Patent Application: US-6106708-A

Abstract:
highly immunoreactive viral peptides are disclosed which are derived from the e protein of major groups of the flavivirus genus by computational analyses . these peptides are used in reliable diagnostic methods for the detection and diagnosis of flavivirus , detecting the presence of antibodies against flavivirus , and to form vaccine composition for the prevention of flavivirus infections in humans .

Description:
analysis of sequence conservation in the flavivirus genus and dengue species sequence conservation of e proteins was analyzed based on structure - guided multiple sequence alignment , neighbor - joining phylogenetic tree , and shannon entropy . twelve e proteins representative sequences were chosen from the major flavivirus groups where the genome polyproteins are available [ 65 including fig1 ]. conserved residues involved in critical interactions have also been identified [ 65 including fig1 ]. the taxonomic groups were defined based on the ictvdb nomenclature of the international committee on taxonomy of viruses [ 1 ]. the e protein sequences were extracted from polyproteins in the uniprot knowledgebase ( uniprotkb ) [ 1 , 4 ]. the multiple sequence alignment was manually edited using the cn3d sequence - structure viewer [ 1 , 5 ], guided by a reference structure alignment in mmdb [ 6 ] using three e protein structures ( 1oke , 1uzg , 1svb ), which were originally deposited in pdb [ 1 , 6 ]. the manual editing involves superimposing and aligning the structures in the structure viewer and then adding individual sequences manually and aligning them with the sequences from the structure . for the c - terminal regions outside the structural alignment (˜ 100 aa ), the sequences were aligned using clustalw [ 1 , 7 ] and then manually verified . a phylogenetic tree was generated using the neighbor - joining program in clustalw with 1 , 000 bootstrap replicates . the bootstrap values indicate the confidence in the estimated tree branches . the sequence conservation of dengue e proteins was further analyzed based on the multiple sequence alignment of 740 full - length proteins from all four serotypes . the conservation in each amino acid position was quantified based on the shannon entropy , as estimated by the nine component dirichlet mixture algorithm [ 1 , 8 ]. the entropy calculation took into account conservative substitutions of amino acids with similar physicochemical properties . selection analysis was studied using maximum likelihood methods to calculate non - synonymous / synonymous ( dn / ds ) nucleotide substitution ratio based on codon alignment and phylogenetic tree . the maximum likelihood methods evaluate the probability that the chosen model has produced the observed data . dna sequences were extracted from the ncbi nucleotide nt database [ 19 ], resulting in four datasets consisting of 146 , 269 , 121 , and 204 sequences for denv1 , denv2 , denv3 , and denv4 , respectively , as well as a fifth dataset totaling 740 sequences with all four serotypes combined . the codon alignment and consensus neighbor - joining tree was derived using mega [ 20 ]. to quantify selective pressure at a given codon site , estimates of synonymous ( ds ) and non - synonymous ( dn ) substitution rates were compared using a statistical test as described earlier [ 21 ]. if ds & gt ; dn ( or , ds & lt ; dn ), then a site was inferred to be negatively ( or positively ) selected . we employed two likelihood based methods — single likelihood ancestor counting ( slac ) and fixed effects likelihood ( fel ) [ 22 ] and considered sites to be “ selected ” when both slac and fel methods yielded a p - value of & lt ; 0 . 01 . slac reconstructs the most likely unobserved ancestral sequences , counts the number of non - synonymous and synonymous changes at every site , and tests whether the number of non - synonymous changes per non synonymous site is significantly different from the number of synonymous changes per synonymous site . fel derives the branch lengths and substitution rate bias ( global ) parameters from the entire alignment , and then directly estimates the ratio of non - synonymous to synonymous rates under a codon - substitution model for each site in a sequence alignment holding all global parameters fixed . the fel method is in general more powerful but also more computationally demanding than