Patent Application: US-83844910-A

Abstract:
a method to prepare inactivate viral vaccine by exposing the virus to a predetermined concentration of an inactivating psoralen , and a preselected intensity of ultraviolet radiation for a time period sufficiently long to render the virus non - infectious but less than that which would result in degradation of its antigenic characteristics .

Description:
according to the present invention , vaccines useful for the inoculation of mammalian hosts , including both animals and humans , against infection by dengue virus are provided . the vaccines are prepared by inactivation of live dengue virus in a medium containing an amount of an inactivating psoralen sufficient to inactivate the virus upon subsequent irradiation with ultraviolet radiation of predetermined intensity ( uva ). degradation of the antigenic characteristics of the live virus is reduced or eliminated by carefully selecting psoralen ( s ) of a pre - determined concentration and exposing the virus to only minimum intensity and duration of uva necessary to inactivate the virus . suitable vaccines may be prepared by combining the inactivated virus with a physiologically - acceptable carrier , typically an adjuvant , in an appropriate amount to elicit an immune response , e . g ., the production of serum neutralizing antibodies , upon subsequent inoculation of the host . the present invention is suitable for producing vaccines against one or all four serotypes of dengue virus as well as other arbovirus . the vaccine may comprise inactivated dengue virus from one or more serotypes . the inactivation of each serotype of dengue virus may be carried out individually , and subsequently combined in the final vaccine or carried out together in a single process . in preparing the present vaccines , sufficient amounts of the virus to be inactivated may be obtained by growing seed virus in a suitable mammalian cell culture . seed virus , in turn , may be obtained by isolation from an infected host . suitable mammalian cell cultures include primary or secondary cultures derived from mammalian tissues or established cell lines such as vero cells , monkey kidney cells , bhk21 hampster cells , lmtk31 cells , and other cells permissive for the desired virus , and which may be grown in vitro as monolayer or suspension cultures . the cell cultures are grown to approximately 80 % saturation density , and infected with the virus at a low multiplicity of infection ( moi ), usually between about 0 . 05 and 0 . 005 , preferably at about 0 . 01 . after adsorbing the viral inoculum to the cells by incubation for a limited period of time at a temperature in the range from 35 ° c . to 40 ° c ., an appropriate growth or maintenance medium is added . the cells are further incubated at about the same temperature , in the presence of about 5 % carbon dioxide in air , until a sufficient amount of virus has been produced . the growth and maintenance medium is conventional mammalian cell culture medium , such as eagle &# 39 ; s minimum essential medium or medium 199 , usually supplemented with additives such as broth prepared from dehydrated standard microbial culture media , fetal bovine serum , fetal calf serum , or the like . psoralens may be used in the inactivation process include psoralen and substituted psoralens , in which the substituent may be alkyl , particularly having from one to three carbon atoms , e . g ., methyl ; alkoxy , particularly having from one to three carbon atoms , e . g ., methoxy ; and substituted alkyl having from one to six , more usually from one to three carbon atoms and from one to two heteroatoms , which may be oxy , particularly hydroxy or alkoxy having from one to three carbon atoms , e . g ., hydroxy methyl and methoxy methyl , or amino , including mono - and dialkyl amino or aminoalkyl , having a total of from zero to six carbon atoms , e . g ., aminomethyl . there will be from 1 to 5 , usually from 2 to 4 substituent , which will normally be at the 4 , 5 , 8 , 4 ′ and 5 ′ positions , particularly at the 4 ′ position . illustrative compounds include 5 - methoxypsoralen ; 8 - methoxypsoralen ( 8 - mop ); 4 , 5 ′, 8 - trimethylpsoralen ( tmp ); 4 ′- hydroxymethyl - 4 , 5 ′, 8 - trimethylpsoralen ( hmt ); 4 ′- aminomethyl - 4 , 5 ′, 8 - trime - thylpsoralen ( amt ); 4 - methylpsoralen ; 4 , 4 ′- dimethylpsoralen ; 4 , 5 ′- dimethylpsoralen ; 4 ′, 8 - dimethylpsoralen ; and 4 ′- methoxymethyl - 4 , 5 ′, 8 - trimethylpsoralen . of particular interest are amt4 , 5 ′, tmp and 8 - mop . different psorlarens may be used individually or in combination in the inactivation process . the psoralens may present in amounts ranging from 1 - 100 μg / ml , preferably from about 5 - 25 μg / ml . in carrying out the invention , the psoralen ( s ) are combined with the viral suspension , conveniently a viral suspension in an aqueous buffered medium , such as those used for storage . the virus concentration is generally about 1 × 10 5 to 1 × 10 6 pfu / ml , and preferably about 3 - 5 × 10 5 pfu / ml . although viral inactivation according to the present invention will normally be carried out in an inactivation medium as just described , it may be desirable to introduce psorlarens to the virus by addition to a cell culture medium in which the virus is grown . the inactivation is then carried out by separating the live viral particles from the culture medium , and exposing the particles to ultraviolet light in an inactivation medium which may or may not contain additional psoralens . when employing psoralens with limited aqueous solubility , typically below about 50 g / ml , it has been found useful to add an organic solvent , such as dimethyl sulfoxide ( dmso ), ethanol , glycerol , polyethylene glycol ( peg ) or polypropylene glycol , to the aqueous treatment solution . for psoralens having limited solubility , such as 8 - mop , tmp , and amp , adding a small amount of such organic solvents to the aqueous composition , typically in the range from about 1 to 25 % by weight , more typically from about 2 to 10 % by weight , the solubility of the psoralen can be increased to about 200 ng / ml , or higher . such increased psoralen concentration may permit the use of shorter irradiation times . also , inactivation of particularly recalcitrant microorganisms may be facilitated without having to increase the length or intensity of ultraviolet exposure . the addition of an organic solvent may be necessary for inactivation of some viruses with particular furocoumarins . the ability to employ less rigorous inactivation conditions is of great benefit in preserving the antigenicity of the virus during inactivation . the psoralen may be added to the viral suspension in a single addition or in multiple additions , where the virus is irradiated between additions . the psoralens may also be added continuously during the entire treatment period , or a portion thereof . usually , the number of additions will be from 1 to 4 . the total amount of psoralen is sufficient to provide a concentration between 1 ng / ml and 100 ng / ml . the time of uv irradiation will vary depending upon the light intensity , the concentration of the psoralen , the concentration of the virus , and the manner of irradiation of the virus receives , where the intensity of the irradiation may vary in the medium . the time of irradiation will be inversely proportional to the light intensity . the total time will usually be at least about 5 minutes and no more than about 30 minutes , generally ranging from about 5 to 10 minutes . the light , which is employed , will generally have a wavelength in the range from about 300 nm to 400 nm . usually , an ultraviolet light source will be employed together with a filter for removing uvb light . the intensity will generally range from about 150 μw / cm2 to about 1500 μw / cm2 , although in some cases , it may be higher . prior to treatment with ultraviolet light , the virus furocoumarin solution is placed upon a bed of ice . the temperature for the irradiation is preferably under 25 ° c . during irradiation , the medium may be maintained still , stirred or circulated . the solution may be either continuously irradiated or be subjected to alternating periods of irradiation and non - irradiation . the circulation may be in a closed loop system or in a single pass system ensuring that the entire sample has been exposed to irradiation . it may be desirable to remove the unexpended psoralen and / or its photobreakdown products from the irradiation mixture . this can be readily accomplished by one of several standard laboratory procedures such as dialysis across an appropriately sized membrane or through an appropriately sized hollow fiber system after completion of the irradiation . alternatively , one could use affinity methods for one or more of the low molecular weight materials to be removed . the inactivated virus may then be formulated in a variety of ways for use as a vaccine . the concentration of the virus will generally be from about 1 × 10 5 to 1 × 10 6 pfu / ml , and preferably about 3 - 5 × 10 5 pfu / ml , as determined prior to inactivation . three psoralen compounds with differing solubility and intercalation properties were selected for viral inactivation testing : 4 ′- aminomethyltrioxsalen hydrochloride ( amt ) ( sigma - aldrich , product number a4330 , cas number 62442 - 61 - 9 ); 8 - methoxypsoralen ( 8 - mop ) ( acros organics , catalog number 214150010 , cas number 298 - 81 - 7 ); and 4 , 5 ′, 8 - trimethylpsoralen ( tmp ) ( acros organics , catalog number 229881000 , cas number 3902 - 71 - 4 ). denv - 1 ( dengue virus serotype 1 ) western pacific 74 strain was selected as the virus for this study . this virus has been used in prior vaccine efforts 21 , 22 and was propagated in aedes albopticus c6 / 36 clonal cell culture . 