Yellow fever disease severity and endothelial dysfunction are associated with elevated serum levels of viral NS1 protein and syndecan-1

Abstract

Yellow fever virus (YFV) infections can cause severe disease manifestations, including hepatic injury, endothelial damage, coagulopathy, hemorrhage, systemic organ failure, and shock, and are associated with high mortality in humans. While nonstructural protein 1 (NS1) of the related dengue virus is implicated in contributing to vascular leak, little is known about the role of YFV NS1 in severe YF and mechanisms of vascular dysfunction in YFV infections. Here, using serum samples from qRT-PCR-confirmed YF patients with severe (n=39) or non-severe (n=18) disease in a well-defined hospital cohort in Brazil, plus samples from healthy uninfected controls (n=11), we investigated factors associated with disease severity. We developed a quantitative YFV NS1 capture ELISA and found significantly increased levels of NS1, as well as syndecan-1, a marker of vascular leak, in serum from severe YF as compared to non-severe YF or control groups. We also showed that hyperpermeability of endothelial cell monolayers treated with serum from severe YF patients was significantly higher compared to non-severe YF and control groups as measured by transendothelial electrical resistance (TEER). Further, we demonstrated that YFV NS1 induces shedding of syndecan-1 from the surface of human endothelial cells. Notably, YFV NS1 serum levels significantly correlated with syndecan-1 serum levels and TEER values. Syndecan-1 levels also significantly correlated with clinical laboratory parameters of disease severity, viral load, hospitalization, and death. In summary, this study points to a role for secreted NS1 in YF disease severity and provides evidence for endothelial dysfunction as a mechanism of YF pathogenesis in humans.

Keywords: NS1; endothelial dysfunction; pathogenesis; syndecan-1; yellow fever.

Fig. 1.. Development of quantitative YFV NS1 ELISA.

(A) Sigmoidal standard curve of YFV NS1 capture ELISA performed with YFJ19 mAb and thirteen different concentrations of recombinant YFV NS1. (B) Comparison of standard curve of YFV NS1 diluted or not in normal human serum (1:10). (C) Specificity of mAb YFJ19 by direct ELISA performed with NS1 (5 ug/mL) from 12 different flaviviruses as follows: YFV; dengue virus serotypes 1 (DENV1), 2 (DENV2), 3 (DENV2), and 4 (DENV4); Saint Louis encephalitis virus (SLEV); West Nile virus (WNV); Zika virus (ZIKV); Wesselsbron virus (WBV); Usutu virus (USUV); Japanese encephalitis virus (JEV); Tick-borne encephalitis virus (TBEV). (D) Cross-reactivity an anti-flavivirus NS1 mAb (2B7) by direct ELISA performed with NS1 (5 ug/mL) from the 12 different flaviviruses as in C. (E) Western blot analysis showing specificity of mAb YFJ19 for YFV NS1.

Fig. 2.. YFV NS1 serum levels in severe and non-severe YF patients.

(A) YFV NS1 levels were determined using an in-house sandwich ELISA, as described in Materials and Methods. Studied individuals were classified in three different groups as described in Materials and Methods: 1) Severe YF (n=39); non-severe YF (n=16); controls (n=5): healthy individuals. (B) YFV NS1 levels in survivors and deceased YFV-infected groups. Mean ranks of groups were compared by Mann-Whitney test (significance level of 0.05). (C) YFV NS1 serum levels in individuals with acute YF according to days since symptom onset, visualized using a LOESS model.

Fig. 3.. sSDC-1 levels in sera of individuals with acute YF and dengue.

(A) The sSDC-1 levels in sera were determined by the Human sSDC-1 ELISA Kit. Studied individuals were classified in three different groups as described in Materials and Methods: 1) Severe YF (n=39); non-severe YF (n=18); controls (n=11): healthy individuals. (B) sSDC-1 serum levels in survivors and deceased YFV-infected groups. (C) Distribution and average (solid lines) of sSDC-1 serum levels in individuals with acute YF according to days since symptoms onset, visualized using a LOESS model. (D) sSDC-1 serum levels in individuals with acute DENV infection. Dengue no leak (n=7): individuals with acute dengue who did not display plasma leakage; Dengue leak (n=7): individuals with acute dengue who displayed plasma leakage; Controls (n=11): same healthy individuals shown in panel A. Mean ranks of sSDC-1 levels of YF or dengue groups were compared by Kruskal-Wallis + Dunn’s multiple comparisons test. Median of sSDC-1 levels in deceased and survived patients were compared Mann Whitney test (significance level of 0.05).

Fig. 4.. Relative TEER of human endothelial cells treated with acute YF human serum samples and IFA of SDC-1 after NS1 treatment.

(A) Confluent monolayers of human endothelial cells cultured in Transwell inserts were treated or not with 10% serum from three different groups: 1) Severe YF (n=24); Non-severe YF (n=10); Controls (n=11): healthy individuals. YFV NS1 (10 ug/mL) was used as positive control. The transendothelial electrical resistance (TEER) was measured from 2 to 10h post-treatment. Graph shows mean ± SD of relative TEER for each treatment group. (B) Area under the curve (AUC) calculation for the different treatments. Mean values of AUC for each group were compared to untreated medium-only control group using one-way ANOVA + Tukey’s test. (C, D) Human endothelial cell monolayers grown in gelatin-coated coverslips were treated with (C) medium only or (D) 10 ug/mL of YFV NS1 for 3h. After fixation, cell surface SDC-1 was stained in red and nuclei in blue. (E) Quantification of SDC-1 protein on to the cells surface was expressed as mean fluorescence intensity (MFI) as shown in panels C and D. NS1 treatment was compared to medium-only treatment by t-test. Asterisks indicate significant difference with p<0.05.

Fig. 5.. Correlation analysis of clinical and laboratory data of study participants during acute YF.

(A) Correlation matrix analyzed by Spearman test using a linear model. Asterisks indicate significant correlations (p<0.05, Spearman r>0.35). (B) Correlation between serum levels of YFV NS1 and sSDC-1. (C) Correlation between serum levels of YFV NS1 and TEER AUC values. (D) Correlation between serum levels of sSDC-1 and TEER AUC values. Abbreviations: TEER, transendothelial electrical resistance; AUC, area under the curve; Hb, hemoglobin; Ht, hematocrit; AST, aspartate transaminase; ALT, alanine aminotransferase; TPI/INR thrombin potential index/international normalized ratio; aPTT, activated partial thromboplastin time; TB, total bilirubin; DB, direct bilirubin; IB, indirect bilirubin.