the slac method [ 22 ]. for this study we reported only those sites , which were concordantly classified with both methods . for the large combined set with all four serotypes , a more stringent cutoff of p & lt ; 10 - 5 was used [ 22 ]. selection analyses were performed using the hyphy program [ 23 ], which took into account nucleotide substitution biases and ds and dn rate variation across sites . to exclude the possibility of erroneous selection inference due to recombination between different denv serotypes , a maximum likelihood test for phylogenetic incongruence [ 24 ] was run to screen for possible recombination in the e protein ; no statistically significant recombination breakpoints were identified in any of the denv datasets . three online prediction programs , netmhc 2 . 1 [ 25 ], mhcpred 2 . 0 [ 26 ] and rankpep [ 27 ], were used to analyze the four serotypes of dengue e proteins . as both mhc - i and - ii molecules are highly polymorphic and the specificity of the alleles is often very different , predictions were performed on multiple supertypes to cover the polymorphic loci . netmhc was used for the prediction for 12 supertypes of mhc - i locus . a binding affinity threshold of 500 nm was used as the cutoff for mhc - i binding [ 26 ]. both mhcpred 2 . 0 and rankpep were used for mhc - ii binding predictions to obtain consensus mhc - ii epitopes . for mhcpred prediction , 3 supertypes of mhc - ii locus were used , with a final binding affinity cutoff of 60 nm for mhc - ii binding to obtain approximately the top 5 % of the binders that correspond to regions of synthetic peptides reported to induce t - cell immune responses in mice [ 28 ]. for rankpep prediction , 50 mhc - ii locus types were used and the cutoff was set at 4 % of top - scoring peptides with above default threshold scores as reported in ref . [ 27 ]. data from individual supertypes of mhc - i or - ii loci were combined for the analysis of each peptide . to ensure better predictive accuracy , only consensus results above the cutoff of both mhcpred and rankpep were considered as mhc - ii binders . the structural features of dengue e proteins were analyzed using known structures in pdb for denv2 , 1oan ( dimer ), 1ok8 ( post - fusion trimer ), and 1 oke ( protein in complex with n - octyl - b - d - glucoside ) [ 8 , 29 ], and the denv3 structure , 1uzg ( dimer ) [ 12 ]. the extent of exposure of amino acid residues was determined by computing the relative accessible surface area ( asa ) using the polyview server [ 30 ]. relative asa of a residue is the ratio of the asa of that residue in the protein to asa of the same residue in the fully extended tripeptide alanine - residue - alanine . based on the value of the relative asa , the residues were grouped as buried ( 0 . 0 - 0 . 60 ) or exposed ( 0 . 61 - 1 . 0 ). to determine interactions across the dimer and trimer interface , the occluded surface ( os ) area was computed using the method of pattabiraman et al . [ 31 ]. residues with os area & gt ; 0 . 5 å 2 were considered as interacting . the conformational rearrangements occurring during the dimer to trimer transition were measured by the changes in the backbone torsion angles u and w between the denv2 e protein dimer ( 1oan ) and trimer ( 1ok8 ). the conformational angles were obtained using the dssp program [ 32 ], and the difference in the backbone torsion angles , du and dw , for each amino acid residue was calculated . sequence conservation of e proteins among dengue species and other members of the genus flavivirus was studied based on structure - guided multiple sequence alignment using 12 representative sequences chosen from the major flavivirus groups where the genome polyproteins are available . the multiple sequence alignment shows 72 completely conserved amino acid residues in the 12 flaviviruses [ 65 including fig1 ]. these residues include cysteines forming the six pairs of disulfide - bonds [ 33 ] that stabilize loop structures in the three structural domains of e protein . other crucial structure - stabilizing interactions by the flavivirus - conserved amino acids include the d98 - k110 salt bridge , several intramolecular hydrogen bonds involving d10 , c30 , g100 , w101 , c105 , l216 , and l218 , and the hydrophobic environment provided by leucines ( l216 , l218 , and l264 ) and v208 . also , completely conserved among flaviviruses are two