Patent Application: US-72913596-A

Abstract:
the present invention relates to the production of vaccines having improved safety , particularly to a process therefor which allows even an aids vaccine to be manufactured , comprising in order , the steps of : a ) treating the virus with a general inactivating agent ; b ) deaggregating the virus with a suitable solvent or detergent ; c ) treating the virus with an rna and / or dna inactivating agent ; and d ) stabilizing the virus with a suitable cross - linking agent .

Description:
it will be appreciated that the present invention is applicable to any virus , particularly the enveloped viruses . however , where a virus is either not particularly dangerous in itself , or can efficiently be inactivated by an alternative one - step procedure , then it would not generally be appropriate to use the method of the present invention . accordingly , it is preferred to use the method of the present invention for the preparation of particularly dangerous viruses , of which hiv and the various strains thereof are especially preferred . in addition , particularly heat and / or chemical resistant viruses , such as scrapie and bse , may be used in vaccines , if prepared by the method of the invention . in addition , as it known that there exists a number of antigenic variants of hiv , it is preferred to apply the process of the invention to a representative selection of strains . in particular , enough strains should be chosen such that an immune response raised from the resulting vaccine will be suitable to target any infectious strain of hiv . selecting various appropriate strains of hiv can be done by any appropriate method known in the art . in particular , it may be appropriate to compare the gp120 protein , one of the major antigenic targets of any immune response against hiv . the method of the present invention may be applied directly to a mixed batch of strains as described above , or may be applied individually to batches of single strains , and the results mixed . 62 - propiolactone provides a useful initial inactivant , allowing safe handling and concentration of the virus , without altering its antigenic properties . however , the initial treatment of the virus with a general inactivating agent may be with any appropriate inactivating agent but , for the purposes of the present invention , it is preferred that such agent is β - propiolactone . the treatment with β - propiolactone may be in any suitable manner known to achieve inactivation , and may be carried out at low temperatures or high temperatures , and either generating β - propiolactone in the reaction , or using pre - prepared β - propiolactone . in general , it is preferred to use pre - prepared β - propiolactone at higher temperatures , such as 37 ° c . or 40 ° c . also , at such high temperatures , it is preferred to repeat the treatment with β - propiolactone before proceeding to the next stage . a suitable concentration of β - propiolactone is 0 . 1 % v / v . this first inactivation stage may be conducted on purified virus , but this is not generally necessary , and may be conducted on crude culture supernatant fluid . however , after such treatment , it is then preferred to purify the virus , and this may by done by any suitable technique , such as by spinning the virus on a sucrose cushion . after purification of the virus , the next stage is to deaggregate the virus . as described above , this is particularly important for a dangerous virus , as any virus which escapes the inactivation is potentially infectious . deaggregation can be achieved by methods known in the art , and suitable solvents and detergents have been described above . the only specific requirement is that as many clumps of virus are broken up as possible . it will be appreciated that it is not possible to guarantee that all clumps are broken up , but it is preferred to use such agents as will ensure maximum disruption of the clumps . in particular , it may be preferable to use a combination of solvents and detergents to maximise the effect . the deaggregation of the clumps of virus also has another beneficial side - effect , in that the substances used to achieve this result also tend to disrupt the virus , which helps in inactivation . further , the compounds also lead to &# 34 ; ballooning &# 34 ; of the virus , which allows access of other substances to the viral core , which would otherwise be protected . &# 34 ; ballooning &# 34 ; is thought to entail expansion of the viral envelope without actually destroying the envelope , thus creating pores through which other substances can freely pass . under selected circumstances , such as those described herein , ballooning is reversible , insofar as the envelope substantially readopts its original proportions . once the viruses have been ballooned , the genetic material can be inactivated . as has been described above , there are various techniques for inactivating rna and dna , and it is preferred to use , in accordance with the present invention , an ethylenimine , such as binary ethylenimine or acetyl - ethylenimine , and , in addition , an rnase and / or dnase . because of its superior penetrating properties ( fellows , 1966 ) and its first order inactivation kinetics , binary ethylenimine is an ideal second stage inactivant to back up the action of β - propiolactone . after this stage , the virus may then be stabilised with a suitable substance , such as formaldehyde or glutaraldehyde . any other suitable substances may also be used , the purpose being to present , as far as possible , a more morphologically intact virus . more particularly , the intention is to present a virus which is as similar as possible to the original virus , but which is completely inactivated . because of the toxicity of saponin and its mixed composition , we investigated the immunostimulatory action of digitonin . using a highly purified , soluble form of digitonin , we found , in preliminary experiments , that mice injected with digitonin - treated influenza virus survived after receiving considerable quantities ( 100 mg / kg ) of digitonin , without ill effect . we have also discovered that digitonin is a particularly useful adjuvant , and this forms a preferred aspect of the present invention , either in combination with the process of the invention , or otherwise . if used alone , digitonin may be used as a conventional adjuvant to enhance the immune response generated by a vaccine . for example , digitonin may be of particular use in influenza vaccines , and need only be added to the vaccine , although it may be incorporated in such a manner as to ensure that it is incorporated into the viral coat , for instance . in the context of the process of the invention , digitonin may be incorporated at any useful stage , but it is preferred to incorporate it after inactivation of the genetic material , and before stabilisation of the virus . the result of using digitonin at this stage is that it is incorporated into the viral envelope , serving further to disrupt the envelope and also to provide an adjuvant in situ . other adjuvants may also be used , as appropriate , such as fca , if the vaccine is for administration to animals . for human applications , alum may be used . also , as described above , it may be possible to use other suitable substances , such as mdp , such as threonyl mdp , in combination with a suitable oil , such as squalene , and , if required , a spreading agent , such as pluronic 121 . where appropriate , it may also be desirable to enhance the adjuvanticity with , for example , iscoms . further , it may be appropriate to employ saponin as an adjuvant , especially where this can be effected in combination with cholesterol - lecithin liposomes . in order to enhance the safety of the vaccines of the present invention , it is generally desirable to assay the various stages of the preparation . suitable assay procedures include the rapid syncytium assay , which may be applied after the initial inactivation step . other safety assays include testing of the vaccine preparation for residual non - inactivated virus by co - cultivation with human pbmn cells . the preparations may also be assayed for any residual intact dna or rna . this may be done by any method known in the art , such as by pcr . it may also be appropriate to test any vaccine prepared to ensure that the full range of antigens is present in the vaccine . this may be done by testing in an animal , for example , and then challenging the animal . however , this tends to be rather empirical , and is not preferred . a more preferred technique would be to provide an elisa test , where antibodies against all of the desired antigens are provided . other appropriate tests will be apparent to those skilled in the art , and such tests may include , for example , immunoblotting . the preparations may also be standardized for immunogenicity by vaccination tests in small or large laboratory animals , if desired . accordingly , the most preferred embodiment of the present invention is as follows : 1 ) adaptation of ` street ` viruses to continuously propagated t - cell line such as cem or h9 . 2 ) inactivation of crude culture supernatant fluid with 2 stages of β - propiolactone ( 0 . 1 % v / v each at 40 ° c . for 18 hrs ) 2a ) subsidiary testing for infectious virus by rapid syncytium assay in cell culture . 