Patent Abstract:
this invention provides flavivirus vaccines that comprise live - attenuated flaviviruses and methods of making and using these vaccines . the flavivirus vaccines described herein possess higher potency due to in situ production of additional immunogens in a way that mimics viral infection and the vaccines have potential for higher potency , reducing costs for production and delivery .

Detailed Description:
as used herein the specification , “ a ” or “ an ” may mean one or more . as used herein in the claim ( s ), when used in conjunction with the word “ comprising ”, the words “ a ” or “ an ” may mean one or more than one . as used herein “ another ” or “ other ” may mean at least a second or more of the same or different claim element or components thereof . furthermore , unless otherwise required by context , singular terms shall include pluralities and plural terms shall include the singular . as used herein , the term “ or ” in the claims is used to mean “ and / or ” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive , although the disclosure supports a definition that refers to only alternatives and “ and / or .” as described herein , the term “ single - cycle flavivirus ” refers to a flavivirus that is unable to produce infectious particles in vaccinated animals due to deletion of its capsid ( c ) gene . as described herein , the terms “ chimera ” and “ chrimeric flavivirus ” refer to a type of flavivirus comprising a live - attenuated virus vaccine derived from one flavivirus that expresses flavivirus glycoproteins from a second flavivirus . as described herein , the terms “ trimera ” and “ trimeric flavivirus ” refer to a type of chimeric flavivirus vaccine that expresses all three flavivirus glycoproteins from a target pathogen in an lav derived from another flavivirus . flaviviruses cause vaccine - preventable diseases that are responsible for considerable morbidity and mortality worldwide . there are only two types of vaccines for flavivirus disease that are currently approved for use in man . these include live attenuated viral vaccines ( lav ), such as the yellow fever virus ( yfv ) strain 17d ( yf 17d strain ; used worldwide ), and inactivated viral vaccines ( inv ), such as a formalin - inactivated preparation of japanese encephalitis virus ( jev ) obtained from the brains of virus - infected mice . existing vaccines for flavivirus diseases need improvement , and no vaccines exist for dengue ( the flavivirus disease with the highest incidence worldwide ) or diseases caused by west nile virus ( wnv ; which is responsible for the largest epidemic of viral encephalitis in united states history ). live - attenuated virus vaccines are considered preferable to inactivated viral vaccines due to their economy of production and greater potency than inactivated viral vaccines . both cost and public health impact are greatly affected by potency , since many inactivated viral vaccines require boosts that are either minimal or unnecessary in the case of live - attenuated virus vaccines . live attenuated virus vaccines currently in use have been produced by empirical attenuation ( by passage in unnatural systems ), but these same methods have failed to produce useful live - attenuated virus vaccines for all four serotypes of dengue and other flaviviruses . alternatives to these empirically attenuated live - attenuated virus vaccines include genetically derived chimeras based on genetic engineering of other live - attenuated virus vaccines to serve as “ vectors ” to deliver the envelope proteins of a second flavivirus , producing an live - attenuated virus vaccine that can protect against infections by the second flavivirus . in the case of yf 17d , this technology , referred to as the chimerivax technology , was first applied to japanese encephalitis virus . briefly , the japanese encephalitis virus prm and e genes were substituted into the yf 17d genome , producing a new live - attenuated virus vaccine ( chimerivax je ) that could protect against japanese encephalitis virus . construction of these chimeric vaccines was based in part on early studies suggesting that the junctions between e and ns1 were the most fruitful places to construct viable intra - viral chimeras with high replicative ability suitable for lav strain formulation , and the belief that much of the protective immunity engendered by flavivirus vaccines is due to the immunity afforded by the e protein , especially when expressed as a viral particle or a sub - viral particle ( svp ). however , it has been known for decades that the flavivirus ns1 protein is also an important immunogen and ns1 immunity can prevent flavivirus - induced disease . and , moreover , as such , a genetically engineered chimera of the type shown in fig1 could be rendered less effective in a host carrying immunity to the ns1 protein produced by previous exposure to the “ vector ” used to create such a chimeric live - attenuated virus vaccine . by extension , multivalent live - attenuated virus vaccines , which seek to induce immunity to multiple flaviviruses ( as is envisioned for the required tetravalent vaccine for dengue ) would likely be further compromised by ns1 “ vector ” immunity ( if all were derived from a single vector ), resulting in competition among the chimeras , producing unequal immunity to all vaccine components . the instant invention 1 ) demonstrates that ns1 immunity induced by a special type of single - cycle live - attenuated virus vaccine derived from west nile virus is able to provide protection from infection , proving the