Patent Application: US-58689406-A

Abstract:
the invention concerns a method of detecting dengue infection by detecting anti - dengue neutralizing substances including anti - dengue antibody by measuring inhibition of dengue binding to dentritic cell - specific intracellular adhesion molecule 3 - grabbing nonintegrin or liver / lymph node - specific intracellular adhesion molecule 3 - grabbing nonintegrin . dc - sign or l - sign can be used expressed in transfected cell lines that normally are not infected by dengue virus . the invention also concerns a method of detecting anti - dengue drugs by their ability to inhibit binding of dengue virus to dc - sign or l - sign .

Description:
important limitations exit for currently available methods analyzing patient serum , such as prnt assay . therefore , assays capable of analyzing large numbers of patient serum for diagnosis of dengue infection is needed . the current invention incorporates the ability of dc - sign and l - sign to mediate dengue infection 5 ( tassaneetrithep , et al , 2003 ) into a virus neutralization assay . table 1 illustrates the ability of dc - sign to facilitate infection into a raji b cell line . in table 1 , infectivity was assessed by flow cytometry as a function of varying inputs of virus and time of harvesting infected cells . p values were obtained from a student &# 39 ; s t test for independent samples against mock - infected cells . moi a 0 . 0005 b 0 . 0001 0 . 00001 p value p value p value dengue 1 48 hrs 13 . 3 % & lt ; 0 . 001 3 . 3 % 0 . 003 0 . 2 % 0 . 091 72 hrs 39 . 0 % & lt ; 0 . 001 26 . 3 % & lt ; 0 . 001 7 . 3 % & lt ; 0 . 001 96 hrs 42 . 5 % & lt ; 0 . 001 30 . 4 % 0 . 005 14 . 8 % & lt ; 0 . 001 dengue 2 48 hrs 12 . 3 % 0 . 682 2 . 6 % 0 . 11 0 . 1 % 0 . 102 72 hrs 31 . 2 % & lt ; 0 . 001 14 . 3 % 0 . 003 2 . 1 % 0 . 111 96 hrs 44 . 3 % & lt ; 0 . 001 35 . 8 % & lt ; 0 . 001 6 . 9 % & lt ; 0 . 001 dengue 3 48 hrs 1 . 0 % 0 . 2 0 . 1 % 0 . 149 0 . 0 % — 72 hrs 8 . 9 % & lt ; 0 . 001 2 . 0 % & lt ; 0 . 001 0 . 6 % 0 . 001 96 hrs 25 . 3 % & lt ; 0 . 001 8 . 3 % 0 . 002 1 . 5 % 0 . 004 dengue 4 48 hrs 10 . 8 % & lt ; 0 . 001 2 . 2 % 0 0 . 0 % 0 . 22 72 hrs 47 . 1 % & lt ; 0 . 001 39 . 1 % & lt ; 0 . 001 4 . 3 % 0 . 37 96 hrs 52 . 1 % & lt ; 0 . 001 42 . 2 % & lt ; 0 . 001 25 . 7 % & lt ; 0 . 001 a moi = multiplicity of infection . b moi of 0 . 001 corresponds to an input of 60 plaque forming units ( pfu ). the inventive method is predicated on the reduction in infectivity of cells expressing dc - sign or l - sign . normally dengue infection - insensitive tumor cells , such as raji b cells can be transfected with dc - sign making them high susceptible to dengue infection . raji b cells expressing dc - sign rapidly accumulate easily detectable amounts of dengue antigen . therefore , discrimination between infection and non - infection is readily discemable within 24 hours ( tassaneetrithep , et al , 2003 ). by utilizing millions of cells with each representing the analog of one potential plaque , the sensitivity of the assay is increased dramatically . the inventive method contemplates any method for measurement of dengue infection , including microscopy . however , a preferred embodiment is the measurement of anti - dengue antibody binding by flow cytometry . flow cytometry is an extremely sensitive measurement technique and further improves assay reliability and amenable to high - throughput analysis . additionally , since only small amounts of serum are required , conservation of resources is improved over currently available methods such as prnt . a . serial dilutions of test sera and control ( non - immune ) sera are added to inocula of virus at a dose pre - determined to yield detectable infectivity ; b . incubate sera / virus mixture for 30 minutes ; c . add incubated sera / virus mixture to dc - sign transfected cells cultured into 96 - well plates ; d . incubate virus / sera added transfected cells overnight ; e . measure intracellular dengue antigen 20 to 24 hours after infection ; f . from measurement of intracellular dengue antigen , determine the percent neutralization of infection of the sera dilutions based on control samples . the percent reduction of infection at each dilution is calculated as the number of positive cells from normal sera sample minus the number of positive cells from the test sample divided by the number of positive cells from the normal sera sample multiplied by 100 . the resultant is a reduction expressed as a percentage . in this manner , any non - specific neutralization