Immunogenic Properties of a BCG Adjuvanted Chitosan Nanoparticle-Based Dengue Vaccine in Human Dendritic Cells

<div><p>Dengue viruses (DENVs) are among the most rapidly and efficiently spreading arboviruses. WHO recently estimated that about half of the world’s population is now at risk for DENV infection. There is no specific treatment or vaccine available to treat or prevent DENV infections. Here, we report the development of a novel dengue nanovaccine (DNV) composed of UV-inactivated DENV-2 (UVI-DENV) and <i>Mycobacterium bovis</i> Bacillus Calmette-Guerin cell wall components (BCG-CWCs) loaded into chitosan nanoparticles (CS-NPs). CS-NPs were prepared by an emulsion polymerization method prior to loading of the BCG-CWCs and UVI-DENV components. Using a scanning electron microscope and a zetasizer, DNV was determined to be of spherical shape with a diameter of 372.0 ± 11.2 nm in average and cationic surface properties. The loading efficacies of BCG-CWCs and UVI-DENV into the CS-NPs and BCG-CS-NPs were up to 97.2 and 98.4%, respectively. THP-1 cellular uptake of UVI-DENV present in the DNV was higher than soluble UVI-DENV alone. DNV stimulation of immature dendritic cells (iDCs) resulted in a significantly higher expression of DCs maturation markers (CD80, CD86 and HLA-DR) and induction of various cytokine and chemokine productions than in UVI-DENV-treated iDCs, suggesting a potential use of BCG- CS-NPs as adjuvant and delivery system for dengue vaccines.</p></div

Evaluation of a Dengue NS1 Antigen Detection Assay Sensitivity and Specificity for the Diagnosis of Acute Dengue Virus Infection

<div><p>Background</p><p>Currently, no dengue NS1 detection kit has regulatory approval for the diagnosis of acute dengue fever. Here we report the sensitivity and specificity of the InBios DEN Detect NS1 ELISA using a panel of well characterized human acute fever serum specimens.</p><p>Methodology/Principal Findings</p><p>The InBios DENV Detect NS1 ELISA was tested using a panel composed of 334 serum specimens collected from acute febrile patients seeking care in a Bangkok hospital in 2010 and 2011. Of these patients, 314 were found to have acute dengue by either RT-PCR and/or anti-dengue IgM/IgG ELISA. Alongside the InBios NS1 ELISA kit, we compared the performance characteristics of the BioRad Platelia NS1 antigen kit. The InBios NS1 ELISA Ag kit had a higher overall sensitivity (86% vs 72.8%) but equal specificity (100%) compared to the BioRad Platelia kit. The serological status of the patient significantly influenced the outcome. In primary infections, the InBios NS1 kit demonstrated a higher sensitivity (98.8%) than in secondary infections (83.5%). We found significant variation in the sensitivity of the InBios NS1 ELISA kit depending on the serotype of the dengue virus and also found decreasing sensitivity the longer after the onset of illness, showing 100% sensitivity early during illness, but dropping below 50% by Day 7.</p><p>Conclusion/Significance</p><p>The InBios NS1 ELISA kit demonstrated high accuracy when compared to the initial clinical diagnosis with greater than 85% agreement when patients were clinically diagnosed with dengue illness. Results presented here suggest the accurate detection of circulating dengue NS1 by the InBios DENV Detect NS1 ELISA can provide clinicians with a useful tool for diagnosis of early dengue infections.</p></div

Heat map display of cytokine and chemokine productions.

<p>The iDCs were treated with BCG-CS-NPS, UVI- DENV (1, 3 and 10 μg) and DNV (1, 3 and 10 μg). Supernatant was collected on day 1, 2 and 3 and cytokine production was measured by Bio-plex assay.</p

Nanoparticle properties investigated by zetasizer.

<p>Ten microliters of CS-NPs, BCG-CS-NPs or DNV were added into 1,000 μl of 0.1 mM NaCl prior to measure their properties by zetasizer.</p

Sensitivity of InBios and Bio-Rad Assays stratified by date after onset of illness in (A) All infections and in (B) Primary and (C) Secondary infections.

<p>Serum samples (total samples, n = 314; primary, n = 51; secondary, n = 260) were tested using the InBios and Bio-Rad NS1 kits. Sensitivity was plotted against day post-onset of illness. p-values were calculated using McNemar's Chi-square test. NA – not applicable. Unable to do statistical analysis when value equals 0. NS – not significant.</p

The expression levels of CD80 (A), CD86 (B) and HLA-DR (C) on various regimens treated iDCs.

<p>The iDCs were treated with mock, BCG-CS-NPs, UVI-DENV (1, 3 and 10 μg) and DNV (1, 3 and 10 μg) for 24, 48 and 72 h. The expressions of CD80, CD86 and HLA-DR were determined as the mean fluorescence intensity (MFI) by flow cytometry. ^a indicates significant difference between mock- and adjuvant-treated culture. *,** and *** indicate significant difference in MFI level of CD80, CD86 and HLA-DR between DNV and UVI-DENV antigen at 1 μg, 3 μg and 10 μg, respectively.</p

The presence of BCG-CWCs and UVI-DENV antigen in different NP preparations.

<p>NPs in each step of the DNV preparation were stained with anti-LAM (A) and anti-DENV (B) antibodies and the mean fluorescence intensity (MFI) were measured by flow cytometry.</p

The mean fluorescence intensity (MFI, A) and the frequency (B) of UVI-DENV positive THP-1 cells.

<p>THP1 cells were stimulated with BCG-CS-NPs (grey bar, white dots), UVI-DENV (white bar, black dots) or DNV (black grid bar) or left unstimulated (negative control, white bar) for 1 or 2 days prior to intracellularly staining with anti-DENV antibody and determined the MFI and the frequency of UVI-DENV positive cells by flow cytometry. * indicates significant differences (<i>p</i><0.05) between UVI-DENV and DNV.</p

Identification of BCG-CWCs localization on BCG-CS-NPs observed with ConA induced aggregation.

<p>ConA solution was added into CS-NPs or BCG-CS-NPs to induce NPs aggregation whereas ConA+MDM was added as an inhibitor of ConA. Aggregation was measured by zetasizer at the designated time point starting from 0, 5, 10 and 20 minutes. * indicates a significant difference in particle sizeof BCG-CS-NPs in the presence or absence of ConA or ConA inhibitor. ** indicates a significant difference in particle size of CS-NPs in the presence or absence of ConA or ConA inhibitor. *** indicates a significant difference in particle size of BCG-CS-NPs and CS-NPs in the presence of ConA.</p

Overall performance characteristics of the InBios and Bio-Rad assays compared to reference standard^a.

a<p>The composite reference standard included samples that were positive either by serology and/or RT-PCR.</p>b<p>CI – confidence interval.</p>c<p>PPV – positive predictive value.</p>d<p>NPV – negative predictive value.</p>e<p>Sensitivity = (true positives)/(total positive by reference standard).</p>f<p>Specificity = (true negatives)/(total negative by reference standard).</p>g<p>Diagnostic Accuracy = (true positives+true negatives)/(total number of samples).</p>h<p>PPV = (true positives)/(total positive by InBios or Bio-Rad assay).</p>i<p>NPV = (true negatives)/(total negative by InBios or Bio-Rad assay).</p><p>Overall performance characteristics of the InBios and Bio-Rad assays compared to reference standard^{<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003193#nt107" target="_blank">a</a>}.</p