importance of ns1 immunity to genetically engineered flavivirus vaccines ; 2 ) describes how to generate a new type of chimeric flavivirus vaccine ( referred to herein as a “ trimera ”) that expresses all three flavivirus glycoproteins from a target pathogen ( in this example japanese encephalitis virus ) in an live - attenuated virus vaccine derived from another flavivirus ( in this example west nile virus ); and 3 ) demonstrates that trimeras produced by this method provide superior immunity to the target pathogen ( in this example japanese encephalitis virus ) than typical chimeras ( table 4 ). in some embodiments of the present invention there is provided a live - attenuated trimeric flavivirus , comprising a first flavivirus encoding glycoproteins from a second flavivirus , comprising membrane precursor gene ( prm ), envelope gene ( e ) and a ns1 protein gene ( ns1 ) from a second flavivirus , wherein the second flavivirus is different from said first flavivirus . further to this embodiments , a representative second flavivirus is : 1 ) a dengue virus , including but not limited to dengue - 1 , dengue - 2 , dengue - 3 , or dengue - 4 virus ; or 2 ) a yellow fever virus , including but not limited to a yf - 17d yellow fever virus ; or 3 ) a west nile virus ; or 4 ) a japanese encephalitis virus . further to this embodiments , a representative first flavivirus is west nile virus , japanese encephalitis virus , yellow fever virus or dengue virus . in another embodiments of the present invention , the live - attenuated trimeric flavivirus contains one or more than one mutation ( s ) in : 1 ) amino acid 18 of the ns4a protein ; 2 ) amino acid 29 of the ns4a protein ; 3 ) amino acid 135 of the ns4a protein ; 4 ) amino acid 47 of the prm protein ; 5 ) amino acid 62 of the capsid protein ; or a combination thereof . further to these embodiments , the live - attenuated trimeric flavivirus contains one or more than one mutation ( s ) consisting of : 1 ) a glycine to arginine mutation at amino acid 18 of the ns4a protein ; 2 ) a valine to isoleucine mutation at amino acid 29 of the ns4a protein ; 3 ) a valine to methionine mutation at amino acid 135 of the ns4a protein ; 4 ) a aspartic acid to asparagine mutation at amino acid 47 of the prm protein ; 5 ) a threonine to serine mutation at amino acid 62 of the capsid protein ; or a combination thereof . in yet another embodiment of the present invention there is provided a method of inducing an immune response to a pathogenic flavivirus infection in a patient . further to these embodiments , the method comprises administering to the patient a live - attenuated trimeric flavivirus discussed supra . further to these embodiments , the patient discussed supra does not have , but is at risk of developing , said flavivirus infection . further to these embodiments , the patient discussed supra has flavivirus infection . in yet another embodiment of the present invention there is provided an immunogenic composition comprising the live - attenuated trimeric flavivirus discussed supra and a pharmaceutically acceptable carrier or diluent . in yet another embodiment of the present invention there is provided a method of increasing the potency and efficacy of a chimeric live attenuated virus vaccine by addition of a ns1 protein gene to the chimeric live attenuated virus vaccine , thereby eliciting better immune responses to e and to the relevant ns1 . the following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion . one skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned , as well as those objects , ends and advantages inherent herein . changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art . large - scale production of replivax for human use requires a cell line that : 1 ) produces high levels of c , supporting high - titer growth of replivax , 2 ) encodes a c gene that cannot productively recombine with the replivax genome to produce a “ live ” virus , and 3 ) can be propagated serially without losing the c gene . high - level c expression was assured by using a non - cytopathic veerep ( 53 ). eliminating the ability of replivax wn to recombine with cell - expressed c to produce a genome capable of spreading in the vaccinated host was addressed by three independent methods that produced a modified c gene ( c *) unable to recombine with replivax wn . these methods were 1 ) the c * gene corresponded to the smallest functional fragment of the west nile virus genome ( the mature c gene ), 2 ) the c * gene contained synonymous mutations in the region overlapping the remnant of c ( trc ) present in replivax wn ; and 3 ) the synonymous mutations in c * ablated the cyclization sequence ( cs ) ( 1 ). stable , long - term expression of c by packaging cells was addressed by fusing c * to a puromycin ( pur ) acetyl - transferase gene ( pac ) driven by the subgenomic promoter of the veerep ( 55 ), producing replicon veerep / pac - ubi - c *. this fusion reduces the chance of “ loss ” of the c gene by intermolecular recombination during propagation of veerep - expressing cells , which has been observed ( 52 ). pur - resistant bhk cells transfected with veerep / pac - ubi - c * ( bhk - c *) were able to produce replivax wn at titers of & gt ; 10 7 infectious units ( iu )/ ml ( fig3 ). importantly , when monolayers of passage 8 ( p8 ) and p70 bhk - c * were infected with stocks of replivax wn , no differences were detected in either infectious focus number or size , demonstrating extraordinary phenotypic stability ( essential for utility as a master cell stock for human vaccine