pairs of residues involved in interactions of domains i and iii , r9 and e368 , which form a salt bridge , and h144 and h317 that are involved in hydrogen bonds with the main chain of the opposite domain across the interface [ 34 ]. the most highly conserved region in flavivirus e protein is 98drgwgngcglfgkg111 , where 13 of the 14 residues are strictly conserved [ 65 including fig1 ]. this peptide , contained in an internal loop between two b strands on domain dii , corresponds to the known fusion motif [ 8 ] involved in denv infectivity . the highly variable region 380igvepgqlkl389 , in the lateral loop on domain iii , has been implicated in receptor - binding of denv2 [ 35 ] and tickborne viruses [ 34 ]. the alignment of representative e protein sequences of the four dengue serotypes reveals a very high degree of sequence conservation as expected due to their close evolutionary relationship [ 65 including fig2 ]. this alignment identified negatively selected sites in each serotype and sites under negative selection pressure only in a specific serotype and not in a merged dataset . a total of 260 residues (˜ 53 % of all residues ) are conserved in the multiple alignment of the four sequences . the conservation spans across the entire sequence length , including complete sequence identity of the fusion motif in d98 - g111 and the two known n - linked glycosylation sites at n67 and n153 [ 9 ] with the asn - x - thr / ser - x potential glycosylation site motif ( x can be any residue except for proline ) [ 65 including fig2 ]. shannon entropy was used to quantify the conservation of each amino acid position in the multiple sequence alignment of 740 e proteins from all four serotypes . the entropy ranges from 0 . 365 to 2 . 42 bits , with scores & lt ; 0 . 6 for highly conserved residues , 0 . 6 - 0 . 9 for conservative amino acid substitutions , and & gt ; 0 . 9 for nonconserved residues . the mean entropy of the full - length protein is 0 . 539 bits , and 55 and 91 % of the positions have entropies of & lt ; 0 . 6 and & lt ; 0 . 9 , respectively [ 65 including fig2 ]. comparisons of the entropy measure of the three structural domains di ( amino acids 1 - 52 , 134 - 191 , 280 - 295 ), dii ( aa 53 - 133 , 192 - 279 ) and diii ( aa 296 - 394 ), and the c - terminal region ( aa 394 - 495 ) reveal regions of different sequence variability . in particular , domain diii has the highest variability , with an entropy mean of 0 . 703 bits and entropies of & lt ; 0 . 6 and & lt ; 0 . 9 for 44 and 86 % of the positions . interestingly , serotype - specific neutralization escape mutant sites in denv e proteins are all located on the surface of domain iii [ 12 ]. twelve sequence regions are highly conserved in denv e proteins , containing five or more consecutive amino acid sites with entropy scores of & lt ; 0 . 6 , namely , n8 - g14 , v24 - d42 , r73 - e79 , v97 s102 , d192 - m196 , v208 - w220 , v252 - h261 , g281 - c285 , e314 - t319 , e370 - g374 , k394 - g399 , and r411 - 5424 , designated as sequence regions i to xii , respectively [ 65 including fig2 ]. estimates of synonymous or silent ( ds ) and non - synonymous or amino - acid altering ( dn ) nucleotide substitution rates at a given position in a codon alignment have become a standard measure of selective pressure , especially in the framework of maximum likelihood phylogenetic methods [ 22 ]. if ds is estimated to be significantly more than dn at a given site , ( a dn / ds value of & lt ; 1 ), this can be taken as evidence of purifying ( negative ) selective pressure on that site . that is , for that particular site amino acid changes are , on average , deleterious ( negative selection ). when the opposite is true , i . e . dn & gt ; ds , ( dn / ds & gt ; 1 ) there is selective pressure to generate and possibly maintain amino - acid polymorphisms , i . e . undergo adaptive change ( positive selection ). this unusual condition may reflect a change in the function of a gene or an immediate change in environmental conditions ( such as a pathogen &# 39 ; s response to an administered drug ) that forces the organism to adapt . to quantify selective pressure , ds and dn were estimated for each site of the alignment of individual dengue serotypes and also for all serotypes combined . the tamura nei ( tn93 ) [ 36 ] model of nucleotide substitution bias ( out of 203 possible models ) was selected