3 ) centrifugation at 19000 rpm for 1 hr onto a sucrose cushion , to achieve virus purification . virus should be morphologically intact . 4 ) deaggregation , ballooning and further inactivation of virus with 0 . 05 % cholate . 5 ) inactivation of &# 34 ; opened &# 34 ; cores ( wherein the term &# 34 ; opened &# 34 ; is used to denote cores which are accessible because the envelope is ballooned ) with binary ethylenimine ( 0 . 01m at 37 ° c . for 18 hrs ). 6 ) inactivation of viral rna and contaminating host cell dna with rnase and dnase . 7 ) &# 34 ; adjuvanting &# 34 ; ( treating so as to provide an adjuvant effect in the final vaccine ) and further virus disruption and inactivation with digitonin . 8 ) stabilisation of virions with mild formaldehyde or glutaraldehyde ( 0 . 05 % on ice for 4 hrs ). 9 ) testing of vaccine preparation for residual non - inactivated virus by co - cultivation with human pbmn cells for 6 weeks . 10 ) analysis by polymerase chain reaction ( pcr ) for residual proviral dna and viral rna ( additional step of reverse transcription to cdna ) using primers specific for viral reverse transcriptase and integrase . 11 ) standardisation of vaccine by elisa and immunoblotting with human post infection antisera , to ensure antigenic survival . 12 ) standardisation of immunogenicity by vaccination tests in small or large laboratory animals . administration of the vaccines and treatments according to the present invention will vary according to the circumstances , taking into account such factors as age , weight and general condition of the patient . the vaccine may be administered as one self - sufficient dose or as a series of doses over a period of time . repetition of dosing either to boost or maintain immunity is also generally desirable at a later time , conveniently about 3 months later , but such booster dosing may be given earlier or at any time during the remainder of the life - time of the patient , and on as many occasions are necessary . pharmaceutical grade saline may be used as a carrier to provide a simple vaccine . however , it may often be preferred to use adjuvants , such as described hereinabove . in general , an adjuvant may be administered together with the vaccine , in the same or different preparations , or separately , at a time different from that of the administration of the vaccine . vaccines according to the present invention will usually be administered by a conventional route such as , for example , by injection by the intravascular , intraperitoneal , intramuscular or subcutaneous routes . other suitable routes may comprise intradermal inoculation or administration via particulate aerosols . such vaccines will normally comprise a pharmaceutically acceptable carrier and optionally an adjuvant , substances to render the vaccine isotonic with the body fluids and such flavourings , emulsifiers and other ingredients as may be required . such vaccines as described above may be sub - divided for separate administration , whether simultaneously or over a period of time , suitably weeks . in general , it will be appreciated that the type of vaccine and its ingredients will be determined by the virus concerned , and will often correspond to existing vaccines , the advantage lying in the safety of the preparation . thus , it will be appreciated that the present invention provides a unique combination of steps enabling a safe vaccine to be prepared from highly dangerous viruses . of course , it will be appreciated that no vaccine can be absolutely guaranteed , but the present invention provides a vaccine whose benefits far outweigh the potential risks . in general , preferred objectives of our procedure for producing a whole hiv vaccine are : 1 ) to incorporate a &# 34 ; cocktail &# 34 ;, or selection , of viruses selected to match circulating wild type or &# 34 ; street &# 34 ; viruses on the basis of epidemiological features and antigenic and nucleotide sequence analysis of gp120 loop regions . alternatively , fewer but &# 34 ; archetypal &# 34 ; ( strains carrying selected distinctive viral characteristics ) virus strains may be used . 2 ) to inactivate the viruses completely using a unique multistage chemical and biological process which will still maintain the structural integrity of the virus and hence the major antigenic determinants of both externally situated glycoproteins and core proteins . 3 ) to ensure appropriate immunogenicity of the inactivated vaccine preparation and to compare the immune response in laboratory animal models with the standard immune response following hiv infection of humans . 