production ). further , sequencing across the c gene of bhk - c * at p24 failed to detect any changes relative to veerep / pac - ubi - c *, confirming stability ( 1 ). to demonstrate that replivax wn did not recombine with the c gene in bhk - c *, replivax wn was serially passaged ( sp ) in bhk - c * cells . after sp30 , undiluted replivax wn was then blind passaged on vero cells ( to remove contaminating replivax ), and the entire second - pass monolayer was stained for viral antigen , revealing no infected cells , thus rigorously demonstrating absence of productive recombination ( 1 ). since bhk cells are not a suitable substrate for human vaccine manufacture , vero - c * cells were produced by applying the methods outlined above to human vaccine production - compatible vero cells ( from s . whitehead , nih ) maintained in serum - free media . these cells , which have maintained their complementing phenotype for dozens of passages , support high levels of production of replivax wn . although replivax titers obtained on vero - c * are lower than those achieved with bhk - c *, the vero - c * did not show cytopathic effect , permitting repeated harvesting {( 1 ) & amp ; fig3 }. these properties indicate that multiple harvests could be used for vaccine preparation , and suggest that replivax harvests will be low in vero dna content , facilitating manufacture and licensure . although the inexpensive antibiotic pur is used during routine c * cell passage , the veerep is maintained in these cells for up to a week in its absence , and pur is always excluded from cultures during replivax growth . taken together , these data demonstrate the high stability of gene expression in these packaging cell lines , lack of infectious virus formation , and high efficiency of replivax packaging into infectious particles in a cell line suitable for preparation of an lav that could be used in man . replivax wn serially passaged 10 times ( sp10 ) in bhk - c * cells produced polymorphic foci of infection on bhk - c * cells , with many foci 3 - to 5 - times larger than those produced by unpassaged replivax wn ( 1 ). furthermore , sp10 replivax wn replicated faster than our original replivax wn , with an endpoint titer twice as high ( 1 ). analyses of the trc through prm region of sp10 replivax wn revealed two mutations . as expected from the heterogeneous nature of the sp10 foci , both mutations were present as mixtures . one mutation consisted of an aag_atg ( k_m ) at position p3 following the ns2b / ns3 cleavage site ( qkkr | ggk ( m ) t ) in trc . the second mutation consisted of an agc_tgc ( s_c ) at a position preceding the prm signal peptidase cleavage site ( s ( c ) vga | vtls ). re - derivation of 2nd - generation replivax wn with either mutation demonstrated that either change produced better - growing replivax wn . the re - derived clone containing the s & gt ; c mutation in the signal peptidase cleavage site was designated replivax wn . 2 , and used for all further studies . altering ns2b / ns3 and signal peptidase cleavage between c and prm has been shown to influence flavivirus particle yield and infectivity ( 56 - 59 ). mouse and hamster studies demonstrated 100 % protection by a single immunization of replivax wn . 2 which produced 90 % neut antibody titers of 1 : 40 at a dose of 4 × 104 iu / mouse and 1 : 160 at a dose of 2 × 105 iu / hamster { the lowest doses tested for each species ; ( 1 )}. these studies also indicated that the second - generation replivax was even more potent than the first ( 1 ). work on a large number of flavivirus vaccine candidates has demonstrated that small animal model potency and efficacy can be predictive of responses in primates . however , in several cases ( notably dna vaccines ), lab animal data has not correlated with eventual utility . to be certain that replivax was a useful platform , a preliminary non - human primate study utilizing the replivax wn . 2 vaccine candidate was undertaken . for this study 4 rhesus macaques { seronegative for all commonly circulating flaviviruses ( 60 )} were inoculated with 106 iu of replivax . evaluation of sera collected from these animals 28 days later revealed 50 % neut titers ( the standard used for non - human primate research ) of 1 : 32 to 1 : 64 , a bit lower than the 50 % neut titers reported for the yf - 17d - based chimerivax - wn vaccine ( 61 ), a result consistent with the fact that chimerivax - wn produced detectable viremia in every monkey included in these published studies ( 61 ). although the single - dose neut titers of the animals were lower than those obtained with live - attenuated virus vaccines capable of producing a viremia , they were similar to those detected in monkeys after receiving two doses of the commercially available japanese encephalitis virus - inactivated viral vaccine ( 62 ). nevertheless , half of the monkeys were boosted with a second dose of replivax wn . 2 . following challenge of these 4 animals and a control , diluent - inoculated macaque with 100 , 000 pfu of wnv ny99 , the animals were tested for viremia on d 1 , 2 , 4 and 6 post - challenge . the single control animal in this experiment displayed viremias of & gt ; 100 pfu / ml on d 1 , 2 , & amp ; 4 , and a viremia at the limit of detection ( 7 . 5 pfu / ml ) on d 6 . among the two singly vaccinated animals , one displayed a viremia at the limit of detection ( 7 . 5 pfu / ml ) on d 1 , and all other sera from this animal and all sera from the remaining three vaccinated animals did not display any detectable viremia on any of the other days tested . thus , this study has clearly documented the efficacy of replivax wn . 