for example , the vector selected may comprise a bacterial plasmid and a strong viral promoter and polyadenylation / transcriptional termination sequence arranged in the correct order to obtain expression of the antigenic peptides . the construction of vectors which include these components and optionally other components such as enhancers , restriction enzyme sites and selection genes , such as antibiotic resistance genes , is well known to persons skilled in the art and is explained in detail in maniatis et al ( 21 ). as it is important to prevent the plasmids replicating within the mammalian host and integrating within the chromosomal dna of the animal , the plasmid will preferably be produced without an original of replication that is functional in eukaryotic cells . the methods and compositions according to the present invention can be used in relation to prophylactic or treatment procedures of all mammals including , for example , domestic animals , laboratory animals , farm animals , captive wild animals and most preferably , humans . the compounds recited above which have been identified by the present inventors as exhibiting the favourable activity of enhancing both humoral and cellular immunogenic activity initiated by dna vaccine administration are known compounds , previously reported in wo94 / 07479 as having immunopotentiatory properties . in particular , the preferred compound 4 -( 2 - formol - 3 - hydroxyphenoxymethyl ) benzoic acid , which is also known as tucaresol , was originally described in ep 0054924 and has been reported as having immunopotentiatory activity and as being useful for treatment of various disorders including hiv , hbv , hcv , tumours and sickle cell anaemia . it is thought that the reported activity of tucaresol can be explained by its ability to form schiff bases and that it can thereby substitute for physiological donors or carbonyl groups and provide a co - stimulatory signal to cd4 th - cells . it was previously reported that tucaresol enhanced cd4 th - cell response , selectively favouring a th 1 - type profile over th 2 ( 22 ), whereas the present inventors have demonstrated that it is capable of enhancing both th 1 and th 2 isotypes of antibody in a murine model . by referring to enhancement of both humoral and cellular immune responses initiated by the antigenic peptide , and caused by the compounds of the present invention , it is intended to convey that both serum antibody levels and cytotoxic t lymphocyte ( ctl ) levels respectively will be raised as a result of administration of the compounds , compared to levels associated with administration of dna sequence encoding for the antigenic peptide alone . such levels can be quantified by methods well known in the art , as will be further explained in the appended examples . the vectors which comprise the dna sequences encoding antigenic peptides can be administered in a variety of manners . it is possible for the vectors to be administered in a naked form ( that is as naked dna not in association with liposomal formulations , with viral vectors or transfection faciltating proteins ) suspended in an appropriate medium , for example a buffered saline solution such as pbs and then injected intramuscularly , subcutaneously , intraperitonally or intravenously , although some earlier data suggests that intramuscular or subcutaneous injection is preferable ( 23 ), ( the disclosure of which is included herein in its entirety by way of reference ). it is additionally possible for the vectors to be encapsulated by , for example , liposomes or within polylactide co - glycolide ( plg ) particles ( 25 ) for administration via the oral , nasal or pulmonary routes . it is also possible , according to a preferred embodiment of the invention , for intradermal administration of the vector , preferably via use of gene - gun ( particularly particle bombardment ) administration techniques . such techniques may involve lyophilisation of a suspension comprising the vector and subsequent coating of the vector on to gold beads which are then administered under high pressure into the epidermis , such as , for example , as described in ( 26 ). the amount of dna delivered will vary significantly , depending upon the species and weight of mammal being immunised , the nature of the disease state being treated / protected against , the vaccination protocol adopted ( i . e . single administration versus repeated doses ), the route of administration and the potency and dose of the adjuvant compound chosen . based upon these variables , a medical or veterinary practitioner will readily be able to determine the appropriate dosage level . it is possible for the dna vector , including the dna sequence encoding the antigenic peptide , to be administered on a once off basis or to be administered repeatedly , for example , between 1 and 7 times , preferably between 1 and 4 times , at intervals between about 1 day and about 18 months . once again , however , this treatment regime will be significantly varied depending upon the