of normal sera is subtracted out and baseline reduction is set at 0 . a reduction of infection at each dilution is analyzed by probit analysis / best - fit quadratic estimation and curve fitting non - linear regression analysis . because some cells , such as raji b cells , express fcr , it is possible that functional internalization of dengue virus complexed to non - neutralizing antibody may be measurable simultaneously with neutralization of infectivity . therefore , the net effect is dependent solely upon the characteristics of the particular serum specimen analyzed . the estimation of net enhancement of infection verses net neutralization of infection can be determined mathematically . as a specific example , neutralization of specific anti - dengue antibody ( 3h5 ) compared to anti - flavivirus antibody ( 4g2 ) and the negative control antibody 15f3 using prc / cmv - dc - sign transfected raji b cells ( geijtenbeek , et al , 2000 ). measurement of dengue infection was by flow cytometry . the assay was conducted in 96 - well plates in duplicate . neutralizing sera and normal sera control was 4 - fold serially diluted in complete growth media ( gm )( rpmi 1640 supplemented with l - glutamine , fetal bovine serum ( fbs ) and penicillin and streptomycin . dengue virus is added to the sera dilutions . the virus is used at a dilution to yield a multiplication of infection ( moi ) that will result in an approximately 10 - 20 % infection rate . the plates were then incubated at 37 ° c . under 5 % co 2 for 30 minutes . in addition to test samples , control samples were prepared containing sera plus media ( in place of virus ). additionally , controls samples for back - tritration were prepared containing several dilutions starting at the 1 : 10 dilution viral dilution yielding an moi giving 10 - 20 % infection rate plus media . subsequent to incubating sera and virus , dc - sign transfected raji b cells were seeded at 1 . 2 × 10 5 per well . the dc - sign transfected raji b cells , sera and virus was incubated for 24 hours at 5 % co 2 at 37 ° c . infectivity was analyzed by measuring intracellular prem - antigen by flow cytometry . intracellular dengue virus antigen was detected by first fixing cells with 4 % paraformaldehyde , permeabilitzed with 0 . 5 % saponin using direct staining with antibody ( e . g . anti - dengue 2 , 3h5 or anti - flavivirus , 4g2 or non - neutralizing antibody , 15sf3 ). samples were analyzing by acquiring approximately 10 , 000 events per sample . the control ( uninfected ) samples were used as background reference and a graphical representation of the percentage of infection verses associated virus dilution was determined . a plot is then constructed whereby percent infection is a function of increasing moi . fig1 shows results of this experiment as percent neutralization verses antibody dilution . fig2 illustrates the effectiveness and specificity of neutralization ( e . g . compare the anti - dengue 3h5 with anti - flavivirus 4g2 ) of two dilutions of antibody compared to negative control . this example illustrates the use of the current inventive method using raji b cell tumor and flow cytometry . however , it is recognized that other the assay may be used by neutralizing the infectivity of dengue to other cell lines transfected with dc - sign . it is also contemplated as part of the current invention that dc - sign can be attached to other surfaces such as microbeads . in this case the percent neutralization is the inhibition of binding of dengue to immobilized dc - sign . 1 . alvarez , c . p ., f . lasala , j . carrillo , o . muniz , a . l . corbi , and r . delgado . 2002 . c - type lectins dc - sign and l - sign mediate cellular entry by ebola virus in cis and in trans . j . viro . 76 : 6841 - 6844 . 2 . baribaud , f ., s . pohlmann , g . leslie , f . mortari , and r . w . doms . 2002 . quantitative expression and virus transmission analysis of dc - sign on moncyte - derived dendritic cells . j . virol . 76 : 9135 - 9142 . 3 . colmenares , m ., a . puig - kroger , o . muniz pello , a . l . corbi and l . rivas . 2002 . dendritic - cell specific icam - 3 grabbing nonintegrin ( dc - sign , cd209 ), a c - type surface lectin in human dendritic cells , is a receptor for leishmania amastigotes . j . biol . chem . 277 : 36766 - 36769 . 4 . chan , l . and m . guan . u . s . pat . no . 5 , 824 , 506 , october 1998 . 5 . curtis , b . m ., s . scharnowske , and a . j . watson . 1992 . sequence and expression of a membrane - 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