2 in non - human primates . by substituting the prm / e genes of japanese encephalitis virus for the corresponding genes in replivax wn , a chimeric replivax expressing japanese encephalitis virus svps was generated . replivax japanese encephalitis virus grew poorly in bhk - c * cells , but quickly adapted to grow more rapidly ( 54 ). an analogous mutation to those described in 3 . 2 was detected in the genome of this passaged variant , and this mutation was used to produce a replivax je . 2 which grew to titers of & gt ; 10 7 iu / ml on bhk - c * cells ( 54 ). studies on svp synthesis from vero cells infected with parental and 2nd - generation replivax japanese encephalitis virus indicated that the single mutation in the ns2b / ns3 cleavage site increased the amount of svps produced by replivax je . 2 in normal cells ( 54 ). mice and hamsters immunized with replivax je . 2 produced high - titer , japanese encephalitis virus - specific neut titers , and these mice were completely protected from lethal japanese encephalitis virus challenge ( 54 ). there is no suitable japanese encephalitis virus model for hamsters , so these replivax je . 2 - vaccinated hamsters were challenged with west nile virus , based on a report demonstrating that an live attenuated virus vaccine for japanese encephalitis virus could protect hamsters from west nile encephalitis ( wne ) ( 64 ). as expected from cross - neut titers and anti - ns1 titers , replivax je . 2 - vaccinated hamsters were completely protected from lethal west nile virus challenge ( 54 ). early ( 7 d post inoculation ) cellular responses of c57 / bl6 mice to replivax wn . 2 vaccination ( 10 4 or 10 5 iu ) were comparable to those produced by west nile virus infection ( 1 , 000 pfu ). splenic lymphocytes from the replivax wn - infected animals produced robust cd4 and cd8 t cell cytokine responses following ex - vivo stimulation with synthetic peptides representing dominant wnv - specific epitopes for each type of lymphocyte { mason & amp ; nikolich - zugich , ohsu , unpublished and ( 34 )}. replivax wn . 2 also elicited a strong cd8 t cell cytotoxic response in these same unpublished studies . these antigen - specific responses were dose - dependent , and replivax wn . 2 proved more potent than live west nile virus , indicating that replivax wn . 2 induced strong antigen - specific t cell responses that likely contribute to its efficacy . mice inoculated with west nile virus vrps ( single - cycle particles with the same external structure at replivax — see 3 . 0 & amp ; 3 . 1 ) produce large amounts of interferon a ( ifn a ) as early as 8 h post injection ( 65 ), and high levels of interferon a mrna , replicon rna , and replicon - encoded antigen were detected in the draining lymph nodes at 24 hour post inoculation ( 65 ). animals inoculated with uv - inactivated vrps did not produce detectable interferon , indicating that infection was needed to trigger this response . since monocyte - derived dendritic cells ( mdcs ) are likely to be involved as initial targets infectious agents such as replivax , and presentation of its encoded antigens to the adaptive immune system , and plasmacytoid dcs ( pdcs ) are likely to be involved in production of interferon a ( which can modulate adaptive immune responses ), studies were performed to test the response of these important cell types to west nile virus infection . to this end , pdc and mdc cultures obtained from pbmc of healthy human donors were incubated with wnv for 24 hour , and interferon a in the cell culture supernatants was detected using elisa . west nile virus elicited the expression of interferon a in both pdc and mdc in a moi - dependent manner , and pdcs produced about ten - times more interferon a on a per - cell basis than the mdcs . ( 66 ). interestingly , in the case of pdcs , uv - west nile virus was equivalent to west nile virus in its ability to induce interferon production , whereas in mdcs , uv - west nile virus was inactive ( 66 ). effective stimulation of innate immunity is likely important for replivax potency and efficacy , since these responses direct the adaptive immune response ; hence interferon induction will be used as a measure for improving replivax . replivax combines many useful aspects of current inactivated viral vaccine and live attenuated virus vaccine technologies , and is hence likely superior to all existing vaccine candidates . prm - e - ns1 replivax je chimeras in both replivax yf and replivax wn genetic backbones replivax japanese encephalitis virus chimera built on a replivax wn backbone grew well in bhk - c * cells and protected hamsters from wne . these replivax - je - vaccinated hamsters were challenged with west nile virus due to lack of a useful hamster model for je , as a test of the cross - protective immunity raised to jev . however , replivax je - elicited immunity to west nile virus ns1 ( derived from the replivax wn backbone ) could have helped to protect against wne , consistent with previous studies showing that ns1 immunity can provide protection . therefore , studies were performed to test the role of ns1 immunity in providing protection from wne in hamsters by comparing vrps ( which express only ns1 ) with replivax wn . 2 ( which expresses both west nile virus prm / e svps and ns1 ). these studies demonstrated that replivax wn . 