for all alignments . to each alignment we fitted one of four models of site - to - site rate variation ( constant : ds = dn = 1 ; proportional : dn is proportional to ds , which varies among sites ; non - synonymous : ds = 1 , dn varies among sites ; and dual : ds and dn vary among sites independently ). strong evidence supporting the dual model of rate variation was observed in each alignment ( shown in supplementary table 51 ). this finding suggests that both ds and dn vary across sites , but there is no simple correlation pattern between the two rates [ 37 ]. ninety negatively selected sites were identified in denv1 , 161 in denv2 , 49 in denv3 , and 57 in denv4 , while 186 sites were found to be under negative selection pressure in the large combined set with all four serotypes [ 65 including fig2 ]. because the signature of negative selection is the relative abundance of synonymous substitutions likely due to functional constraints , most negatively selected sites were shown to correspond to conserved residues or substitution of an amino acid by another with similar chemical properties ( conservative substitutions ). altogether it was demonstrated that 138 out of 186 ( 74 %) negative sites in the merged set have low entropy scores of & lt ; 0 . 6 bits . furthermore , 14 sites ( c3 , c60 , r73 , t189 , f213 , a267 , f306 , t319 , s376 , f392 , k394 , s424 , g445 , and v485 ) are negatively selected in at least three of the four serotypes as well as in the merged dataset ( underlined residues ); most are highly conserved and a few have conservative substitutions . only 6 out of 186 ( 3 %) negative sites in the merged set were identified as having & gt ; 0 . 9 bits entropy scores ( s95 , p143 , t180 , s300 , v309 , and y488 ). note that while these sites are not conserved within the dengue species , they all correspond to negatively selected sites in one or two specific serotypes , possibly reflective of selective sweeps that have become fixed in individual serotypes and are now maintained by purifying selection ( negative selection ). finally , several serotype specific negatively selected sites were also identified ( 19 in denv1 , 48 in denv2 , 7 in denv3 , 16 in denv4 ). most notable are the 5 ( e383 and q386 - l389 ) denv2 specific negative sites in the receptor - binding region [ 65 including fig2 ]. although there are several reports on the role of positively selected sites in pathogen - host interaction , such as evading host immunity [ 38 - 42 ], there are few reports of experimental validation of the functional significance of negatively selected sites . it has been shown that epitopes consisting of negatively selected sites perform better as vaccines than ones containing positively selected sites [ 43 , 44 ]. the underlying assumption is that because negatively selected sites are less likely to change , due to functional constraints , vaccines or diagnostic targets directed against them may be more effective . no extant positively selected sites were detected in any of the dengue serotypes in this study . our findings agree with previous selection studies where constant ds across all sites was assumed a priori and the data were not stratified based on genotypes and passage types [ 45 , 46 ]. comparable analysis of e gene sequences from other flaviviruses , such as st . louis encephalitis virus , west nile virus and yellow fever virus , also did not detect any positive selection [ 47 ]. in order to screen for possible selection on amino - acid residues prior to the divergence of serotypes , we estimated dn and ds along the four tree branches separating individual serotype clades in the joint phylogeny using a fixed effects likelihood method [ 22 ]. five sites with evidence of ancient positive selection were suggested ( p £ 0 . 