5 ) to formulate the vaccine for both intramuscular and oral administration . the combination of steps in the correct order is unique , as is the use of detergent ballooning and adjuvant ( digitonin ) incorporation into the virion lipid . the most important novel features of our procedure are probably : 1 ) multiple inactivation steps -- current inactivated vaccines are killed using single chemical agents . our method ideally utilises five independent inactivation steps ( 4 chemical and 1 biological ) each one of which is individually able to inactivate the virus . 2 ) correct sequence of chemicals -- the order of inactivating steps is particularly important . inactivating agents each have a specific target ( virus protein , nucleic acid , virus envelope ) and need to be used in the correct sequence , which we have established experimentally . for example , in the only published paper ( rowlands , 1972 ) a reverse sequence of two of the agents was used . 3 ) new adjuvants -- digitonin , a saponin - like molecule , disrupts the lipid membrane of retroviruses . digitonin in the virus preparation both disrupts and inactivates virus , while also enhancing immunogenicity . 4 ) virus deaggregation and ballooning by cholates -- the use of detergents ( eg cholates ), not to totally disrupt virus , but to deaggregate them and cause ballooning , hence allowing access of nucleic acid inactivating agents such as binary ethylenimine , is a preferred aspect of the invention . we have shown that rna - containing viruses with lipid envelopes may be uniquely ballooned with the correct concentrations of detergent . 5 ) nucleic acid destruction -- incorporation of rnase and dnase steps to inactivate contaminating proviral dna from virus infected cells and virion rna . 6 ) enhanced penetration of inactivating agents -- the further partial disruption of the virus by digitonin and disturbance of the lipid bilayer allows enhanced penetration of binary ethylenimine . 7 ) &# 34 ; presentation shape &# 34 ; to the immune system -- formaldehyde or mild glutaraldehyde fixation allows presentation of antigens in a correct &# 34 ; presentation shape &# 34 ; ( the 3 - dimensional conformational features of the virus are substantially the same as untreated virus ) for recognition by the immune system . the following examples illustrate the invention , and are not intended to limit the invention in any manner whatsoever . the following experiment is based on the assumption that a single dose of influenza vaccine would not induce protective immunity . accordingly , if immunity can be demonstrated after the administration of only one dose , then it is extremely likely that any additional substance that had been incorporated into the vaccine has acted as an adjuvant . digitonin was tested for adjuvant activity in an experimental protocol , using the known adjuvant , alhydrogel , for comparison . digitonin and alhydrogel were included in influenza vaccine preparations to investigate whether the combination could enhance immunity to a protective level . 14 g balbc mice were immunised i . p . with a subunit influenza vaccine at 4 and 0 . 4 μg per mouse . four weeks post immunisation , approximately 50ld 50 of the homologous influenza virus was administered to each mouse , intranasally under light ether anaesthesia . non - immunised mice were included as controls . deaths were scored daily . results are shown in the accompanying table . table______________________________________effect of subunit influenza vaccine versusinfection with lethal influenza virus no . of dead micevaccine preparation ( 5 mice per cage ) ______________________________________subunit vaccine alone 4 μg 4subunit vaccine alone 0 . 4 μg 4subunit vaccine plus alhydrogel 4 μg 1subunit vaccine plus alhydrogel 0 . 4 μg 3alhydrogel alone 4digitonin ( 0 . 1 %) alone 4digitonin ( 0 . 01 %) alone 5subunit vaccine plus digitonin ( 0 . 1 %) 4 μg 0subunit vaccine plus digitonin ( 0 . 1 %) 0 . 4 μg 0subunit vaccine plus digitonin ( 0 . 01 %) 0 . 4 μg 0______________________________________ the results clearly demonstrate that one dose of non - adjuvanted vaccine offers no protection against infection . alhydrogel had little or no adjuvant activity with the lower dose vaccine , and could not save all mice even at the higher dose . by contrast , digitonin saved all mice at all concentrations tested , even with the 0 . 4 μg vaccine . table 1______________________________________inactivation of concentrated hiv withβ - propiolactone . virus was concentrated by peg precipitation to give ahigh titre . virus was treated with variousconcentrations of β - propiolactone at room temperaturefor 24 hours then assayed for residual infectivity . log 10 tcid of virus per ml concentration of bpl______________________________________8 . 0 0 . 0 % 5 . 0 0 . 01 % 2 . 0 0 . 05 % 1 . 0 0 . 5 % 0 . 0 1 . 0 % 0 . 0 2 . 