2 provided complete protection from both morbidity and mortality , whereas vrps provided 100 % protection from mortality , but failed to keep all hamsters from becoming ill ( see table 1 ). although these studies cannot exclude that other aspects of the immune response ( notably cellular immunity ) help replivax wn to supply superior immunity over vrps , these data strongly suggest that ns1 - specific humoral immunity participates in replivax efficacy . these studies have two profoundly important implications for chimeric replivax development . first , inclusion of ns1 in a chimera would be expected to produce a better vaccine against the target agent . second , and equally importantly , this finding indicates that ns1 immunity could interfere with the re - use of chimeric vaccines ( since previous immunity to ns1 could interfere with subsequent vaccination with a chimera sharing the ns1 protein present in the first replivax ). this same rationale would argue that tetravalent live attenuated virus chimeras could compete with each other following vaccination , producing an “ interference ” that might help to explain previously reported unequal immune responses to all four serotypes of dengue virus following a single immunization of a tetravalent dengue live attenuated virus vaccine preparation ( containing chimeras ) in non - human primates ( 67 ). interference is not a new idea ; it has been discussed before in the context of chimeric yf - 17d - based vaccines . interestingly , the possibility that yfv immunity ( again likely due to ns1 immunity ) could prevent immunization with yf - 17d - based chimera expressing denv2 prm / e , was supported by one non - human primate study ( 68 ) but refuted in a human trial ( 69 ). to determine the feasibility of adding ns1 from a “ foreign ” flavivirus to a flavivirus live attenuated virus vaccine , previously described replivax japanese encephalitis virus live attenuated virus vaccine construct { containing the attenuating , single - cycle phenotype conferred by using a deleted capsid ( c ) gene } was selected . addition of ns1 was accomplished by precise fusion of the last codon of ns1 of jev to the first codon of ns2a of west nile virus . initially , this construct did not replicate well in cell culture ( fig4 ), but upon repeated passage in cells encoding the c gene needed to trans - complement the c - deletion in this ns1 - expressing replivax japanese encephalitis virus chimera ( referred to hereon as triplivax ), produced a triplivax with a mutation in the ns4a gene ( at position 295 in the ns4a gene ) that grew well ( fig4 ). introduction of this change into the original triplivax japanese encephalitis virus construct confirmed its ability to confer the high - growth phenotype ( results not shown ). to test the comparative efficacy of triplivax japanese encephalitis virus ( created by passage ) and replivax je , mice were vaccinated with these constructs , bled 21 days later , and then challenged one week later with approximately 30 50 %- lethal - doses ( ld50 ) of the beijing strain of japanese encephalitis virus . these same studies included groups of mice inoculated with vrps encoding the original west nile virus ns1 protein as well as chimeric west nile virus - derived vrps encoding the japanese encephalitis virus ns1 protein . table 3 shows the data obtained from sera collected from these mice 21 days following inoculation . as expected all constructs encoding the japanese encephalitis virus ns1 protein produced higher titers of antibodies to ns1 . most remarkable however are the findings that co - expression of the japanese encephalitis virus ns1 with the japanese encephalitis virus prm / e cassette resulted in an lav , which elicited much higher levels of antibodies to the japanese encephalitis virus e ( see shaded areas of table 2 ). most importantly , the enhanced japanese encephalitis virus e - specific immunity was observed in both the elisa assay , and in the functionally important neutralization ( neut ) assay . in this assay , triplivax japanese encephalitis virus produced much higher neut titers than replivax japanese encephalitis virus at both the 1 × 10 4 and 4 × 10 4 doses ( table 2 ). when the animals from the experiment shown in table 3 were challenged with japanese encephalitis virus , all of the vaccinated groups displayed significantly better protection ( p = 0 . 001 by fisher &# 39 ; s exact test using a 2 - tailed comparison ) than the diluent group ( table 3 ). triplivax japanese encephalitis virus also appeared to protect better than replivax japanese encephalitis virus , but these results were not statistically significant with this small number of animals ( table 3 ). the experiment displayed in tables 3 and 4 also shows that the jev ns1 protein , when expressed as part of a vrp , produced higher antibody titers to the ns1 antigen in elisa ( table 2 ) and provided better protection from jev challenge ( table 3 ). to obtain additional information on the utility / superiority of triplivax japanese encephalitis virus versus replivax je , groups of 9 or 10 mice were immunized with lower doses of the two vaccines ( 2 . 5 × 10 3 and 6 . 25 × 10 2 iu ) and challenged with 30 ld50 of japanese encephalitis virus on 4 - week post immunization . as shown in table 5 , 20 % and 50 % of mice immunized with 2 . 5 × 10 3 and 6 . 25 × 10 2 iu of replivax japanese encephalitis virus died in 21 days , respectively . on the other hand , 2 . 5 × 10 3 iu of triplivax japanese encephalitis virus provided 100 % protection and 6 . 