Use of a Blockade-of-Binding ELISA and Microneutralization Assay to Evaluate Zika Virus Serostatus in Dengue-Endemic Areas.

Zika virus (ZIKV) serological diagnostics are compromised in areas where dengue viruses (DENV) co-circulate because of their high levels of protein sequence homology. Here, we describe the characterization of a Zika blockade-of-binding ELISA (Zika BOB) and a Zika microneutralization assay (Zika MN) for the detection of ZIKV nonstructural protein 1 (NS1)-specific antibodies and ZIKV neutralizing antibodies, respectively. Zika BOB and Zika MN cutoffs were established as 10 and 100 endpoint titers, respectively, using samples collected pre- and post-virologically confirmed ZIKV infection from subjects living in DENV-endemic areas. Specificity of the assays was equally high, whereas sensitivity of Zika BOB was lower than that of Zika MN, especially in samples collected > 6 months post-infection. Immunosurveillance analysis, using combined results from both Zika BOB and Zika MN, carried out also in DENV-endemic regions in Colombia, Honduras, Mexico, and Puerto Rico before (2013-2014) and after (2017-2018) ZIKV introduction in the Americas suggests unapparent ZIKV seroprevalence rates ranged from 25% to 80% over the specified period of time in the regions investigated.

Development of an anti-dengue NS1 IgG ELISA to evaluate exposure to dengue virus.

Dengue virus infection elicits immune responses to multiple viral antigens including antibodies to dengue non-structural protein 1 (NS1) which are rapidly induced and detected within days of infection. The recombinant, live, attenuated, tetravalent dengue vaccine (CYD-TDV; Sanofi Pasteur) uses the yellow fever vaccine virus as a back-bone but expresses dengue virus pre-membrane and envelop proteins. Since CYD-TDV does not express dengue NS1, we evaluated the utility of dengue NS1-specific IgG antibodies as biomarkers of dengue exposure in CYD-TDV recipients and controls. We optimized and evaluated a quantitative anti-dengue NS1 IgG enzyme-linked immunosorbent assay (ELISA). Parameters assessed included: accuracy, dilutability/linearity, precision, limit of quantitation and specificity. The assay specificity was further evaluated using Japanese Encephalitis virus, West Nile virus, Yellow Fever virus or Zika virus positive sera samples collected following confirmed infection or vaccination. Receiver-operating-characteristics (ROC) curves as well as sensitivity and specificity for discriminating previous dengue exposure were assessed using 1250 reference samples. Overall, the anti-dengue NS1 IgG ELISA was able to discriminate previous dengue exposure from non-exposure before vaccination with CYD-TDV (ROC area under the curve > 0.9). Assessment of paired samples from 2511 vaccinated participants showed high overall agreement (93%) between pre-vaccination and post-vaccination dengue serostatus classification based on the anti-dengue NS1 IgG ELISA. However, misclassification of dengue serostatus was observed after vaccination likely due to a combination of asymptomatic dengue infections, assay variability and a modest effect of CYD-TDV on the anti-dengue NS1 IgG ELISA readout.

Development of antibody biomarkers of long term and recent dengue virus infections.

Dengue virus (DENV) infections elicit antibody responses to the non-structural protein 1 (NS1) that are associated with protection against disease. However, the antibody isotypes and subclasses involved, and their kinetics have not been extensively studied. We characterized the antibody responses to DENV NS1 by enzyme-linked immunosorbent assay (ELISA) in a longitudinal cohort of 266 confirmed dengue cases in Recife, Northeast Brazil. Samples were collected during the febrile phase and up to over 3 years after onset of symptoms. The antibodies investigated [IgA, IgM, total IgG (all subclasses measured together) and each subclass (IgG2, IgG3 and IgG4) measured separately] had distinct kinetic profiles following primary or secondary DENV infections. Of interest, most of these antibodies were consistently detected greater than 6 months after onset of symptoms, except for IgG3. Anti-dengue NS1-specific IgG was consistently detected from the acute phase to beyond 3 years after symptom onset. In contrast, anti-dengue NS1-specific IgG3 was detected within the first week, peaked at week 2-3, and disappeared within 4-6 months after onset of symptoms. The mean duration of the IgG3 positive signal was 149 days (ranging from 126 to 172 days). In conclusion, anti-dengue NS1-specific IgG and IgG3 are potential biomarkers of long-term and recent (less than 6 months) DENV infections, respectively.