Serum From Melioidosis Survivors Diminished Intracellular Burkholderia pseudomallei Growth in Macrophages: A Brief Research Report.

Melioidosis is a neglected tropical disease with high mortality rate. It is caused by the Gram-negative, CDC category B select agent Burkholderia pseudomallei (B. ps) that is intrinsically resistant to first-line antibiotics. An antibody-based vaccine is likely to be the most effective control measure. Previous studies have demonstrated significant mechanistic roles of antibodies in protection against death in animal models, but data from human melioidosis is scarce. Herein, we used in-vitro antibody-dependent cellular phagocytosis and growth inhibition assays to assess the mechanism of protective antibodies in patients with acute melioidosis. We found that serum from patients who survived the disease enable more live B. ps to be engulfed by THP-1 derived macrophages (median 1.7 × 103 CFU/ml, IQR 1.1 × 103-2.5 × 103 CFU/ml) than serum from patients who did not survive (median 1.2 × 103 CFU/ml, IQR 0.7 × 103-1.8 × 103, p = 0.02). In addition, the intracellular growth rate of B. ps pre-opsonized with serum from survivors (median 7.89, IQR 5.58-10.85) was diminished when compared with those with serum from non-survivors (median 10.88, IQR 5.42-14.88, p = 0.04). However, the difference of intracellular bacterial growth rate failed to reach statistical significance when using purified IgG antibodies (p = 0.09). These results provide new insights into a mechanistic role of serum in protection against death in human melioidosis for antibody-based vaccine development

Chikungunya virus was isolated in Thailand, 2010

Chikungunya fever (CHIKF) is an acute febrile illness caused by a mosquito-borne alphavirus, chikungunya virus (CHIKV). This disease re-emerged in Kenya in 2004, and spread to the countries in and around the Indian Ocean. The re-emerging epidemics rapidly spread to regions like India and Southeast Asia, and it was subsequently identified in Europe in 2007, probably as a result of importation of chikungunya cases. On the one hand, chikungunya is one of the neglected diseases and has only attracted strong attention during large outbreaks. In 2008–2009, there was a major outbreak of chikungunya fever in Thailand, resulting in the highest number of infections in any country in the region. However, no update of CHIKV circulating in Thailand has been published since 2009. In this study, we examined the viral growth kinetics and sequences of the structural genes derived from CHIKV clinical isolates obtained from the serum specimens of CHIKF-suspected patients in Central Thailand in 2010. We identified the CHIKV harboring two mutations E1-A226V and E2-I211T, indicating that the East, Central, and South African lineage of CHIKV was continuously circulating as an indigenous population in Thailand. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11262-014-1105-5) contains supplementary material, which is available to authorized users

Human Immune Responses to Melioidosis and Cross-Reactivity to Low-Virulence Burkholderia Species, Thailand1.

Melioidosis is a neglected tropical disease with an estimated annual mortality rate of 89,000 in 45 countries across tropical regions. The causative agent is Burkholderia pseudomallei, a gram-negative soil-dwelling bacterium. In Thailand, B. pseudomallei can be found across multiple regions, along with the low-virulence B. thailandensis and the recently discovered B. thailandensis variant (BTCV), which expresses B. pseudomallei-like capsular polysaccharide. Comprehensive studies of human immune responses to B. thailandensis variants and cross-reactivity to B. pseudomallei are not complete. We evaluated human immune responses to B. pseudomallei, B. thailandensis, and BTCV in melioidosis patients and healthy persons in B. pseudomallei-endemic areas using a range of humoral and cellular immune assays. We found immune cross-reactivity to be strong for both humoral and cellular immunity among B. pseudomallei, B. thailandensis, and BTCV. Our findings suggest that environmental exposure to low-virulence strains may build cellular immunity to B. pseudomallei

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

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

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