25 × 10 2 iu of triplivax japanese encephalitis virus produced 90 % protection from death . for all four of these vaccinated groups , all of the mice that survived did not show any measurable manifestations of jev - induced disease , and none of the surviving mice displayed a challenge - induced weight loss ( table 5 ). on the other hand , a large fraction ( 3 out of 9 ) of the mice that were given diluent and survived infection displayed considerable weight loss in the challenge period ( table 5 ). thus , triplivax japanese encephalitis virus is a better vaccine than replivax japanese encephalitis virus based on antibodies it elicits to e , antibodies it elicits to ns1 , and efficacy data in a mouse model for japanese encephalitis virus . by extension , addition of ns1 to any type of chimeric live attenuated virus vaccine would be expected to improve its potency and efficacy . a mortality in these groups indicate the % of mice that died in the 21 - day observation period that followed challenge with 30ld 50 of jev 4 weeks post vaccination . b morbidity indicates the sum of animals that died during the 21 - day observation period and the animals that displayed a loss in weight of at least 20 % during the observation period . improving triplivax je by accumulation of adaptive mutations that occurred in multiple passages as described above , the anti - ns immunity conferred by single - cycle flaviviruses can protect animals from lethal flavivirus challenge . since a portion of this immunity was likely due to the ns1 protein , it was reasoned that incorporation of ns1 into a chimeric lav , such as replivax , would make the vaccine more effective . prior to construction of a prm / e / ns1 chimeric replivax je , a prm / e / ns1 chimeric live virus named trimera je was prepared in order to assess the replicational competence of prm / e / ns1 chimerization without the complicating factor of c - transcomplementation required for propagation of replivax chimeras . initially , the trimera je rna was introduced into bhk ( veerep / pac - ubi - wnns1ns2a ) cells expressing wnv ns1 as well as wild - type bhk cells . progeny trimera je was recovered from both cell lines ( data not shown ), indicating that trans - complementation with the authentic wnv ns1 was not essential for propagation of this trimera je . based on this result , wild - type bhk cells were used in all subsequent experiments using trimera je , to simplify the manipulations . initially trimera je grew poorly and produced small foci on bhk cells , but following 14 sequential passages , a derivative population of trimera je capable of producing larger foci was obtained . sequence analyses revealed that the blind - passed trimera je population contained four amino acid changes in c , prm and ns4a genes ( table 6 ). once it was confirmed that prm / e / ns1 chimerization was not lethal in the context of an intact viral genome , we constructed a prm / e / ns1 chimeric single - cycle je vaccine , which we named triplivax je , by replacing the wnv ns1 gene of replivax je with the jev ns1 gene . when introduced into bhk ( veerep / pac - ubi - c *) cells , triplivax je displayed small foci , similar to the foci observed with trimera je on wild - type bhk cells ( fig6 ). to obtain a better - growing derivative of this single - cycle virus , triplivax je was blind - passed in bhk ( veerep / pac - ubi - c *) cells . following 15 blind - passages , a triplivax je population capable of producing larger foci was obtained ( fig6 b ). to further characterize the phenotype of these viruses , the numbers of cells forming individual foci were counted . the foci of un - passaged triplivax je ( p0 ) contained 15 . 8 ± 8 . 8 ( n = 15 foci ) cells , while those of blind - passed triplivax je ( p15 ) contained significantly larger number of cells ( 47 . 1 ± 19 . 9 ; n = 15 foci ; p & lt ; 0 . 001 ). this better - growing triplivax je had a mutation ( v29i in ns4a ) identical to that found in trimera je ( table 6 ), suggesting that the v29i mutation in ns4a was responsible for the growth improvement of both types of prm / e / ns1 chimeric constructs . examination of the effects of the ns4a mutation in trimera je and triplivax je on their growth in order to examine the effects of the ns4a mutation found in blind - passed trimera je and triplivax je , the v29i mutation was introduced into the parental trimera je and triplivax je , producing new variants designated as trimera je - ns4a * and triplivax je - ns4a *, respectively . consistent with the passaged versions of the original constructs described above , trimera je - ns4a * produced foci that were larger than the parental trimera je and similar to those produced by blind - passed trimera je ( fig6 a ). as expected , trimera je - ns4a * grew better than parental trimera je and its growth kinetic was similar to that of blind - passed trimera je ( fig7 a ). thus , this ns4a mutation was responsible for the growth improvement of the passaged derivative of trimera je . similar examinations were performed using triplivax je - ns4a *. the foci produced by triplivax je - ns4a * on bhk ( veerep / pac - ubi - c *) cells were larger than parental triplivax je ( fig6 b ) and the mean number of cells forming a focus ( 45 . 8 ± 23 . 