0 % ______________________________________ table 2______________________________________inactivation of unconcentrated hiv byβ - propiolactone . virus in tissue cuiture supernatant was treated withvarious concentrations of β - propiolactone at room tempfor 24 hours then assayed for residual infectivity . log 10 tcid of virus per ml concentration of bpl______________________________________5 . 0 0 . 00 % 4 . 0 0 . 002 % 2 . 0 0 . 004 % 0 . 0 0 . 008 % 0 . 0 0 . 016 % 0 . 0 0 . 03125 % 0 . 0 0 . 0625 % 0 . 0 0 . 125 % 0 . 0 0 . 25 % 0 . 0 0 . 5 % 0 . 0 1 . 0 % 0 . 0 2 . 0 % ______________________________________ table 3______________________________________time course of inactivation of hiv withβ - propiolactone . concentrated hiv was treated with 0 . 2 % β - propiolactoneand the reaction stopped at various time intervals byincubation at 37 ° c . for 30 mins to hydrolyse theβ - propiolactone . log 10 tcid of virus / ml time ( hours ) ______________________________________8 . 0 06 . 0 0 . 55 . 0 14 . 0 22 . 0 32 . 0 41 . 0 50 . 0 6______________________________________ table 4______________________________________inactivation of hiv with binary ethylenimine . concentrated hiv was treated with differentconcentrations of binary ethylenimine at 37 ° c . for 24hours . residual binary ethylenimine was neutralisedwith sodium thiosulphate and the virus tested forinfectivity . log 10 tcid of virus / ml concentration of bei ( mm ) ______________________________________8 . 0 08 . 0 0 . 17 . 0 0 . 54 . 0 50 . 0 100 . 0 200 . 0 50______________________________________ table 5______________________________________time course of inactivation of hiv with binaryethylenimine . concentrated hiv was treated with 10mm binaryethylenimine . aliquots were taken at various timeintervals , neutralised with sodium thiosulphate andassayed for residual infectivity . log tcid of virus / ml time ( hours ) ______________________________________8 . 0 06 . 0 15 . 0 24 . 0 33 . 0 42 . 0 51 . 0 60 . 0 70 . 0 8______________________________________ preliminary studies on the immunogenicity of the vaccine were carried out in rats , guinea pigs and rabbits . animals were intravenously immunised with 100 μg ( total protein in 0 . 5 ml pbs ) per animal of either : animals were boosted on days 10 and 60 and serum samples were taken on days 0 , 19 , 52 and 72 . serum antibodies were assayed by solid phase elisa to whole virus and gp120 and by radioimmunoassay to gp120 derived peptides . table 6______________________________________serum responses to whole hiv rf and hiv iiibderived gp120 ( expressed as endpoint titres ). antibody titrevaccine animal whole rf iiib gp120______________________________________ rat 1 45 , 000 1 , 000 rat 2 400 , 000 1 , 200 rat 3 400 , 000 1 , 000vaccine 1 rat 4 100 , 000 & gt ; 10 , 000 ( bpl only ) guineapig 1 700 , 000 25 , 000 guineapig 3 74 , 000 25 , 000 guineapig 4 74 , 000 1 , 800 rabbit 1 20 , 000 20 , 000 rabbit 2 74 , 000 & gt ; 25 , 000 rat 5 450 , 000 400 rat 6 250 , 000 160 rat 7 16 , 000 & gt ; 10 , 000vaccine 2 rat 8 800 , 000 & gt ; 10 , 000 ( complete guineapig 5 10 , 000 3 , 000process ) guineapig 6 100 , 000 25 , 000 guineapig 7 450 , 000 10 , 000 guineapig 8 80 , 000 3 , 600 rabbit 3 74 , 000 6 , 000______________________________________ radio immunoassay studies on the guineapig sera showed responses to the v2 and v3 loop areas of hiv rf gp120 in both groups of animals ( vaccine 1 and vaccine 2 ). ii ) empirical formula -- c 3 h 4 o 2 , mol . wt . 72 . 1 iv ) solubility -- 37 % v / v at 25 ° c . in water ( slowly hydrolyses ). miscible with ethanol ( reacts ), acetone , ether , chloroform , and probably most polar organic solvents and liquids . v ) stability -- polymerises on storage , hydrolyses to 3 - hydroxy propionic acid , a non - toxic derivative , approx . 18 % per hour at 25 ° c ., more rapidly at elevated temperatures . iii ) substitution of alkyl groups at one of ring carbon atoms increases rate of ring opening . iv ) substitution at ring nitrogen decreases rate of ring opening unless alkyl group contains an electronegative group ( e . g . acetyl ) when rate is increased . v ) common derivatives have low boiling points e . g . 56 . 7 ° c . for ei . vapour pressure high enough to be an inhalation hazard . vii ) ethylenimine is known to react with α and epsilon amino , imidazole , carboxyl , sulphydryl and phenolic groups of proteins , inorganic phosphate , glycero and hexose phosphates and amino groups of adenine and thiamine . viii ) ethylenimine can be prepared by the cyclisation of bromoethylamine hydrobromide under alkaline conditions : ## str1 ## 2 ) anderson , k . p ., et al ., j . inf . dis . 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