001 ): n83 , p132 , e174 , q293 , and l458 . mhc - i peptide binding prediction using netmhc [ 25 ] identified several potential binding regions for each of the four e proteins . however , the overall mhc - i binding affinity was low and the number of high binders was small . low predicted mhc - i binding was further confirmed using mhcpred ( data not shown ). these results are consistent with the observation that e proteins mainly induce antibody response to denv , while non structural protein 3 ( ns3 ) mainly induces t - cell immune responses to denv [ 48 ]. many mhc - ii binding peptides ( th - cell epitopes ) were predicted by both mhcpred and rankpep [ 27 ] above the affinity threshold ( 56 , 64 , 50 , and 53 peptides for denv1 , denv2 , denv3 , and denv4 , respectively , had affinities ranging from 1 to 60 nm ) [ 65 including fig2 ]. of these , 11 peptides have been identified in regions common to all four serotypes and represent immunogenic consensus sequence epitopes in the denv species . high - affinity mhc - ii binding peptides among the top - ranking predictions have been identified that are unique to one of the four serotypes . the predicted binding peptides common to all serotypes generally are present in more conserved regions with low - entropy and / or negatively selected sites , except the last two consensus binding peptides occurring in the c - terminal transmembrane region . on the other hand , most predicted serotype - specific binding peptides are in variable regions with lower sequence conservation . interestingly , the most highly variable domain diii contains only predicted serotype - specific binding peptides , but no consensus binding peptides common to all four serotypes [ 65 , including fig2 ]. to cross - validate the predicted results , we further mapped the 64 predicted mhc - ii binders of denv2 e protein to regions covered by synthetic peptides that were previously determined to mimic th - cell epitopes and to elicit antibody responses in three different mouse strains [ 28 ] ( amino acid regions of peptides in supplementary table s2 ). as 15 of the predicted peptides are not completely covered within regions of the synthetic peptides , the comparison was based on the remaining 49 predicted peptides . we noted that 39 of the 49 ( 80 % true positive ) predicted mhc - ii peptides were matched with 16 synthetic peptides that experimentally tested positive for th - cell epitopes ; while the remaining 10 binders ( 20 % false positive ) correlated to synthetic peptides that did not elicit an immune response either in vitro or in vivo [ 28 ]. conversely , the computational methods predicted all but one of the 17 synthetic peptides shown to induce an immune response ( table s2 ), yielding a 94 % ( 16 / 17 ) recall rate . the predictive accuracy observed here is consistent with the benchmarking results of epitope prediction programs [ 49 ]. consistent with the notion that the variable surface residues are likely to be responsible for the serotype - specific immunogenic variation , we identified four specific sequence regions ( 329dgs331 , 342lekrh346 , 360ekds363 , and 383epg385 ) that also match with predicted serotype specific th - cell epitopes and / or neutralizing mab - binding regions and experimentally determined th - cell epitopes [ 28 ]. these serotype - specific th - cell binders , coupled with the predicted consensus th - cell binders , reveal dengue immunogenic properties at both the species and serotype levels [ 65 including fig2 ]. several structural computational analyses were used to assign functional roles to amino acid residues in dengue e proteins [ 65 including fig3 ]. based on the relative accessible surface area ( asa ), buried residues that are important to maintain the structural integrity of the protein and exposed residues that may provide clues about protein interaction and immunogenicity were identified . the dimmer ( pre - fusion ) and trimer ( post - fusion ) structures of denv2 e protein each have about 60 exposed residues ( relativeasa & gt ; 0 . 6 ), half of which remain exposed on both the dimmer and the trimer surfaces . the solved structures for denv2 and denv3 show that there are minor structural differences at the viral surface of the two serotypes . it has been suggested that the nonconserved residues exposed on the viral protein surface may be involved in differential antibody binding [ 12 ]. among 43 non - conserved residues across the four serotypes ( entropy & gt ; 0 . 9 bits ), 19 are exposed on the surface of either oligomer , including 5 in the dimer only ( k157 , p243 , q293 , e343 , and e360 ), 1 in the trimer only ( l342 ), and 13 in both the dimer and trimer ( n83 , k88 , k122 , e174 , d203 , q227 , s274 , s300 , d329 , r345 , h346 , d362 , and g385 ). domain iii alone has 9 exposed and non - conserved residues , including 6 exposed in both the dimer and trimer . a total of 128 interface residues critical for dimerization and trimerization based on the occluded surface area were identified . in particular , it was noted that several residues in the fusion motif , d98 , l107 , f108 , and k110 , are involved in interactions at both the dimer and trimer interfaces of denv2 e proteins , as well as the dimer interface of denv3 e protein ( not shown ). it was further noted that most of these interface residues are highly conserved within the dengue species . approximately 73 % ( 94 residues ) of all interface residues either have entropy scores & lt ; 0 . 