7 ) was similar to those of blind - passed triplivax je . however , side - by - side growth curves of triplivax je - ns4a * and its parental triplivax je , demonstrated that the new construct did not reach the titers obtained with the blind - passed triplivax je ( fig7 b ). these results suggested that an additional mutation in the highly passaged triplivax je ( possibly the other substitution in ns4a ( g18r )) could be responsible , in part , for the higher growth rate of the passage - 15 triplivax je . to determine if the v29i substitution altered the growth properties of replivax je and replivax wn , replivax je - ns4a * and replivax wn - ns4a * were constructed . by contrast to the effects of the mutation observed in trimera je and triplivax je , replivax je - ns4a * and replivax wn - ns4a * exhibited identical growth kinetics to those of the parent replivax je and replivax wn , respectively ( data not shown ). taken together , these data suggest that the growth improving properties of the v29i substitution in ns4a were only evident in chimeric viruses that encoded both the ns1 and prm / e cassettes of jev . in order to examine the effects of the ns4a mutation found in blind - passed trimera je and triplivax je , the v29i mutation was introduced into the parental trimera je and triplivax je , producing new variants designated as trimera je - ns4a * and triplivax je - ns4a *, respectively . consistent with the passaged versions of the original constructs described above , trimera je - ns4a * produced foci that were larger than the parental trimera je and similar to those produced by blind - passed trimera je ( fig6 a ). as expected , trimera je - ns4a * grew better than parental trimera je and its growth kinetic was similar to that of blind - passed trimera je ( fig7 a ). thus , this ns4a mutation was responsible for the growth improvement of the passaged derivative of trimera je . similar examinations were performed using triplivax je - ns4a *. the foci produced by triplivax je - ns4a * on bhk ( veerep / pac - ubi - c *) cells were larger than parental triplivax je ( fig6 b ) and the mean number of cells forming a focus ( 45 . 8 ± 23 . 7 ) was similar to those of blind - passed triplivax je . growth curves of triplivax je - ns4a * revealed more growth compared to its parental triplivax je , although triplivax je - ns4a * did not reach the titers obtained with the blind - passed triplivax je ( fig7 b ). these results suggested that an additional mutation in the highly passaged triplivax je ( possibly the other substitution in ns4a ( g18r )) could be responsible , in part , for the higher growth rate of the passage - 15 triplivax je . to evaluate triplivax je as a vaccine candidate in animal models , immunogenicity and protective efficacy of triplivax je were compared to replivax je . in this experiment , blind - passed triplivax je which contained mutations in ns4a was used . at 21 days post - immunization , all mice immunized with either triplivax je or replivax je elicited detectable neutralizing antibodies ( table 7 ). all three groups immunized with triplivax je developed higher neutralizing antibody titers than groups immunized with similar doses of replivax je ( table 7 ). as expected , mice immunized with either je - ns1 - vrps or wn - ns1 - vrps failed to develop detectable neutralizing antibodies . antibody levels against jev e , jev ns1 and wnv ns1 were also assessed by elisa using individual sera . as expected from the neutralization data in table 7 , triplivax je - immunized mice showed higher levels of anti - jev e antibodies than replivax je at all three doses tested ( fig8 ). again , as expected from the neutralization data in table 7 , almost no immune responses against jev e were detected in je - ns1 - vrp - and wn - ns1 - vrp - immunized groups . unexpectedly , equivalent levels of antibody responses against jev ns1 were observed both in triplivax je - and replivax je - immunized groups , although je - ns1 - vrp - immunized mice developed higher anti - jev ns1 immune responses than wn - ns1 - vrp - immunized mice ( fig8 ). on the other hand , replivax je - immunized mice developed significantly higher anti - wnv ns1 immune responses than triplivax je - immunized mice ( fig8 ). almost no immune responses against wnv ns1 were observed in triplivax je - immunized groups . wn - ns1 - vrp - immunized mice showed higher immune responses against wnv ns1 than je - ns1 - vrp - immunized mice . these results suggested that triplivax je is a superior vaccine candidate to replivax je , since it induced better anti - e immune responses , although levels of antibodies to jev ns1 were comparable . to compare protective efficacy of triplivax je and replivax je , the mice were challenged with 30 ld 50 of jev beijing p3 strain at 28 days post - immunization . more than 90 % of mice immunized with either triplivax je or replivax je survived the challenge . single doses of 4 × 10 4 or 1 × 10 4 iu of triplivax je provided 100 % protection , whereas 4 × 10 4 or 1 × 10 4 iu of replivax je provided 90 % protection . both 2 . 5 × 10 3 iu of triplivax je and replivax je showed 90 % protection ( table 7 ). there were no significant differences in mortality or morbidity between groups immunized with triplivax je and replivax je . when both vrp - immunized groups were compared , je - ns1 - vrp immunization provided 80 % protection ( 20 % morbidity ), while wn - ns1 - vrp immunization provided only 22 % protection ( 78 % morbidity ) ( table 7 ). these results showed again the contribution of anti - ns immunity ( in particular ns1 ) to the protection animals from jev disease . to further evaluate the potential superiority of triplivax je as a vaccine to prevent je , a lower dose regimen of replivax je and triplivax was utilized . to this end , mice were immunized once with 2 . 5 × 10 3 or 6 . 25 × 10 2 iu of triplivax je or replivax je . interestingly , even this very low - dose immunization induced detectable neutralizing antibody titers ( 46 to 99 ), but there were no significant differences in neutralization titers detected between the responses to this low dose of triplivax je and replivax je at 21 days post - immunization ( table 5 ). in accordance with the serological data obtained from the evaluation of a high dose regimen described above , 2 . 