size and species of animal concerned , the disease which is being treated / protected against , the amount of dna administered , the route of administration , the potency and dose of adjuvant compound selected and other factors which would be apparent to a skilled veterinary or medical practitioner . the adjuvant compound specified herein can similarly be administered via a variety of different administration routes , such as for example , via the oral , nasal , pulmonary , intramuscular , subcutaneous , intradermal or topical routes . this administration may take place between about 14 days prior to and about 14 days post administration of the dna sequence , preferably between about 7 days prior to and about 7 days post administration of the dna sequence , more preferably between about 24 hours prior to and 24 hours post administration of the dna sequence , and particularly preferably , substantially simultaneous with administration of the dna sequence . by “ substantially simultaneous ” what is meant is that administration of the compound is preferably at the same time as administration of the dna sequence , or if not , at least within a few hours either side of dna sequence administration . in the most preferred treatment protocol , the compound will be administered substantially simultaneously to administration of the dna sequence , and then again on approximately a daily basis for up to 14 days post dna sequence administration , preferably daily for the 3 days following initial administration . obviously , this protocol can be varied as necessary , in accordance with the type of variables referred to above . once again , depending upon such variables , the dose of administration will also vary , but may , for example , range between about 0 . 1 mg per kg to about 100 mg per kg , where “ per kg ” refers to the body weight of the mammal concerned . this administration of the adjuvant compound would preferably be repeated with each subsequent or booster administration of the dna sequence . most preferably , the administration dose will be between about 0 . 1 mg per kg to about 10 mg per kg , preferably between about 1 mg per kg and about 5 mg per kg . while it is possible for the adjuvant compounds to be administered in the raw chemical state , it is preferable for administration in the form of a pharmaceutical composition . that is , the compounds will preferably be combined with one or more pharmaceutically or veterinarily acceptable carriers , and optionally other therapeutic ingredients . the carrier ( s ) must be “ acceptable ” in the sense of being compatible with other ingredients within the formulation , and not deleterious to the recipient thereof . the nature of the formulations will naturally vary according to the intended administration route , and may be prepared by methods well known in the pharmaceutical art . all methods include the step of bringing into association a compound of the invention ( the adjuvant compound ) with an appropriate carrier or carriers . in general , the formulations are prepared by uniformly and intimately bringing into association the compound with liquid carriers or finely divided solid carriers , or both , and then , if necessary , shaping the product into the desired formulation . formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules , cachets or tablets each containing a pre - determined amount of the active ingredient ; as a powder or granules ; as a solution or a suspension in an aqueous liquid or a non - aqueous liquid ; or as an oil - in - water liquid emulsion or a water - in - oil emulsion . the active ingredient may also be presented as a bolus , electuary or paste . a tablet may be made by compression or moulding , optionally with one or more accessory ingredients . compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free - flowing form such as a powder or granules , optionally mixed with a binder , lubricant , inert diluent , lubricating , surface active or dispersing agent . moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent . the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient . formulations for injection via , for example , the intramuscular , intraperitonile , or subcutaneous administration routes include aqueous and non - aqueous sterile injection solutions which may contain antioxidants , buffers , bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient ; and aqueous and non - aqueous sterile suspensions which may include suspending agents and thickening agents . the formulations may be presented in unit - dose or multi - dose containers , for example , sealed ampoules and vials , and may be stored in a freeze - dried ( lyophilised ) condition requiring only the addition of the sterile liquid carrier , for example , water for injections , immediately prior to use . extemporaneous injection solutions and suspensions may be prepared from sterile powders , granules and tablets of the kind previously described . formulations suitable for pulmonary administration via the buccal or nasal cavity are presented such that particles containing the active ingredient , desirably having a diameter in the range of 0 . 