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.

A nonsense mutation in TLR5 is associated with survival and reduced IL-10 and TNF-α levels in human melioidosis.

BACKGROUND: Melioidosis, caused by the flagellated bacterium Burkholderia pseudomallei, 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 TLR5 genetic variant that truncates the receptor with clinical outcomes and with immune responses in melioidosis. METHODOLOGY/PRINCIPAL FINDINGS: We genotyped TLR5 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, P = 0.04) and with lower 90-day mortality (OR 0.25, 95% CI 0.07-086, P = 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, P = 0.06); and the adjusted OR for 90-day mortality was 0.27 (95% CI 0.08-0.97, P = 0.04). c.1174C>T was associated with a lower rate of bacteremia (P = 0.04) and reduced plasma levels of IL-10 (P = 0.049) and TNF-α (P < 0.0001). We did not find an association between c.1174C>T and IFN-γ ELISPOT (T-cell) responses (P = 0.49), indirect haemagglutination titers or IgG antibodies to bacterial flagellin during acute melioidosis (P = 0.30 and 0.1, respectively). CONCLUSIONS/SIGNIFICANCE: This study independently confirms the association of TLR5 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.

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.

Monoclonal antibody targeting chikungunya virus envelope 1 protein inhibits virus release.

Chikungunya virus (CHIKV) causes an acute clinical illness characterized by sudden high fever, intense joint pain, and skin rash. Recent outbreaks of chikungunya disease in Africa and Asia are a major public health concern; however, there is currently no effective licensed vaccine or specific treatment. This study reported the development of a mouse monoclonal antibody (MAb), CK47, which recognizes domain III within the viral envelope 1 protein and inhibited the viral release process, thereby preventing the production of progeny virus. The MAb had no effect on virus entry and replication processes. Thus, CK47 may be a useful tool for studying the mechanisms underlying CHIKV release and may show potential as a therapeutic agent.

Sequence variation of dengue type 2 virus isolated from clinical cases in Thailand.

Dengue fever (DF) and dengue hemorrhagic fever (DHF) are caused by mosquito-borne dengue virus (DENV) infection leading to death in tropical and subtropical countries. In Thailand, all 4 serotypes of DENV are circulating. The most severe cases of DF and DHF are primarily introduced by secondary infections. Epidemiological studies have demonstrated that approximately 20% of the primary infection cases were caused by DENV-1 and -3, while the cases of DENV-2 or -4 accounted for less than 3%. For this reason, DENV-2 and -4 from primary infections have not been well studied. In this study, the sequence diversity of the envelope gene of 8 DENV-2 clinical isolates from primary/secondary infections was analyzed. DENV-2 from primary infections were highly heterogeneous in individual patients, whereas those from secondary infections were homogeneous. Phylogenetic analysis demonstrated that the heterogeneous population of DENV-2 from primary infections was composed of closely related quasispecies. Homogenous DENV-2 could be derived from selection of a particular viral population in secondary infections. The degree of sequence diversity of DENV-2 varied, and thus quasispecies may be involved in the progression of DENV infection.

Characterization of chikungunya virus infection of a human keratinocyte cell line: role of mosquito salivary gland protein in suppressing the host immune response.

The chikungunya virus (CHIKV) is a mosquito-borne virus that has recently re-emerged in several countries. On infection, the first vertebrate cells to come into contact with CHIKV are skin cells; mosquitoes inoculate the virus together with salivary gland protein into host skin while probing and feeding on blood. However, there is little known about the susceptibility of human skin cells to CHIKV infection. To clarify this, we investigated the kinetics of CHIKV in the human keratinocyte cell line, HaCaT. CHIKV actively replicated in HaCaT cells, with virus titers in the supernatant increasing to 2.8 × 10(4) plaque-forming units (PFU) ml(-1) 24h post infection. CHIKV infection suppressed production of interleukin-8 (IL-8) in HaCaT cells. The function of IL-8 is to recruit immune cells to virus-infected sites, a process known as chemotaxis. Furthermore, we assessed the role of mosquito salivary gland protein in CHIKV infections by comparing the levels of CHIKV gene expression and chemokine production in HaCaT cells with and without salivary gland extract (SGE). SGE enhanced both the expression of the CHIKV gene and the suppression effect of CHIKV on IL-8 production. Our data suggest that the HaCaT cell line represents an effective tool for investigating the mechanism of CHIKV transmission and spread in skin cells. At the mosquito bite site, CHIKV works together with SGE to ensure the virus replicates in skin cells and escapes the host immune system by suppression of IL-8 production.

Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies derived from dengue patients at acute phase of secondary infection.

Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab')2 fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection.