| Dengue, caused by dengue virus (DENV); is a major public health problem in over 100 tropical and subtropical countries and occurres almost every year in southern China, especially in the southeast coastal provinces. DENV is classified into four serotypes, DENV-1,-2,-3, and -4. An infection with any of these serotypes can cause clinical manifestations including dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Subsequent heterologous infections may increase the risk of the developing DHF or DSS. Since protection against heterologous serotype infections is both partial and transient, people who live in epidemic areas may be susceptible to four DENV infections in their lifetime. Thus, the cocirculation of virus serotypes in a community is the most common risk factor associated with the emergence of DHF or DSS, the most severe forms of dengue. Since no vaccine is available that elicits long-term protective immunity against all four DENV serotypes and early identification of multiple DENV infections in the acute phase of the illness is important for reducing morbidity and mortality associated with DHF and DSS, as well as for epidemiological control in areas where multiple flaviviruses are endemic. No single diagnostic assay in isolation is adequately sensitive and specific enough to diagnose all acute cases of dengue. DENVspecific RT-PCR and virus isolation relies heavily on experienced technicians and specialized laboratory equipment, and sometimes provides false-positive results due to contamination. DENV serology is a simple and robust approach to diagnosis, but this method is not sensitive in the very early stages of disease and strictly and lacks specificity because of cross-reactivity with other flaviviruses. So the development of a DENV vaccine and an early diagnostic method remain a global public health challenge. A better understanding of how viral components correlate with immune protection and pathogenic determinants is likely to contribute to the development of an effective vaccine and diagnostic method. In the present study, we mapped the B-cell epitopes on viral proteins which correlate with protection and diagnostic marker.Immunization with NS1 or passive administration of anti-NS1 MAbs can protect mice against a lethal virus challenge. NS1 has also been identified as being both a group-specific and serotype-specific determinant and as an early serotyping diagnostic marker. EIII of Envelope (E) protein is a relative independence and integrity of the region, which can be isolated after trypsin treatment of the E protein, and on the surface of DENV virions. It can mediate receptor binding and membrane fusion. Antibodies against EIII of E protein is the most powerful blockers to virus infection. This region has been identified as a candidate targets for vaccine development. A better understanding of how viral components correlate with immune protection and pathogenic determinants is likely to contribute to the development of an effective vaccine. In this study, we produced MAbs against those proteins. The B cell epitopes on those DENV proteins were mapped by those MAbs and antisera from small animals immunized by those DENV proteins, respectively, using competition binding assays and Pepscan analysis. This may be useful for developing an early diagnostic method and designing DENV vaccines.The purpose of this study is to:(1) comprehensively analysis the B cell epitopes of DENV-1 NS1 protein; (2) precisely map the neutralizing B cell epitopes in Eâ…¢of the envelope protein of DENV-1; which are useful for DENV vaccine and early diagnosis.The research is divided into three parts: â… . Production and characterization of monoclonal antibodies and hyperimmune serum against DENV NS1 protein or DENV-1 EIIIA combination of purified recombinant NS1 protein derived from each DENV serotype and with the corresponding DENV was used to immunize BALB/c mice for the production of hybridomas and hyperimmune serum, respectively. A total of 149 hybridoma cell lines from 27 mice immunized with the four DENV serotypes (9 mice with DENV-1 NS1,4 mice with DENV-2 NS1,4 mice with DENV-3 NS1, and 10 mice with DENV-4 NS1) that stably produced MAbs were established on the basis of their strong reactivity with both recombinant NS1 protein and DENV-infected cell lysates as antigen in the ELISA. Immunofluorescence assays (IFA) showed that all MAbs recognized the native NS1 antigen in DENV-infected cells. Most of the MAbs were identified as IgG1 isotype. The serotype specificity and cross-reactivity of the MAbs was further characterized by testing their reactivity with each virus serotype by ELISA and Western blot analysis. Of 149 MAbs,25 MAbs,20 MAbs,15 MAbs, and 15 MAbs reacted exclusively with the DENV-1 NS1, DENV-2 NS1, DENV-3 NS1, and DENV-4 NS1 proteins, respectively, in ELISA plus Western blotting or IFA assays. The remaining 74 MAbs showed various patterns of cross-reactivity with the four DENV serotypes.The purified recombinant DENV-1 EIII also was used to immunize BALB/c mice for the production of