5 ml aliquots of denv - 1 viral culture supernatant at a concentration of 3 . 4 × 10 5 plaque - forming units ( pfu )/ ml were transferred into 60 × 15 mm petri dishes . four test groups of dishes were made . a single psoralen compound ( either amt , 8 - mop , or tmp ) was added to each dish in the first three groups , while the fourth group had no psoralen added and served as control . dishes were exposed to uva radiation at 365 nm ( uvab - 18 lamp , ultralum , inc ., claremont , calif .) for 0 , 1 , 5 , 10 , or 20 minutes at an intensity of 200 or 1000 μw / cm 2 . specimens were then re - titered in bhk cell culture to assess for plaque formation . table 1 shows the summary of vaccine candidate selection results . amt , 8 - mop , and tmp were added to denv - 1 and exposed to uva radiation . control specimens exposed to no uva light produced a titer of 5 . 63 × 10 5 pfu / ml . no detectable pfus were noted following 10 minutes of 200 μw / cm2 of uva to amt - containing denv - 1 supernatant as well as following 5 minutes of exposure to 1000 μw / cm2 . the former was chosen as the candidate for in vitro testing based on its lower overall energy exposure . once an effective psoralen / uva dose combination was identified , the inactivated virus was purified using a centri - sep columns ( princeton separation , catalog # cs - 901 , adelphi , n . j .). 10 mm of phosphate - buffered saline ( pbs ) and 10 % aluminum hydroxide gel ( alhydrogel ) ( sigma , product number a 8222 ) were added to purified inactivated virus , with a final equivalent inactivated denv - 1 concentration of 3 × 10 5 pfu / ml ( 10 ng ). three groups of seven study - naïve , adult swiss - webster outbred ( cfw ) mus musculus mice were selected from the naval medical center detachment ( nmrcd ) mouse colony . no mice possessed anti - dengue antibody at baseline as determined by enzyme - linked immunoassay ( eia ), and all were 300 g or greater in mass . all procedures were conducted in accordance with protocols approved by the nmrcd institutional animal care and use committee . all mice were euthanized humanely at the conclusion of the experiment . all injections and blood sampling were performed by trained personnel using ketamine ( 100 mg / ml )/ acepromazine ( 5 mg / ml )/ xylazine ( 20 mg / ml ) as an intraperitoneal injection administered at a starting dose of 0 . 1 ml / 100 g body mass and titrated to effect . group a received 0 . 05 ml of vaccine candidate ( 5 ng ) injected intradermally into the tail on days 0 , 14 , and 28 . group b received 0 . 1 ml of vaccine candidate ( 10 ng ) injected on the same days . group c received control injections of 10 % alhydrogel and pbs . blood samples were obtained under anesthesia from the retro - orbital sinus of the mice on days 0 , 14 , 28 , 59 , and 90 . eia for detection of anti - denv - 1 igm and igg in mouse sera a capture eia was standardized in order to detect anti - denv - 1 igm in mice . 96 - well format plates were coated overnight at 4 ° c . with goat anti - mouse igm . at the end of incubation and after plate washing , mouse sera diluted to 1 / 100 were added to the plates and incubated for one hour at 37 ° c . afterwards , denv - 1 westpac 74 antigen derived from inactivated whole virus was added and again incubated for one hour at 37 ° c . next , rabbit hyperimmune sera with anti - denv specificity and anti - rabbit peroxidase conjugate were added and incubated for one more hour at 37 ° c . the plates were washed after incubation time . finally , abts substrate ( kpl , inc ., catalog number 50 - 62 - 01 , gaithersburg , md ., usa ) was added to develop color . after 30 minutes , the plates were read using a spectrophotometer at a wavelength of 405 nm . to detect anti - denv - 1 igg , 96 - well format plates were coated overnight at 4 ° c . with denv - 1 antigen . after the plate wash process , mouse sera diluted to 1 / 100 was added to the plates and incubated for one hour at 37 ° c ., followed by the addition of anti - human igg conjugate ( also incubated for one hour ) at 37 ° c . finally , abts substrate was added for color development . after 30 minutes , the plates were read using a spectrophotometer at a wavelength of 405 nm . for igg and igm eias , 0 . 