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. 2023 Feb 28;15(3):654.
doi: 10.3390/v15030654.

Identification of Zika Virus NS1-Derived Peptides with Potential Applications in Serological Tests

Carlos Roberto Prudencio  1 , Vivaldo Gomes da Costa  2 , Leticia Barboza Rocha  3 , Hernan Hermes Monteiro Costa  1 , Diego José Belato Orts  1 , Felipe Rocha da Silva Santos  1 , Paula Rahal  2 , Nikolas Alexander Borsato Lino  2 , Pâmela Jóyce Previdelli da Conceição  2 , Cintia Bittar  2 , Rafael Rahal Guaragna Machado  4 , Edison Luiz Durigon  4 , João Pessoa Araujo Jr  5 , Juliana Moutinho Polatto  3 , Miriam Aparecida da Silva  3 , Joyce Araújo de Oliveira  3 , Thais Mitsunari  3 , Lennon Ramos Pereira  4 , Robert Andreata-Santos  4 , Luís Carlos de Souza Ferreira  4   6 , Daniela Luz  3 , Roxane Maria Fontes Piazza  3
Affiliations

Identification of Zika Virus NS1-Derived Peptides with Potential Applications in Serological Tests

Carlos Roberto Prudencio et al. Viruses. .

Abstract

Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

Keywords: NS1 protein; Zika virus; diagnosis; epitopes; immunoassays; monoclonal antibody; peptides.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Detection of antigen-specific serum antibody responses in ZIKV-NS1 immunized animals. Microplates coated with ZIKV-NS1 were reacted with (A) rabbit or (B) mice serum samples diluted from 1/100 to 1/6400. (C) Reactivity of selected hybridomas by ELISA. Microplates coated with ZIKV-NS1 (black), DENV-NS1 (white), or different anti-IgG isotypes (IgG2a—light grey, IgG2b—dark grey) were reacted with hybridomas supernatant 1A4, 4A11, 2F2. (D) Captured ELISA reactivity of ZIKV-NS1. Microplates coated with rabbit pAb were reacted with ZIKV-NS1 (black columns) or DENV-NS1 (white columns) proteins at concentrations indicated in the figure. The reaction was captured after incubation with mAb 2F2.
Figure 2
Figure 2
Peptide binding array profiles of anti-ZIKV NS1 mAb 2F2 and rabbit sera. (A) Mouse monoclonal 2F2 antibody; (B) rabbit polyclonal anti-ZIKV-NS1 sera were incubated in an NS1-based peptide microarray. The signal was captured after staining with secondary and control antibodies, as well as read-out at scanning intensities of 7/7 (red/green).
Figure 3
Figure 3
Three-dimensional visualization of identified epitopes of ZIKV-NS1 protein. Ribbon diagram representation. (A) NS1 protein is represented in grey, and three selected epitopes to the mAb 2F2 are colored red (position 248–266). (B) NS1 protein is represented in grey, and six selected epitopes to rabbit pAb are colored yellow (position 248–266, position 261–276, position 277–295, position 297–314, position 306–324, and position 339–352).
Figure 4
Figure 4
Three-dimensional visualization of selected epitopes of NS1 protein of ZIKV. (A) Ribbon diagram representation. NS1 protein is represented in grey, and six selected epitopes are colored red (ZKvROX1), blue (ZKvROX2), green (ZKvROX3), yellow (ZKvROX4), pink (ZKvROX5), and cyan (ZKvROX6). (B) The surface map of NS1 protein.
Figure 5
Figure 5
Reactivity of serum antibodies against the peptides (ZKvROX1-ZKvROX6) and ZIKV-NS1 by dot blot assays. (A) Serial dilution of ZKvROX1 against mAb 2F2. (B) Reactivity of all peptides (25 μg/mL) against the mAb 2F2. (C) Rabbit pAb immunized with the recombinant ZIKV-NS1 (left strip) or SARS-CoV-2 RBD protein as negative control (right strip). (D) Mouse immunized with DNA plasmid containing full ZIKV-NS1 (left strip) or mock group (right strip). (E) Human ZIKV and DENV negative sera (S1–S5). (F) Human sera from patients with DENV natural infections (S6–S10). (G) Human sera from patients with ZIKV natural infections (S11–S15).
Figure 6
Figure 6
Reactivity of selected peptides by ELISA. (A) ELISA using the pool of the six peptides (P1–P6) and ZIKV-NS1 protein using serum from ZIKV infected patients or healthy individuals serum samples. (B) Reactivity of the peptides individually (ZKvROX1-ZKvROX6) tested by ELISA by using sera from infected or negative patients for ZIKV. Statistically significant differences among human IgG levels and peptides in ELISA were determined by a Mann–Whitney test. p values < 0.05 indicated statistically significant difference (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
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