5 to 7 microns , are delivered into the bronchial tree of the recipient . possibilities for such formulations are that they are in the form of finely comminuted powders which may conveniently be presented either in a piercable capsule , suitably of , for example , gelatine , for use in an inhalation device , or alternatively , as a self - propelling formulation comprising active ingredient , a suitable liquid propellant and optionally , other ingredients such as surfactant and / or a solid diluent . self - propelling formulations may also be employed wherein the active ingredient is dispensed in the form of droplets of a solution or suspension . such self - propelling formulations are analogous to those known in the art and may be prepared by established procedures . they are suitably provided with either a manually - operable or automatically functioning valve having the desired spray characteristics ; advantageously the valve is of a metered type delivering a fixed volume , for example , 50 to 100 μl , upon each operation thereof . in a further possibility , the active ingredient may be in the form of a solution for use in an atomiser or nebuliser whereby an accelerated airstream or ultrasonic agitation is employed to produce a find droplet mist for inhalation . formulations suitable for intranasal administration generally include presentations similar to those described above for pulmonary administration , although it is preferred for such formulations to have a particle diameter in the range of about 10 to about 200 microns , to enable retention within the nasal cavity . this may be achieved by , as appropriate , use of a powder of a suitable particle size , or choice of an appropriate valve . other suitable formulations include coarse powders having a particle diameter in the range of about 20 to about 500 microns , for administration by rapid inhalation through the nasal passage from a container held close up to the nose , and nasal drops comprising about 0 . 2 to 5 % w / w of the active ingredient in aqueous or oily solutions . examples of appropriate formulations which comprise the adjuvant compounds according to the present invention are provided within wo94 / 07479 , the disclosure of which is included herein by reference , in its entirety . in a preferred embodiment of the invention , it is possible for the vector which comprises the dna sequence encoding the antigenic peptide to be administered within the same formulation as the adjuvant compound . in a particularly preferred embodiment the adjuvant compound is prepared in a form suitable for gene - gun administration , and is administrated via that route substantially simultaneous to administration of the dna sequence . for preparation of formulations suitable for use in this manner , it may be necessary for the adjuvant compound to be lyophilised and adhered onto , for example , gold beads which are suited for gene - gun administration . even if not formulated together , it may be appropriate for the adjuvant compounds to be administered at or about the same administration site as the dna sequence . other details of pharmaceutical preparations can be found in ( 24 ), the disclosure of which is included herein in its entirety , by way of reference . the present invention will now be described further , with reference to the following non - limiting examples : lymphoid cells from tcr - transgenic mice , expressing a receptor specific for an ovalbumin antigen were transferred into normal syngeneic mice . these mice were then immunised subcutaneously with ovalbumin peptide with or without adjuvants . three days later , regional lymph node cells were removed and their proliferative response to ovalbumin peptide was measured by means of incorporation of tritiated thymidine into dna . this provides a measure of the degree of specific t - cell priming that occurred in vivo in response to immunisation . immunisation with ovalbumin peptide alone (◯) resulted in a significant but low level of t - cell priming compared with mock immunisation (●). bacterial lipopolysaccharide ( lps ) ( ), complete freunds adjuvant ( cfa ) (□), and bacillus calmette geurin ( hk - bcg ) (⊕), all provided significant enhancement of this response . in the absence of an immunising antigen , administration of bacterial lipopolysaccharide ( lps ) (▴), complete freunds adjuvant ( cfa ) (▪), and bacillus calmette geurin ( hk - bcg ) (▾), had minimal effects on subsequent proliferation in response to ova peptide . lymphoid cells from tcr - transgenic mice , expressing a receptor specific for an ovalbumin antigen were transferred into normal syngeneic mice . these mice were then immunised subcutaneously ( by means of gene gun ) with plasmid dna ( pvac1 ) or with plasmid dna construct encoding ovalbumin ( pvac1 . ova ) with or without adjuvants given subcutaneously . three days later , regional lymph node cells were removed and their proliferative