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. 2017 Oct 15;6(4):14.
doi: 10.3390/antib6040014.

Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies

Leticia Barboza Rocha  1 , Rubens Prince Dos Santos Alves  2 , Bruna Alves Caetano  3 , Lennon Ramos Pereira  4 , Thais Mitsunari  5 , Jaime Henrique Amorim  6 , Juliana Moutinho Polatto  7 , Viviane Fongaro Botosso  8 , Neuza Maria Frazatti Gallina  9 , Ricardo Palacios  10 , Alexander Roberto Precioso  11 , Celso Francisco Hernandes Granato  12 , Danielle Bruna Leal Oliveira  13 , Vanessa Barbosa da Silveira  14 , Daniela Luz  15 , Luís Carlos de Souza Ferreira  16 , Roxane Maria Fontes Piazza  17
Affiliations

Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies

Leticia Barboza Rocha et al. Antibodies (Basel). .

Abstract

Dengue nonstructural protein 1 (NS1) is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2), which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV) protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.

Keywords: NS1; Zika virus; amino acid sequences; antibody recognition; dengue virus; mAbs.

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Conflict of interest statement

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Figures

Figure 1
Figure 1
Characterization of nonstructural protein 1-specific (NS1) monoclonal antibodies (mAbs) reactivity by enzyme-linked immunosorbent assay (ELISA). Reactivity of mAbs to heated-treated or intact rNS1, as solid phase-bound antigens. The mAbs 4F6 (A), 4H2 (B), 2H5 (C) and 4H1BC (D) were serially diluted (log2) from an initial concentration of 2.5 µg/mL. Each well was adsorbed with 400 ng of rNS1. Heat denaturation was performed at 100 °C for 10 min. Statistical analyses were performed by two-way variance analysis followed by Bonferroni’s post-test. (*** p < 0.01; ** p < 0.1; * p < 0.5).
Figure 2
Figure 2
Reactivity of NS1-specific mAbs to dengue-serotype 2-infected Vero cells. Cells were infected with a multiplicity of infection (MOI) of 0.5, fixed, permeabilized and treated with each of the tested mAbs 48 h post infection. Then, cells were labeled with Alexa fluor® conjugated goat-anti mouse IgG. The negative controls: Mock-infected cells treated with a pool of mAbs anti-NS1 (A) and DENV2-infected cells labeled only with secondary antibody (B); Tested mAbs: (C) 4F6; (D) 4H2; (E) 2H5 and (F) 4H1BC. Magnification of 200×.
Figure 3
Figure 3
Three-dimensional structural model of a NS1 dimer and regions corresponding to epitopes recognized by 4F6 mAb. The NS1 3D model was generated by the program Python Molecular (PyMOL) in green. The sequence 25VHTWTEQYKFQPES38 is highlighted in yellow.
Figure 4
Figure 4
Three-dimensional structural model of a NS1 dimer and regions corresponding to epitopes recognized by 4H2 mAb. The NS1 3D model was generated by the program PyMOL in green. The sequence 127ELHNQTFLIDGPETAEC143 is highlighted in red and white. In the detail the red structure represents the novel nine-amino acid sequence described herein.
Figure 5
Figure 5
Three-dimensional structural model of a NS1 dimer and regions corresponding to epitopes recognized by 2H5 and 4H1BC mAbs. The NS1 3D model was generated by the program PyMOL in green. The sequence 193AVHADMGYWIESALNDT209 is highlighted in blue.
Figure 6
Figure 6
Reactivity of NS1-specific mAbs to zika virus-infected Vero cells. Cells were infected with a MOI of 0.05. 72 h post infection, cells were fixed, permeabilized and treated with each of the tested mAbs. Then, cells were labeled with FITC-conjugated goat-anti mouse IgG. Tested mAbs: (A) 4F6; (B) 4H2; (C) 2H5 and (D) 4H1BC. Magnification of 200×.
Figure 7
Figure 7
Reactivity of 4H2 mAb to Vero cells infected with DENV of different serotypes. Vero cells were infected with a MOI of 0.5, fixed, permeabilized and treated with mAb 4H2 48 h post infection. Then, cells were labeled with Alexa fluor® conjugated goat-anti mouse IgG. The negative controls: (A) Mock infected cells treated with a pool of mAbs anti-NS1 and (B) DENV-infected cells labeled only with secondary antibody; Tested DENV serotypes: (C) DENV1; (D) DENV3 and (E) DENV4. Magnification of 200×.
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