hybridomas and hyperimmune serum. A total of 37 hybridoma cell lines that stably produced MAbs were established on the basis of their strong reactivity with both recombinant DENV-1 EIII protein and DENV-1-infected cell lysates as antigen in the ELISA. IFA showed that all MAbs recognized the native E antigen in DENV-infected cells. Most of the MAbs were identified as IgGl isotype. The serotype specificity and cross-reactivity of the MAbs was further characterized by testing their reactivity with each virus serotype by ELISA and Western blot analysis. The neutralization efficiency to DENV-1,2,3,4 of 37 MAbs against EIII of DENV-1 was characterized by Plaque Reduction Neutralization Test (PRNT).36 MAbs can neutralize DENV infectivity. Of those 36 MAbs,17 MAbs reacted exclusively with the DENV-1 E protein in ELISA plus Western blotting or IFA assays. 9 MAbs reacted exclusively with four DENV serotypes in ELISA plus Western blotting or IFA assays.The remaining 10 MAbs showed various patterns of cross-reactivity with the four DENV serotypes.These MAbs provides a good tool for mapping B-cell epitopes of those DENV proteins.â…¡. Comprehensively mapping of B-cell epitopes of nonstructural protein 1 from DENV-1We mapped B-cell linear epitopes on NS1 using 149 monoclonal antibodies with DENV serotype specificity and cross-reactivity as well as antisera from 27 mice immunized with the four DENV serotypes. Epitope recognition analysis was performed using a set of 15-mer sequential overlapping peptides that spanned the entire NS1 protein from DENV-1. Among the 25 DENV-1 serotype-specific MAbs, most of those MAbs showed a strong reaction with three regions of NS1 that are DENV-1 serotype-specific epitopes, namely amino acid residues 1-15,71-85, and 338-352. We compared NS1 sequences in different members of the flavivirus family by aligning NS1 residues 1-15,71-85,111-125, and 338-352 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences of peptide 1 (residues 1-15), peptide 8 (residues 71-125), and peptide 35 (residues 338-352) were completely conserved among DENV-1 serotypes, but different among the flaviviruses, suggesting that three regions of NS1, i.e. peptide 1 (residues 1-15), peptide 8 (residues 71-125), and peptide 35 (residues 338-352), were DENV-1 serotype specific epitopes. We also identified five group-specific B-cell epitopes, namely amino acid residues 21-35,111-125,191-205,261-275, and 291-305, that were highly conserved among isolates of the four DENV serotypes. NS1 sequences from different members of the flavivirus family were compared by aligning NS1 residues 21-35,111-125,191-205,261-275, and 291-305 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. This comparison revealed that the amino acid sequences in these regions were conserved among the four DENV serotypes, but different among the other flaviviruses (WNV, YFV, and JEV) and the amino acid sequences 25VHTWTEQYKFQ35,112KYSWKSWGKAK122,193AVHADMGYWIES204, 266GPWHLGKLE274, and 294RGPSLRTTT302 were similar among the serotypes, indicated that these regions of NS1 are group-specific B-cell epitopes. Peptide 1 (residues 1-15) showed high immunoreactivity, i.e. a high mean absorbance/cut-off ratio, with sera from DENV-1 NS1-immunized mice. This suggests that the region of NS1 comprising residues 1-15 is a serotype-specific immunodominant epitope. Peptide 3 (residues 21-35) reacted with sera from DENV-1-and DENV-2-immunized mice; the high mean absorbance/cut-off ratio was different from that of sera from DENV-3-and DENV-4-immunized mice, suggesting that this region is a DENV-1 and DENV-2 serotype-specific immunodominant epitope. Peptides 12 (residues 111-125), 20 (residues 191-205), and 27 (residues 261-275) had high cross-immunoreactivity with sera from mice immunized with each of the four DENV serotypes. This suggests that the three regions are group-specific immunodominant epitopes among the four DENV serotypes. Taken together, these data suggest that the most immunoreactive epitopes on the NS1 protein involve amino acid residues 1-15,21-35,111-125, 191-205, and 261-275. These novel immunodominant serotype-and group-specific B-cell epitopes of DENV NS1 may aid the development of new dengue vaccines and diagnostic assays.â…¢. Characterization of neutralizing B cell epitopes on envelope protein domainâ…¢of DENV-1The neutralizing B-cell epitopes on Eâ…¢of DENV-1 E protein were mapped by 36 neutralizing MAbs against DENV-1 Eâ…¢. Epitope recognition analysis was performed using two sets of sequential overlapping peptides (16-mer and 12-mer) that spanned the entire Eâ…¢from DENV-1 E protein, respectively. Among the 17 DENV-1 serotype-specific neutralizing MAbs, two of those MAbs reacted two regions of DENV-1 E that were DENV-1 serotype-specific neutralizing B-cell epitopes, namely amino acid residues 329-348, and 381-392. We compared E sequences in different members of the flavivirus family by aligning E protein residues 329-348, and 381-392 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that only the amino acid sequence of 