response to ovalbumin peptide was measured by means of incorporation of tritiated thymidine into dna . this provides a measure of the degree of specific t - cell priming that occurred in vivo in response to immunisation . a control group were immunised with ovalbumin peptide in complete freunds adjuvant which produced substantial t - cell priming (♦). dna immunisation with pvac1 . ova ( ) produced significant t - cell priming compared with immunisation with the empty vector (◯). however , in contrast to the conventional peptide immunisation , none of the adjuvants were able to enhance this response to dna immunisation . lps (▴), heat - killed listeria monocytogenes ( hklm ) (▾), gm - csf (⋄), and complete freunds adjuvant ( cfa ) (⊕), all failed to enhance the response to dna vaccination . [ an anomalous high response was seen in with the empty vector control using hklm as adjuvant ]. the effect of tucaresol on immunisation with a plasmid dna coding for the mycobacterial heat shock protein 65 ( m . hsp65 ) antigen was analysed , and compared the effect of tucaresol to that of plasmids expressing the cytokines gm - csf and ifnγ . groups of mice were immunised intramuscularly ( i . m .) with 20 μg of a plasmid ( p3 ). significant amounts of antibodies to m & gt ; hsp65 could be detected in sera from p3m . 65 immunised mice , but not in the p3 immunised ones ( fig3 a ). the antibody titres were markedly increased when 1 mg of tucaresol was administered subcutaneously simultaneously with the m . hsp plasmid 9p3m . 65 , t ). in contrast , no increase in the specific antibody response was detected in a group of mice immunised with the control plasmid and tucaresol ( p3 , t ), excluding the possibility that a general increase in non - specific cross - reactive antibodies due to the high degree of immuno - potentiation associated with tucaresol administration accounted for the observed effect . ( fig3 a ). we also compared in the same experiment the effect of tucaresol with that of injecting plasmids expressing the cytokines gm - csf and ifnγ . the p3m . 65 plasmid was administered alone or in equimolar combination with either a gm - csf expressing plasmid ( p3m . 65 , g ), an ifnγ expressing plasmid ( p3m . 65 , γ ), or a mixture of gm - csf and ifnγ expressing plasmids ( p3m . 65 , gγ ). the anti - m . hsp65 titres were markedly increased when the gm - csf plasmid was included in the immunisation as compared with to p3m65 . in contrast to the potentiating effect of the gm - csf plasmid on the m . hsp65 specific antibody response , there was insignificant antibody response when the ifnγ expressing plasmid was included in line with previously published data . combining both cytokine plasmids seemed to antagonise the enhancing effect of the gm - csf plasmid , reducing the response to levels essentially similar to that observed when immunising with p3m . 65 only ( data not shown ). collectively , these results demonstrate that tucaresol has a potent capacity to enhance the antibody response induced by genetic immunisation with the m . hsp65 antigen , comparable to the effect of the gm - csf plasmid . we have analysed the isotype of the anti - m . hsp65 antibodies . p3m . 65 immunised mice produced significant amounts of igg2a anti - m . hsp65 antibodies . this titre was significantly ( p = 0 . 003 ) increased in mice receiving tucaresol ( fig3 b ). this clear enhancement of a th1 associated antibody response was unique as immunisation with p3m . 65 , g , p3m . 65γ or p3m . 65 gγ could not exert such an effect . the inclusion of the ifnγ expressing plasmid in the immunisation did not enhance the igg2a anti - m . hsp65 antibody response . a th2 associated anti - m . hsp65 igg1 antibody response could not be detected in a significant amount of mice receiving p3m . 65 , g , p3m . 65γ or p3m . 65 gγ . this response was induced in a significant amount ( p = 0 . 003 ) however , in mice receiving p3m . 65 , t and to a lesser extent ( p = 0 . 027 ) in pm . 65g immunised mice as compared to p3 immunised mice ( fig3 c ). taken together , our data prove that a significant increase in the specific antibody response as a result of genetic immunisation could be achieved by the administration of tucaresol . although this administration strongly enhanced the th1 associated response , it did also induce a th2 associated antibody response . this is the first time tucaresol is reported to enhance the production of a specific antibody response . we next analysed the effect of tucaresol on the proliferative t - cell response induced by vaccination with pdna expressing the epstein barr virus ( ebv ) nuclear antigen number 4 ( ebna - 4 ). groups of mice were immunised i . m . with control plasmid p3 or with the ebna - 4 expressing plasmid ( e4 ) or with e4 plus treatment with tucaresol ( e4 , t ). a minimal proliferative response was detected in the splenocyte cultures from e4 immunised mice when stimulated with the syngeneic ebna - 4 transfected carcinoma line ( s6c - e4 ). interestingly , a much stronger proliferative