5 × 10 3 iu of triplivax je - immunized mice induced higher anti - e and anti - jev ns1 immune responses than replivax je - immunized mice ( fig9 ). to evaluate efficacy , these mice were challenged with 30 ld 50 of jev beijing p3 strain at 28 days post - immunization . mice immunized with 2 . 5 × 10 3 iu of triplivax je exhibited 100 % protection ( 0 % mortality ) and mice immunized with 6 . 25 × 10 2 iu of triplivax je exhibited 90 % protection ( 10 % mortality ) ( table 5 ). in contrast , mice immunized with 2 . 5 × 10 3 iu of replivax je exhibited 80 % protection ( 20 % mortality ) and mice immunized with 6 . 25 × 10 2 iu of replivax je exhibited only 50 % protection ( 50 % mortality ). although the differences in protection between these groups were not significant , the trend between them supported the contention that triplivax je is a superior vaccine to replivax je . triplivax je displayed reduced immune interference caused by pre - existing anti - ns1 immunity as described above , anti - ns1 immunity plays an important role in flavivirus protection . this fact raised a concern that pre - existing anti - ns1 immunity could result in immune interference . since many chimeric flavivirus vaccines in development have utilized prm / e chimerization strategy , and thus share the same nonstructural protein backbone ( including ns1 ), anti - ns1 immunity induced by either infection or immunization could interfere with subsequent immune responses induced by a vaccine sharing the same ns1 . in these cases , prm / e / ns1 chimerization utilized in triplivax je could reduce interference with vaccine potency / efficacy . to test this hypothesis , mice were immunized with wn - ns1 - vrp to provide an initial vaccination that elicited a strong ns1 - specific immunity in the absence of any significant anti - e immune responses . at 20 days post - vaccination immune responses against wnv ns1 were seen in this group , but no detectable immune responses against jev e were observed , whereas control , mice ( inoculated with l15 media only ) did not display any responses to either antigen ( data not shown ). at 21 days post - immunization , all mice were then vaccinated with either 4 × 10 4 iu of triplivax je or replivax je . twenty one days later ( 42 days post first vaccination ), sera were collected and serological analyses were conducted . animals immunized first with l15 media followed by triplivax je or replivax je , developed good jev e - specific elisa antibody responses with group titers of 6580 and 4651 , respectively ( table 8 ). pre - existing ns1 immunity ( produced by initial immunization with wn - ns1 - vrps ), reduced the jev e - specific antibody responses elicited by either triplivax je or replivax je ( table 8 ). however the titer in triplivax je - immunized animals was 3408 which represented only a 48 % reduction in titer compared to that in animals immunized first with l15 , whereas in replivax je - immunized mice the titer was 1150 which represented a 75 % reduction in elisa antibody titer relative to that achieved in the group that was immunized first with l15 media . comparison of the elisa antibody levels found in individual animals in these groups revealed that both groups that were first immunized with wn - ns1 - vrps exhibited lower je e - specific immune responses relative to both groups of animals that were inoculated with l15 ( fig1 ). however , the elisa antibody levels of the groups of mice that were immunized first with wn - ns1 - vrps revealed that a preponderance of animals then vaccinated with replivax je demonstrated barely detectable anti - e responses ( p = 0 . 0002 ; vs animals immunized with replivax je in the absence of wn - ns1 - vrps ), whereas nearly all animals first immunized with wn - ns1 - vrps and then immunized with triplivax je had more robust anti - e responses , with many reaching antibody levels comparable to those observed in non - primed animals ( p = 0 . 0074 ; vs animals immunized with triplivax je in the absence of wn - ns1 - vrps ). these results demonstrate that previous immunization with wn - ns1 - vrps reduced the potency of triplivax je to a lesser extent than it reduced the potency of replivax je . the superiority triplivax je over replivax je in overcoming pre - existing ns - 1 immunity was further illustrated by examination of the neutralizing antibody titers ( table 8 ). in the absence of a wn - ns1 - vrp immunization , triplivax je and replivax je elicited neutralizing antibody titers of 155 and 120 , respectively . however , mice given wn - ns1 - vrps and then immunized with triplivax je developed a 121 neutralizing antibody titer , whereas mice given wn - ns1 - vrps and then immunized with replivax je only developed a 71 neutralizing antibody titer . a side - by - side repeat comparison of the ability of these pooled sera to neutralize jelucvrps confirmed the superior potency of the triplivax je relative to replivax je in animals that had high - titer antibodies to the wnv ns1 ( data not shown ). to further confirm these trends , neutralizing antibody titers were measured in individual sera obtained from these animals ( fig1 b ). as described above for the individual elisa data ( fig1 a ) and elisa titers and neutralization titers obtained with pooled sera ( table 8 ), mice given wn - 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