381-392 was completely conserved among DENV-1 serotypes, but different among the flaviviruses, suggesting that E protein amino acid sequence of 381-392 was a DENV-1 serotype specific neutralizing B-cell epitope. Among the 9 DENV group-specific neutralizing MAbs,7 of those MAbs reacted one region of DENV-1 E that was a DENV group-specific neutralizing epitope, namely amino acid residues 309-320. E sequences from different members of the flavivirus family were compared by aligning E residues 309-320 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. This comparison revealed that the amino acid sequences in these regions were conserved among the four DENV serotypes, but different among the other flaviviruses (WNV, YFV, and JEV) and the amino acid sequences 310KEVAETQHGT319 was similar among the serotypes, indicated that these regions of E is group-specific neutralizing B-cell epitopes. We further defined contact residues on E protein residues 309-320 of a panel of those MAbs and found that substitution of residues E309, V312, A313 and V320 in DENV-2,-3,-4 isolates, were antigenically silent. These novel neutralizing B-cell epitopes of DENV E may aid the development of dengue vaccines.Summarization:We identified several immunodominant B-cell epitopes on NS1 using a panel of MAbs and antisera raised in mice against the four DENV serotypes. We identified three regions of NS1 that are DENV-1 serotype-specific B-cell epitopes, namely amino acid residues 1-15,71-85, and 338-352. We compared NS1 sequences in different members of the flavivirus family by aligning NS1 residues 1-15,71-85, 111-125, and 338-352 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences of peptide 1 (residues 1-15), peptide 8 (residues 71-125), and peptide 35 (residues 338-352) were completely conserved among DENV-1 serotypes, but different among the flaviviruses, suggesting that three regions of NS1, i.e. peptide 1 (residues 1-15), peptide 8 (residues 71-125), and peptide 35 (residues 338-352), were DENV-1 serotype specific B-cell epitopes. Peptide 1 (residues 1-15) showed high immunoreactivity with sera from DENV-1 NS1-immunized mice. This suggests that the region of NS1 comprising residues 1-15 is a serotype-specific immunodominant B-cell epitope. We also identified five group-specific B-cell epitopes, namely amino acid residues residues 21-35,111-125, 191-205,261-275,and 291-305. We compared NS1 sequences in different members of the flavivirus family by aligning NS1 residues 21-35,111-125,191-205, 261-275, and 291-305 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences in these regions were conserved among the four DENV serotypes, but different among the other flaviviruses (WNV, YFV, and JEV) and the amino acid sequences 25VHTWTEQYKFQ35,112KYSWKSWGKAK122, 193AVHADMGYWIES204,266GPWHLGKLE274, and 294RGPSLRTTT302 were similar among the serotypes, indicated that these regions of NS1 were highly conserved among the four DENV serotypes and were group-specific B-cell epitopes. Peptides 12 (residues 111-125),20 (residues 191-205), and 27 (residues 261-275) had high cross-immunoreactivity with sera from mice immunized with each of the four DENV serotypes. This suggests that the three regions are group-specific B-cell immunodominant epitopes among the four DENV serotypes. These novel immunodominant serotype-and group-specific B-cell epitopes of DENV NS1 may aid the development of new dengue vaccines and diagnostic assays.We also characterized the neutralizing B-cell epitopes on Eâ…¢of DENV-1 E protein using a panel of neutralizing MAbs against Eâ…¢of DENV-1 E protein. We identified E residues 381-392 that is a DENV-1 serotype-specific neutralizing epitope. We compared E sequences in different members of the flavivirus family by aligning E residues 381-392 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences were completely conserved among DENV-1 serotypes, but different among the flaviviruses, suggesting that E residues 381-392 was a DENV-1 serotype specific neutralizing B-cell epitope. We also identified a group-specific neutralizing B-cell epitope, namely-amino acid residues residues 309-320 on E protein. We compared E sequences in different members of the flavivirus family by aligning E residues 309-320 from DENV-1,-2,-3,-4, WNV, YFV, and JEV. Alignment revealed that the amino acid sequences in the region were conserved among the four DENV serotypes, but different among the other flaviviruses (WNV, YFV, and JEV) and the amino acid sequences 310KEVAETQHGT319 was similar among the serotypes, indicated that these regions of E protein was highly conserved among the four DENV serotypes and was a group-specific neutralizing B-cell epitope. We further defined contact residues on residues 309-320 of a panel of those group-specific neutralizing MAbs and found that substitution of residues E309, V312, A313 and V320 in DENV-2,-3,-4 isolates, were antigenically silent. Those neutralizing B-cell epitopes of DENV E protein may aid the development of dengue vaccines.
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