6 or are negatively selected , and only 4 % ( 5 residues ) are non - conserved with entropy scores & gt ; 0 . 9 . a few [ 18 ] interface residues are among the completely conserved residues in all 12 flaviviruses . the interface residues represent potential candidates for mutation experiments that may alter oligomerization . this region may also be a potential target for inhibitors that prevent oligomerization [ 65 including fig3 ]. there are significant conformational rearrangements in the main chain during the dimer to trimer transition . the plot of the difference in the backbone torsion angles ( du and dw ) shows several regions with major conformational changes , such as 1 - 19 , 242 - 246 , 289 - 298 , and 343 - 350 . many residues change from buried to exposed during the transition , suggesting the importance of these residues in the fusion mechanism . for example , buried residues m1 , h244 , k246 , g254 , g330 , and k344 in the dimmer become exposed in the trimer after significant conformational changes in the main chain , while residues including s16 , q52 , q167 , s169 , p243 , d290 , q293 , s331 , and e343 change from exposed to buried during trimerization [ 65 including fig3 ]. to estimate the relative accuracy of our analyses , computational data on sequence conservation , negative selection , structural features , and t - cell epitopes were compared with each other and also with published , experimentally determined functional sites [ 65 including fig4 ]. such integrated analysis allowed identification of sites that are exposed in the dimer and are also negatively selected ( e . g . n37 , n67 , k88 , e195 , p217 , g266 , d290 , s300 , d362 , f373 , and e383 ). other sites were identified , such as 266gat268 and 445gaafs449 , which ( a ) belong to the group of three or more consecutive sites under negative selection pressure in denv2 , ( b ) have a residue that is negatively selected in at least three of the serotypes ( as observed in the merged dataset ), and ( c ) are also part of a predicted epitope . overall , our computational results are in agreement with experimental information ( see supplementary table s3 ). the high affinity mhc - ii binding peptides predicted here correlated to 80 % of the synthetic peptides shown experimentally to induce t - cell immune responses [ 28 ]. the correlation between computationally predicted data and available experimental information suggests that the computational approaches used here relate rather well to biological features . development of diagnostics with low rates of false negatives and of vaccines difficult to circumvent ( by nature or by man ) would benefit by identifying the amino acid sites that should remain unaltered in spite of natural changes or artificial modification of dengue virus . we integrated all the computational results described above in this study and searched for sites in e protein that were ( a ) conserved , ( b ) consensus t - cell epitopes , and ( c ) exposed . a site - by - site analysis of the sequence of e protein revealed , as expected , that different features were distributed throughout the sequence . rather unexpectedly , however , we observed six sites that had more than one feature in a confined region of e protein . the sites having several features might be of particular importance to the viral genome and , therefore , unlikely to change without profound effects on infectivity and / or virus propagation . we considered these six singular sites ( n37 , q211 , d215 , p217 , h244 , k246 ) as potential candidate regions of the e protein for diagnostics and vaccine development . it was noted that 2 sites ( n37 , p217 ) are exposed in both dimer and trimer , 2 sites ( q211 , d215 ) are exposed only in the dimer , and 2 sites ( h244 , k246 ) are exposed only in the trimer . out of these six sites , h244 and k246 undergo conformational change between dimer and