ADE levels of recombinant DENVs or DENV-2 strain 16681 exposed to D30-plasma or HuMAbs.

<p>A heat-inactivated D30-plasma sample (A) or purified human MAbs derived from patient D30 (B) were serially diluted 10-fold in RPMI-1640 medium and incubated with either DENV-2 16681 or individual recombinant DENVs for 30 min at an MOI of 0.1. Virus-antibody complexes were added to K562 cells and incubated for a further 2 h; maintenance medium was then added (without washing the cells) to yield a FBS final concentration of 2%. Cells and supernatants were collected on Day 3 post-infection. Virus titers in the supernatants were determined in focus-forming immunoassays in Vero cells. Results are expressed as the mean ± SD from two independent experiments performed in triplicate (*<i>p<0.05</i> and **<i>p<0.01</i>, unpaired two-tailed Student t-test, n = 3 per point). Statistically significant differences between data points are indicated by # (# <i>p<0.05</i>, ## <i>p<0.01</i>).</p

List of primers used in the study.

<p>List of primers used in the study.</p

DENV serotypes, viremia titer, and anti-DENV antibody isotypes in DENV-infected patients.

a<p>ID, patient identification.</p>b<p>Dengue disease was determined by examining the clinical symptoms of patients according to the WHO criteria.</p>c<p>DENV serotype was determined by RT-PCR with universal and serotype-specific primers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092173#pone.0092173-Yenchitsomanus1" target="_blank">[31]</a>.</p>d<p>Viremia titers were determined in a focus-forming immunoassay in semi-adherent K562 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092173#pone.0092173-Kurosu1" target="_blank">[30]</a>.</p>e<p>IgM in patient serum was detected by Dengue Virus IgM Capture DxSelect. An index value of >1.00 was interpreted as positive (POS) and an index value of <1.00 was interpreted as negative (NEG).</p>f<p>IgG in patient serum was detected by Dengue Virus IgG DxSelect. An index value of >1.00 was interpreted as positive (POS) and an index value of <1.00 was interpreted as negative (NEG).</p>g<p>Cases with an IgM/IgG index ratio of ≤1.2 were diagnosed as secondary infections <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092173#pone.0092173-Shu1" target="_blank">[32]</a>.</p>h<p>Patient’s blood specimen was used for huMAb preparation as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092173#pone.0092173-Guy1" target="_blank">[24]</a>. ND, not detectable.</p

Construction of recombinant DENVs based on virus samples from patient plasma.

<p>RNA was extracted from the plasma of patient D30 and the prM-E encoding region of DENV was amplified by RT-PCR and cloned into the pCR4Blunt-TOPO vector for DNA sequence analysis. Representative prM-E region variants were selected and used to construct full-length DENV cDNA clones based on plasmid pmMW/R05-624. The resultant full-length cDNAs containing D30-derived variants were used as templates for RNA synthesis, and <i>in vitro</i>-transcribed viral RNAs were transfected into C6/36 cells. Supernatants from transfected cells were passaged once in C6/36 to obtain adequate quantities of viruses. EDI, envelope domain I. EDII, envelope domain II. EDIII, envelope domain III. TM, transmembrane. The recombinant DENV clone in brackets was excluded from further study due to an inadequate titer.</p

Replication kinetics of recombinant DENVs.

<p>DENV-2 16681 or individual recombinant DENVs were used to infect C6/36 (A) and Vero cells (B) at an MOI of 0.001, or K562 cells (C) at an MOI of 0.1. After 2 hours of incubation, the supernatants were removed and cells were washed twice with plain medium before the addition of maintenance medium supplemented with 2% FBS. For infected C6/36 and Vero cells, the supernatants were harvested daily. For infected K562 cells, both the culture medium and infected cells were harvested and centrifuged. Virus titers in the supernatants were determined in focus-forming assays in Vero cells. Results are expressed as mean ± SD of triplicate experiment (*<i>p<0.05</i> and **<i>p<0.01</i>, unpaired two-tailed Student’s t-test, n = 3 per point). Statistically significant differences between data points are indicated by # (# <i>p<0.05</i>, ## <i>p<0.01</i>).</p

Low ADE when using serum and viruses from DENV-patients.

<p>Serum samples (DENV-1, DENV-2, and DENV-3) from HTD dengue patients with DF and DHF symptoms were ultracentrifuged to precipitate DENV virions which were used in assays without any subsequent passage in cells. The supernatant fractions were heat-inactivated at 56°C for 30 minutes and then serially diluted 10-fold. The dilutions were mixed for 30 minutes at 37°C with the precipitated virions from autologous plasma at an MOI of 0.02. The virus-antibody complexes were added to K562 cells and incubated for 2 hours at 37°C before the addition of maintenance medium supplemented with 2% FBS. The cells were then incubated for a further 3 days. Supernatants were harvested for virus titration by focus-forming immunoassay in Vero cells, and the results are expressed as FFU/ml. The mean ± SD of triplicate experiment is shown. ‘No Ab’ means virus infection in the absence of plasma. The ‘No Ab’ value was used as a baseline for calculating virus infection enhancement.</p

High levels of ADE when using patient plasma and laboratory-culture adapted DENV.