response to s6c - e4 was obtained with splenocytes from mice immunised i . m . with the e4 and treated s . c . with tucaresol ( fig4 ). ebna - 4 vaccinia infected stimulators ( s6c - ve4 ) also induced a higher proliferative response than s6c - e4 in the splenocytes from both e4 immunised and the e4 , t immunised mice as previously reported . proliferation was calculated as simulation index ( si ) using the formula : si = splenocyte proliferation towards s6c - ebna - 4 transfectant ( ebna - 4 vaccinia infected )/ splenocyte proliferation towards s6c - gpt ( tk - vacccinia infected ) control transfectant . again , this response was antigen focused as there was no detectable proliferation in the splenocytes from control immunised mice ( p3 ) towards s6c - e4 or ebna - 4 vaccinia infected cells above that toward s6c - gpt or control tk - vaccinia infected s6c - gptv respectively ( fig4 ). pdna immunisation is associated with a predominant th - 1 response as characterised by production of th1 cytokines including ifnγ . to assess the capacity of tucaresol to enhance this th - 1 response , mice were immunised i . m . with p3 , e4 or e4 with simultaneous administration of tucaresol s . c . little , if any ifnγ was produced by splenocytes from mice immunised with e4 only in response to specific stimulation with s6c - e4 as compared to production from control splenocytes from p3 immunised mice or the production in response to s6c - gpt . interestingly , splenocytes from form mice immunised with e4 and treated with tucaresol produced the highest amounts of ifnγ in response to specific in vitro stimulation with s6c - e4 but did not in response to control stimulation with s6c - gpt . we were unable to detect the th2 cytokine il - 4 production in response to specific stimulation in splenocytes cultures from any group ( data not shown ). we therefore conclude that tucaresol administration together with pdna vaccination is a highly efficient way of promoting a specific th1 dominated cytokine response . to investigate the effect of tucaresol on the specific cytotoxic t - cell ( ctl ) response induced by pdna vaccination , we have immunised hla a2 transgenic mice twice with either p3 m . 65 , p3m . 65γ or p3m . 65 , t . two weeks after the last immunisation splenocytes form the immunised mice were stimulated once with the hla - a2 restricted peptide epitope derived from the mycobacterial hsp65 molecule , and the cultures tested for specific ctl activity against the hla - a2 / kb jurkat ( jk - a2 / kbds ) cell line unpulsed or pulsed with the cognate peptide or with a control hla a2 restricted influenza peptide . splenocytes from mice immunised with p3m . 65 and tucaresol ( p3m . 65 , t ) developed high ctl activity against target cells pulsed with the cognate m . hsp65 epitope while their lytic activity against jk - a2 / kb cells unpulsed or pulsed with the control influenza peptide was much lower ( fig6 ). in contrast , splenocytes from mice immunised with the p3m . 65 without any co - stimulatory agent were almost totally inactive . the inclusion of the ifnγ plasmid together with the p3m . 65 vaccine ( p3m . 65γ ) enhanced the ctl activity of the splenocytes as compared to the activity of splenocytes derived from the mice immunised with p3m . 65 only , but this cytotoxicity was only 30 – 50 % of that observed with splenocytes form mice treated with tucaresol . we therefore conclude that tucaresol is a very efficient agent to enhance the development of specific ctl when given together with pdna vaccination . effect of tucaresol on tumour outgrowth inhibition in vivo following immunisation with a plasmid expressing the epstein barr virus nuclear antigen 4 ( ebna - 4 ) epstein barr virus has been implicated in some cancers . we analysed the effect of tucaresol on tumour outgrowth inhibition in vivo as a result of pdna vaccination with a plasmid expressing ebna - 4 ( fig7 ). mice were immunised by intramuscular injection with 40 μg of either control mock plasmid pcdna3 ( p3 ), plasmid expressing ebna - 4 ( e4 ) or plasmid expressing ebna - 4 followed 1 , 2 , 3 and 4 days later with administration of 200 μg of tucaresol s . c .— i . e . 800 μg total tucaresol per mouse ( e4t ). this immunisation schedule was repeated 1 month and 2 months after the initial immunisation . two weeks after the last immunisation mice were challenged with 104 s6c - e4 tumour cells s . c . mice were sacrificed when the tumour reached a 20 mm diameter . fig7 shows that tucaresol significantly enhances the ability of ebna - 4 to inhibit tumour outgrowth . effect of tucaresol on ctl cytokine response induced by gene gun dna immunisation in mice . the production of interferon gamma ( ifnγ ) by cytotoxic t lymphocytes ( ctl ) is a key measure of cell - mediated immune responses important in the elimination of viral infection . c57bl / 6 mice were immunised by gene gun with dna plasmids encoding a / pr8 / 34 influenza virus nucleoprotein ( pvac1 . pr ) at two dose levels ( 10 & amp ; 100 ng ) or empty vector . spleens were collected 