10 od was determined as the cut - off value for positive antibody . adjusted optical density ( od ) values were determined by the difference of the od of the control antigen - coated well subtracted from the corresponding viral antigen - coated well . plaque reduction neutralization test ( prnt ) assays were performed using bhk - 21 cell in 24 - well polystyrene plates as described previously 23 . briefly , serial dilutions ( 1 / 40 , 1 / 80 , 1 / 160 and 1 / 640 ) of heat - inactivated mouse serum were prepared and mixed with an equal volume of a working dilution of denv - 1 west pac 74 ( 10 - 20 pfu / 50 μl ) and then incubated at 4 ° c . overnight . at the end of incubation , the diluted mixture was inoculated into 24 - well plates containing 3 × 10 5 pfu / ml of a bhk cell suspension . the infected plates were incubated for 3 h at 37 ° c . under 5 % c02 atmosphere . then the wells were overlaid with a viscous medium that containing carboxymethyl cellulose . plates were incubated for 7 days at 37 ° c . under 5 % c02 atmosphere . after removal from the incubator , the medium was dumped by inverting plates over a receptacle containing sodium hypochlorite , and plates were rinsed gently under tap water and fixed and stained with 0 . 5 ml , per well , of a solution that contains naphthol blue - black . the plaques were counted and 50 % plaque reduction was determined by probit analysis . fig2 ( a - c ) and fig4 show serologic responses in subject mice . all mice were seronegative for anti - denv - 1 antibodies by eia and prnt 50 at baseline . following the initial vaccine dose at 14 days , mice in group a ( low - dose vaccine ) had detectable igg in 6 / 7 ; of this group , one mouse additionally had both a positive igm and prnt 50 . group b ( high - dose vaccine ) mice had detectable igg in 7 / 7 and a positive prnt 50 in 2 / 7 . interestingly , no group b mice had detectable igm . at day 28 ( following 2 vaccine doses ), all group a mice had detectable anti - denv - 1 igg , with 3 / 7 having a positive prnt 50 and 2 of those 3 having detectable igm . at 59 days ( following all 3 vaccine doses ), 5 / 7 mice in group a had positive prnt 50 , but detectable igg and igm declined to 6 / 7 and 1 / 7 , respectively . at 90 days , one mouse in this group had died ; of the remaining 6 mice , 5 / 6 still had a positive prnt 50 although mean titers had declined . 5 / 6 still had positive igg and 1 / 6 a positive igm , with declining od measurements as compared with those at 59 days . 7 / 7 group b mice showed detectable igg and positive prnt 50 at days 28 and 59 . again , the presence of anti - denv - 1 igm was rare , with a single mouse having showing a detectable igm by eia at day 28 only . at day 90 , all 7 mice retained a positive igg , with no detectable igm . 6 / 7 retained a positive prnt 50 at day 90 , with declining mean titers noted but to a lesser magnitude than in group a . group c ( control ) mice received only adjuvant and pbs injections on days 0 , 14 , and 28 . no detectable anti - denv - 1 igm , igg , or positive prnt 50 assays were noted at days 0 , 14 , 28 , 59 , or 90 . based on the above findings , amt - inactivated denv - 1 is immunogenic in mus musculus , raising the possibility of psoralen / uva inactivation as a method for dengue vaccine development . natural infection with dengue viruses produces a lasting sterile immunity to that particular serotype , although not to the others . several questions remain following this pilot study . the waning of antibody titers in treated mice over 90 days suggests that this particular formulation , while immunogenic , may benefit from strategies to augment its response ( e . g ., different adjuvants , higher vaccine doses , or alternate routes of administration ). testing in non - human primates will provide additional data about the necessary vaccine dose and duration of serologic response , as well as immunity following in vivo dengue virus challenge . in vivo immunogenicity testing of psoralen - inactivated dengue - 1 virus vaccine candidate in aotus nancymaae monkeys in a previous experiment , it is demonstrated that denv - 1 can be deactivated using 10 μg / ml of 4 - aminomethyltroxsalen ( amt ) with 10 minutes of exposure to uva at a dose of 200 μw / cm2 . 10 mm of phosphate - buffered saline ( pbs ) and 10 % aluminum hydroxide gel ( alhydrogel ) were added to purified inactivated virus , with a final equivalent inactivated denv - 1 concentration of 3 × 10 5 pfu / ml ( 10 ng ). two groups of seven adult aotus nancymaae monkeys were selected from the naval medical center detachment ( nmrcd ) colony . no monkeys possessed anti - dengue antibody at baseline as determined by enzyme - linked immunoassay ( eia ). all procedures were conducted in accordance with protocols approved by the nmrcd institutional animal care and use committee . vaccinated monkeys received vaccine candidate ( 10 ng ) on days 0 , 14 , and 28 with 10 % aluminum hydroxide adjuvant and pbs . the agent was given as 5 divided doses intradermally at the base of the back under anesthesia by trained veterinary staff . control monkeys received equivalent doses of adjuvant and pbs on days 0 , 14 , and 28 . blood samples were obtained under anesthesia on days 0 , 14 , 28 , and 62 . capture eias were standardized and performed in order to detect anti - denv - 1 igm and igg in both groups . for igg and igm eias , 0 . 