trimer forms [ 65 including fig4 ]. peptides for creating diagnostics and vaccines against dengue virus ( any subtype ) include : seq id no : 1 : maknkptld ; seq id no : 2 : kltnttt ; seq id no 3 : gldfnem ; seq id no : 4 : wlvhrqwfldlplpw ; seq id no : 5 : fknphakkq ; seq id no : 6 : altgateiq ; seq id no : 7 : imdlekrhvl ; seq id no : 8 : gvepgqlk ; seq id no : 9 : gaafsgvsw ; and seq id no : 10 : vgivtlyl . peptides for creating diagnostics and vaccines against yellow fever virus include : seq id no : 11 : wivdrqwaqdltlpw and seq id no : 12 : fepphaati . peptides for creating diagnostics and vaccines against west nile include seq id no : 13 : flvhrewfmdlnlpw and seq id no : 14 : feephatkq . peptides to create diagnostics and vaccines against tick borne encephalitis virus include : seq id no : 15 : wqvhrdwfndlalpw and seq id no : 16 : fgaphavkm . in this study , the sequence alignment of the flavivirus e proteins and the entropy measure and negative selection results for dengue e proteins have allowed us to identify sites and regions that are conserved across the flavivirus genus or within the dengue species , as well as variable regions that reflect serotype specific functional constraints . such analyses of conserved sites at the different taxonomic levels allow us to differentiate residues of general importance to the infectivity of flaviviruses from residues that may be specific to the dengue viruses . for example , among the 12 dengue - conserved sequence regions , regions i - iv and vi - viii are also highly conserved in other flaviviruses , encompassing over half of the 72 completely conserved residues in flavivirus e proteins [ 65 including fig4 ]. these regions also cover 16 of the 18 denv2 dimer or trimer interface residues that are completely conserved in all 12 flaviviruses . on the other hand , sequence region v ( d192 - m196 ) is dengue species specific . interestingly , region v overlaps with a 13 - amino acid sequence region that contains 11 negatively selected sites identified to be under functional constraints . such selection pressure analysis of codon - based dna alignments is ideal for identifying functionally important residues in serotypes that may not be reflected in the amino acid alignments at the species and serotype level [ 50 ]. several dengue - specific sites were identified . for example , while the n153 glycosylation site is conserved in several flaviviruses , the n67 site is unique to denv . it appears that dengue viruses are heterogeneous in their use of the glycosylation sites [ 51 ] and the precise function of the second glycosylation site is still under investigation [ 52 ]. the significance of this site is not known , although it has been noted that the loss of the n67 glycosylation site may result in a higher ph threshold for conformational change [ 53 ]. we have identified variable residues exposed on the surface of the e protein that are likely to be responsible for the immunogenic variation among dengue serotypes . especially notable is the sequence region 342lekrh346 in structural domain iii , which consists of five surface exposed residues ( 1 in dimer , 2 in trimer and 2 in both dimer and trimer ) in the beginning of a region ( aa 343 - 350 ) that undergoes major conformational changes during the dimer to trimer transition , with e343 becoming buried and k344 becoming exposed . four ( l342 , e343 , r345 , h346 ) of the five residues are not conserved among the serotypes and have entropy scores & gt ; 0 . 9 . another variable and exposed region is within the 10 - aa receptor - binding region ( 1380 - l389 ) that contains 4 exposed residues , including the 383epg385 triad critical for mab binding [ 12 ]. recent progress in molecular - based vaccine strategies , such as recombinant subunit dengue vaccines , has provided hope for the control of the disease [ 6 ]. the phenomenon of antibody dependent enhancement of dengue disease has spurred attempts to develop a tetravalent dengue vaccine that produces neutralizing antibodies against all four serotypes [ 54 ]. large - scale analysis of antigenic diversity of t - cell epitopes for dengue virus [ 55 ] indicates that there are limited numbers of antigenic combinations in e protein sequence variants , and that short regions of the protein are sufficient to capture the antigenic diversity of t - 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