14 days post immunisation and splenocytes re - stimulated in vitro with an np peptide ( 10 μm ), recognised only by cd8 cytotoxic t - cells , together with recombinant human il - 2 ( 50 ng / ml ). ifnγ positive cells per 10e6 splenocytes were detected by elispot assay which measures the number of individual cells producing cytokine ( mean ± s . e . m . ; n = 3 mice ). tucaresol was administered to mice at the time of immunisation either subcutaneously ( s . c .) ( 2 × 1 mg ) at the site of intra - epidermal dna vaccination , or by oral gavage ( 15 mg / kg ) daily for 5 days beginning on the day of immunisation . subcutaneous tucaresol produces a small but significant increase in the ctl cytokine response to immunisation while oral tucaresol produces a doubling of the response . using epstein barr virus nuclear antigen 4 ( ebna - 4 ) ( fig8 b ) mice were immunised by gene gun with dna plasmids encoding ebna - 4 ( e4 ) or empty vector ( p3 ). 2 ug of dna was administered per shot , giving two non overlapping shots per mouse . tucaresol was administered to the mice ( e4t ) in 200 ug amounts subcutaneously on days 1 , 2 , 3 and 4 . mice were boosted after two months with the same immunisation and treatment schedule . two weeks later splenocytes were stimulated in vitro with tumour cells expressing the ebna - 4 antigen ( s6c - e4 ) or with antigen negative tumour cells ( s6c - gpt ) for 72 h . supernatants were then collected and ifn - y titres were determined by specific elisa . tucaresol dramatically increased the production of ifnγ . effect of tucaresol on lytic ctl response induced by gene gun dna immunisation lysis of target cells by cd8 ctl is a principal mechanism in the elimination of viral infections by the immune system . lytic ctl can be measured using target cells carrying viral peptide and labelled with europium . c57bi / 6 mice were immunised with plasmid dna ( 10 ng ) encoding the influenza virus nucleoprotein ( np ) with and without tucaresol given sub - cutaneously ( sc ). mice were killed 14 days post - immunisation and restimulated in - vitro ( 5 days ) with irradiated splenocytes pulsed with virus ( a / pr8 / 34 ). standard europium release techniques were used to determine specific lysis of mhc - matched target cells ( el4 cells ) pulsed with h - 2 d b - restricted np peptide . non - specific lysis was less than 15 % for all controls . tucaresol was given subcutaneously ( 1 mg ) at the site of intrepidermal gene - gun immunisation . c57b1 / 6 mice were immunised with plasmid dna ( 10 ng ) encoding the influenza virus nucleoprotein ( np ) with and without tucaresol given orally . mice were killed 14 days post - immunisation and restimulated in - vitro ( 5 days ) with irradiated splenocytes pulsed with virus ( a / pr8 / 34 ). standard europium release techniques were used to determine specific lysis of el4 cells pulsed with h - 2d b - restricted np peptide . non - specific lysis was less than 15 % for all controls . tucaresol ( 15 mg / kg ) was given by oral gavage once daily for 5 days beginning on the day of immunisation . we introduce herein a simple and very effective approach to enhance pdna immunisation , based on providing a co - stimulatory signal to t - cells via schiff base formation . several of our observations demonstrate that this method is able to circumvent the problem of limitation in efficacy which commonly is encountered as a result of pdna vaccination . these include an observed induction of a specific immune response in the majority of the immunised animals , while the approaches of using cytokine encoding vectors was considerably less efficient in that regard ( data not shown ). the enhancement was also associated with a significant quantitative increase in the response as compared to giving the pdna vaccination alone or in combination with the pdna expressing cytokine genes , and was observed both as increased specific antibody titres and as enhanced proliferative and cytotoxic t - cell responses . the induction of an adequate immune response requires the participation of multiple components of the immune system , and pdna immunisation fulfils this requirement as it induces both humoral and cellular responses including ctl responses , all of which were found to be enhanced by tucaresol . moreover , while other modes of enhancing pdna immunisation will lead to an antibody of a t - cell biased immune response , co - injection of tucaresol resulted in a general enhancement of both types of specific immunity including the enhancement of th1 and th2 associated antibody responses . the combination of pdna vaccination and tucaresol can therefore be considered in conditions where either a cellular of antibody based immune response would be beneficial for the host . there was a marked increase in the production of specific antibody production as also measured by the highest igg : igm ratio as compared to the other immunisation procedure ( data not shown ). this points out the potential advantage of using this procedure during the production of monoclonal