10 od was determined as the cut - off value for positive antibody . adjusted optical density ( od ) values were determined by the difference of the od of the control antigen - coated well subtracted from the corresponding viral antigen - coated well . plaque reduction neutralization test ( prnt ) assays were performed using bhk - 21 cells . serial dilutions ( 1 / 40 , 1 / 80 , 1 / 160 and 1 / 640 ) of heat - inactivated mouse serum were prepared and mixed with an equal volume of a working dilution of denv - 1 west pac 74 ( 10 - 20 pfu / 50 μl ) and then incubated at 4 ° c . overnight . at the end of incubation , the diluted mixture was inoculated into 24 - well plates containing 3 × 105 pfu / ml of a bhk cell suspension . after preparation and 7 days of incubation at 37 ° c ., the plaques were counted and 50 % plaque reduction was determined by probit analysis . on day 132 , both groups of monkeys were injected with 1 ml of 1 . 1 × 104 pfu / ml of denv - 1 west pac 74 virus as challenge . physical examinations and blood sampling were performed on days 133 , 142 , 146 , and 160 . anti - denv - 1 igm and igg were measured to determine response to primary infection or amnestic response in both groups . on days 133 - 142 sera were for denv1 by vero cell culture isolation . results of the challenge study are shown in table 2 . when administered intradermally to aotus nancymaae monkeys , amt - inactivated denv - 1 was immunogenic at 14 , 28 , and 62 days , with detectable igm and igg by eia and with neutralizing antibodies as assessed by 50 % plaque reduction neutralization ( prnt50 ). following experimental challenge with denv - 1 , vaccinated monkeys showed an amnestic response to infection with a rapid increase in igg titers . control monkey , by comparison , showed an increase in igm without detectable igg until day 10 after challenge . control animals demonstrated an average of 3 . 66 days of viremia post challenge , while vaccinated animals only demonstrated 0 . 71 days for an 81 % reduction of days of viremia in vaccinated vs . control animals ( table 3 ). in this study , psoralen - inactivated dengue - 1 viruses are immunogenic in a non - human primate model . there are multiple candidate vaccines in development for dengue virus infections , including dna vaccines , live attenuated viruses , chimeric vaccines based on an attenuated yellow fever virus , and recombinant subunit vaccines . an inactivated virus vaccine generally will not induce cell - mediated immunity , but experimental data suggests that antibodies and other humoral factors may play a more important role than cell - mediated factors in the prevention of dengue infections . psoralen inactivation has the potential advantage of being relatively inexpensive and could serve as a boosting component of a prime - boost dengue vaccine strategy . in conclusion , amt - inactivated denv - 1 is immunogenic in aotus nancymaae monkeys . monkeys receiving amt - inactivated denv - 1 further demonstrate an amnestic response to subsequent live denv - 1 challenge and protected immunized animals against denv1 infection . in order to determine the optimal inactivation conditions for selected denv - 2 , 3 , and 4 virus strains using similar procedure as in example 1 . perform in vitro viral inactivation using mouse sera similar to example 2 , and in vivo immunogenicity testing of psoralen - inactivated dengue - 1 virus vaccine candidate in aotus nancymaae monkeys as in example 3 . to extend the use of amt photo - inactivation to other viruses for the use as vaccines , yellow fever ( yf ), west nile virus ( wnv ), saint louis encephalitis ( sle ) and venezuelan equine encephalitis ( vee ) viruses were photo - inactivated with amt and uva . the virus used are : 1 . yf ( fmd 1240 ) c3 / 36 p - 3 jan . 23 , 2008 titer = 5 . 4 × 10 5 pfu / ml ( bhk - 21 sep . 21 , 2009 [ day 6 ]) 2 . wnv ( cdc ) m29539 ( ny99 - 35262 - 11 ) vero - 3 sep . 17 , 2008 ; titer = 3 . 07 × 10 5 pfu / ml ( llcmk2 [ day 4 ]) 3 . sle tbh - 28 cdc m29777 smb vero - 3 sep . 17 , 2008 ; titer = 6 . 8 × 10 4 pfu / ml ( bhk - 21 sep . 18 , 2009 [ day 3 ]) 4 . vee tc 83 tvb 5215 vero - 4 aug . 26 , 2009 titer = 5 . 45 × 10 9 pfu / ml ( vero - 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