<p>Ultracentrifugation supernatants of patient sera were heat-inactivated at 56°C for 30 min, diluted 10-fold, and pre-mixed at an MOI of 0.02 with laboratory culture-adapted DENV-1, -2, and -3 for 30 minutes at 37°C. Virus-antibody complexes were added to K562 cells and incubated for 2 hours at 37°C. Maintenance medium supplemented with 2% FBS was then added before a further incubation for 3 days. Supernatants were harvested for virus titration in focus-forming immunoassays in Vero cells. Results are expressed as the mean ± SD of triplicate experiments. ‘No Ab’ means virus infection in the absence of plasma. The ‘No Ab’ value was used as a base line for calculating virus infection enhancement.</p

Low Levels of Antibody-Dependent Enhancement in Vitro Using Viruses and Plasma from Dengue Patients

<div><p>Background</p><p>The majority of dengue patients infected with any serotype of dengue virus (DENV) are asymptomatic, but the remainder may develop a wide spectrum of clinical symptoms, ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). Severe cases occur more often in patients who experience a secondary infection with a different virus serotype. A phenomenon called antibody-dependent enhancement (ADE) has been proposed to explain the onset of these severe cases, but the exact mechanism of ADE remains unclear.</p><p>Methodology/Principal Finding</p><p>Virus neutralization and ADE assays were performed using ultracentrifugation supernatants of acute-phase sera from patients with secondary infections or human monoclonal antibodies (HuMAbs) as anti-DENV antibodies. Virus sources included infectious serum-derived viruses from the ultracentrifugation precipitates, laboratory-culture adapted DENV, or recombinant DENVs derived from patient sera. In contrast to the high levels of ADE observed with laboratory virus strains, low ADE was observed with autologous patient-derived viruses, when patient sera were used to provide the antibody component in the ADE assays. Similar results were obtained using samples from DF and DHF patients. Recombinant-viruses derived from DHF patients showed only minor differences in neutralization and ADE activity in the presence of HuMAbs or plasma derived from the same DHF patient.</p><p>Conclusion/Significance</p><p>Serum or plasma taken from patients during the acute phase of a secondary infection showed high levels of ADE, but no neutralization activity, when assayed in the presence of laboratory-adapted virus strains. By contrast, serum or plasma from the same patient showed high levels of neutralization activity but failed to induce significant ADE when the assays were performed with autologous virus. These results demonstrate the significance of the virus source when measuring ADE. They also suggest that repeated passage of DENV in cell culture has endowed it with the capacity to induce high levels of ADE.</p></div

A nonsense mutation in <i>TLR5</i> is associated with survival and reduced IL-10 and TNF-α levels in human melioidosis

<div><p>Background</p><p>Melioidosis, caused by the flagellated bacterium <i>Burkholderia pseudomallei</i>, is a life-threatening and increasingly recognized emerging disease. Toll-like receptor (TLR) 5 is a germline-encoded pattern recognition receptor to bacterial flagellin. We evaluated the association of a nonsense <i>TLR5</i> genetic variant that truncates the receptor with clinical outcomes and with immune responses in melioidosis.</p><p>Methodology/Principal findings</p><p>We genotyped <i>TLR5</i> c.1174C>T in 194 acute melioidosis patients in Thailand. Twenty-six (13%) were genotype CT or TT. In univariable analysis, carriage of the c.1174C>T variant was associated with lower 28-day mortality (odds ratio (OR) 0.21, 95% confidence interval (CI) 0.05–0.94, <i>P</i> = 0.04) and with lower 90-day mortality (OR 0.25, 95% CI 0.07–086, <i>P</i> = 0.03). In multivariable analysis adjusting for age, sex, diabetes and renal disease, the adjusted OR for 28-day mortality in carriers of the variant was 0.24 (95% CI 0.05–1.08, <i>P</i> = 0.06); and the adjusted OR for 90-day mortality was 0.27 (95% CI 0.08–0.97, <i>P</i> = 0.04). c.1174C>T was associated with a lower rate of bacteremia (<i>P</i> = 0.04) and reduced plasma levels of IL-10 (<i>P</i> = 0.049) and TNF-α (<i>P</i> < 0.0001). We did not find an association between c.1174C>T and IFN-γ ELISPOT (T-cell) responses (<i>P</i> = 0.49), indirect haemagglutination titers or IgG antibodies to bacterial flagellin during acute melioidosis (<i>P</i> = 0.30 and 0.1, respectively).</p><p>Conclusions/Significance</p><p>This study independently confirms the association of <i>TLR5</i> c.1174C>T with protection against death in melioidosis, identifies lower bacteremia, IL-10 and TNF-α production in carriers of the variant with melioidosis, but does not demonstrate an association of the variant with acute T-cell IFN-γ response, indirect haemagglutination antibody titer, or anti-flagellin IgG antibodies.</p></div

Immune response measurements by <i>TLR5</i> c.1174C>T genotype.

<p>Immune response measurements by <i>TLR5</i> c.1174C>T genotype.</p