antibodies . the marked ability of tucaresol to enhance pdna induced specific t - cell responses to hsp65 , influenza np and ebna - 4 , as detected by proliferation , cytokine production and cytotoxicity , is of particular importance . protective immunity to mycobacterial infection is dependent on both cd4 + t - cells with the capacity to secrete macrophage activating cytokines , including ifnγ and on cytotoxic cd8 + cells which can eliminate infected macrophages . protective immunity to ebv infection is dependent on cd4 + and cd8 + t - cell responses , and t - cell based immunotherapy against post - transplant transplant lymphoproliferative disorders has already proven to be efficient . since pdna vaccination combined with tucaresol favors both cd4 and cd8 mediated responses , as shown here , this is an attractive mode of vaccination to be applied in new t - cell vaccines against intra - cellular bacteria and viruses . tucaresol is a chemically well - defined molecule which has already been clinically tested . this should simplify the approval procedure of this drug in new pdna - based vaccination protocols . furthermore , as it was shown to be systematically active there is no need for local co - administration of pdna and tucaresol , as shown here by combining intramuscular pdna immunisation with subcutaneous injection of tucaresol . the combination of intradermal “ ballistic ” delivery of pdna vaccination with oral administration of tucaresol may prove a very attractive mode of immunisation particularly under conditions where parenteral immunisations should be avoided due to risks of blood - borne infections or cultural stigmata associated with injections . in summary , we present herein data that show for the first time the utility of using a schiff base forming drug as a simple and effective method to augment the specific immune response induced by pdna vaccination . these data are applicable for both clinical and industrial settings . all genes were inserted in the pcdna3 vector ( invitrogen bv , nv leek , the netherlands ). mycobacterium bovis hsp65 cdna was excised from plasmid prib1300 ( kindly provided by dr . r . v d zee , utrecht university , utrecht , the netherlands ) using eco ri and sal i and sub - cloned in the eco ri and xho i sites of pcdna3 mcs . the identity and orientation of the gene in the resulting plasmid ( p3m . 65 ) were confirmed with restriction mapping . expression and production of hsp protein was detected in the lysate of cos - 7 transfected with p3m . 65 by lipofection using lipofectine ( life technologies , paisley , scotland ). lysates were electrophoresed on 12 % sds - page gel , followed by western blotting on pvdf membrane ( biorad , ca ) and immuno - detection by anti - mycobacterial hsp65 specific monoclonal antibody dc - 16 ( kindly provided by dr . juraj ivanyi , london , u . k .). this was then detected with a secondary alkaline phosphatase conjugated goat anti - mouse ig ( southern biotech , al ) and the blot was developed using the western blue substrate system ( promega , madison , wis .). sera from immunised mice were collected and used in direct elisa as described earlier . recombinant mycobacterial hsp65 ( kindly provided by dr r . v d zee ) was used at a concentration of 4 μg / ml carbonate buffer to coat wells of 96 well plate ( maxisorp , nunc , denmark ) overnight at 4 ° c . sera were added in duplicate at 1 : 100 dilution , incubated overnight at 4 ° c . binding antibodies were detected using igg ( preabsorbed against mouse igm ), igg1 and igg2a specific alkaline phosphatase conjugated goat anti - mouse sera ( southern biotech ). hla - a2 * 0201 / kb transgenic mice ( kindly provided by dr l . sherman , scripps laboratories , san diego , calif .) used in this study have been described . these mice express a chimeric mhc class ii molecule i which the α1 and α2 domains are of the hla - a * 0201 molecule while the α3 trans - membrane and cytoplasmic domains are of the mouse h2 kb molecule . this construction permits the binding site of the mouse cd8 molecule on the t - cell to interact with the α3 domain of the chimeric molecule . the surface expression of the hla - a * 0201 / kb was confirmed using hla - a * 0201 specific fitc - conjugated monoclonal antibody ( one lambda , ca ) and assessed by flow cytometry using facscan ( becton dickinson & amp ; co ., mountain view , calif .). c575bi / 6 mice have been described . these mice have a defined influenza virus nucleoprotein ctl epitope . aca ( h - 2f ) mice were purchased from jackson ( jackson laboratory , bar harbor , main ). mice were propagated and held in our sp environment in mtc animal house at the karolinska institute . genetic immunisation was accomplished by intra - muscular immunisation . plasmids were prepared form lb ampicillin e . coli culture using qiagen plasmid giga kit ( qiagen gmbh , hilden , germany ). concentrations and purity were determined using spectrophotometer and analytical gel electrophoresis . mice were injected in the regenerating tibialis - anterior muscle according to the method of davis et al using 20 μg pdna / 100 ul / muscle of either control plasmid ( p3 ), ebna - 4 expressing plasmid plus control plasmid p3 ( e4 ), p3m . 65 expressing plasmid plus control plasmid p3 ( p3m . 65 ), p3m . 65 plus gm - csf expression plasmids ( p3m . 65g ), or p3m . 65 plus ifnγ expression plasmid ( 9p3m . 65γ ). plasmids were mixed in equal molar quantities . alternatively , immunisation was carried out by gene gun according to the method of fynan et al ( 27 ) using 10 ng or 100 ng of dna plasmid encoding a / pr8 / 34 influenza virus nucleoprotein ( pvac1 . pr ) or empty vector , or 2 ug of plasmid encoding ebna - 4 ( e4 ) or control vector p3 . tucaresol treated mice were immunised with e4 , p3m . 65 , or pvac1 . pr ( e4 , t p3m . 65 , t and pvac1 . np pr ( tuc sc ) respectively ) and either injected at the same time with 1 mg tucaresol each sub - cutaneously or received daily injections of tucaresol 200 μg per mouse for a period of 4 days also sub - cutaneously . mice received boosting two weeks after priming with the same dose . jurkat a * 0201 / kb ( jk - a2 / kb ), a human t - cell leukaemia 0hla - a * 0201 negative cell line stably transfected with hla - a80201 / kb chimeric gene ( kindly provided by dr . w . m . kast , loyola university , maywood ill .). the s6c cell line was derived from a spontaneous mammary adeno - carcinoma that has been originated in an aca mouse . s6c - gpt and s6c - e4 are control plasmid and ebna - 4 transfectant respectively ( kindly provided by dr . george klein , mtc , karolinska institute , stockholm ). all cell lines were maintained by passing in vivo in syngeneic aca mice and in vitro in rpmi 1640 supplemented with 10 % fetal bovine serum ( fbs ), 100 u / ml penicillin , 100 μg / ml streptomycin , 50 μm 2 - mercaptoethanol , and 2 mm l - glutamine . splenocytes were harvested from immunised mice . a single cell suspension was prepared and cells were re - suspended in imdm supplemented with 10 % fbs and l - glu and antibiotics . mixed splenocytes and tumor cell cultures ( mstc ) were prepared by mixing 3 × 10 6 tumor cells per ml . cultures were incubated for 5 days at 37 ° c . in 7 . 5 % co 2 . one μci of tritium labelled thymidine was added to each well of u - shaped bottom 96 well plates . cells were further incubated for 18 hours in the same conditions as above and harvested and the amount of incorporated tritium labelled thymidine was measured using beta plate reader ( wallac , turku , finland ). the test was done in triplicates and the stimulation index ( si ) was calculated using the formula : si = splenocyte proliferation towards the s6c - ebna - 4 transfectant ( or ebna - 4 vaccinia infected )/ splenocyte proliferation toward s6c - gpt ( or tk - vaccinia infected ) control transfectant . mixed splenocytes ( from e4 immunised mice ) and tumor cell cultures ( mstc ) were prepared by mixing 3 × 10 6 splenocytes plus 3 × 105 tumor cells per ml . supernatants were collected after 72 hours of culture and were tested for the presence of interferon gamma ( ifnγ ) and il - 4 using commercially available matched antibody pairs for mouse cytokines elisa ( immunokontact , bioggio , switzerland ) according to the manufacturer &# 39 ; s instructions . peptide specific ctl lines were prepared in 12 - well plates as follows . splenocytes , from immunised or control non - immunised mice were plated at 6 × 10 6 per well and co - cultured with 3 × 10 6 peptide pulsed ( 5 μg per ml p4 ) syngeneic splenocytes . after 6 – 8 days cell - mediated cytotoxicity was measured by 51 cr release as follows . one million target cells were incubated at 37 ° c . in the presence of 200 μci sodium 51 cr chromate ( amersham , uk ) for 1 hour , washed three times and re - suspended in compete medium at 10 5 cells / ml in the presence of absence of 10 μg of the relevant ( p4 ) or irrelevant ( influenza np 58 – 66 ) peptide . the test was performed by incubating 5 × 10 3 target cells at different effector to target ratios in triplicate wells at a final volume of 200 μl in v - bottomed 96 well plates . cells were incubated for 4 hours at 37 ° c . after which supernatants were harvested and used to determine specific lysis using the following equation : percent specific release = 100 ×( experimental release − spontaneous release )/( maximum release − spontaneous release ). it is to be understood that the present invention has been described by way of example only , and that modifications and / or alterations thereto , which would be obvious to a skilled person based upon the disclosure herein are also considered to fall within the scope and spirit of the invention , as defined in the appended claims . 1 . donnelly j . et al , “ dna vaccines ” annu . rev . immunol . 1997 , 15 : 617 – 48 . 2 . donnelly , j . j ., friedman a ., martinez d ., montogomery , d . l ., shiver , j . w ., motzel , s . l ., ulmer , j . b ., liu , m . a . 1995 . preclinical efficacy of a prototype dna vaccine - 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