PCR combined with lateral flow dipstick assay (PCR-LFD) for a rapid diagnosis of melioidosis.

BACKGROUND: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. Septicemic melioidosis patients have a high mortality rate within 48 hours. OBJECTIVE: To develop a polymerase chain reaction (PCR) combined with a lateral flow dipstick (LFD) assay for detection of B. pseudomallei in blood samples. METHODS: The PCR with wcbG gene primers and a PCR-LFD test were developed. The specificity and sensitivity were determined using the B. pseudomallei and other bacterial DNAs. They were evaluated using 43 B. pseudomallei positive blood samples and another 43 blood samples positive for other microbial infections. RESULTS: The detection limit of the PCR-LFD test was 50 fg of bacterial gDNA or 1.0 CFU per 200 µl of blood. All B. pseudomallei were positive while B. thailandensis and selected gram-negative bacterial strains were negative. The PCR-LFD gave all positives with all 43 B. pseudomallei culture positive patient blood samples and all negative with 43 blood samples that were culture positive for K. pneumoniae, E. gallinarum, E. faecium, E. coli, S. aureus, A. baumannii, A. hydrophila, S. haemolyticus, S. pneumoniae, P. aeruginosa, E. cloacae, S. hominis, E. aerogenes, P. mirabilis, C. neoformans, C. albicans, A. caviae, E. faecalis and K. variicola. CONCLUSION: The developed PCR-LFD assay provided 100% sensitivity and 100% specificity compared to the conventional blood culture. The technique took only 1.5 hours that is easy and quick to perform compared to the 3-7 days of culture method. The new method of PCR with LFD could facilitate the detection to be a semi-point-of-care testing (POCT).

Early Activation of iNKT Cells Increased Survival Time of BALB/c Mice in a Murine Model of Melioidosis.

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. High interferon gamma (IFN-γ) levels in naive mice were reported to mediate protection against B. pseudomallei infection. Invariant natural killer T (iNKT) cells can produce and secrete several cytokines, including IFN-γ. When iNKT cell-knockout (KO) BALB/c mice were infected with B. pseudomallei, their survival time was significantly shorter than wild-type mice. Naive BALB/c mice pretreated intraperitoneally with α-galactosylceramide (α-GalCer), an iNKT cell activator, 24 h before infection demonstrated 62.5% survival at the early stage, with prolonged survival time compared to nonpretreated infected control mice (14 ± 1 days versus 6 ± 1 days, respectively). At 4 h after injection with α-GalCer, treated mice showed significantly higher levels of serum IFN-γ, interleukin-4 (IL-4), IL-10, and IL-12 than control mice. Interestingly, the IFN-γ levels in the α-GalCer-pretreated group were decreased at 4, 24, and 48 h after infection, while they were highly increased in the control group. At 24 h postinfection in the α-GalCer group, bacterial loads were significantly lower in blood (no growth and 1,780.00 ± 51.21, P < 0.0001), spleens (no growth and 34,300 ± 1,106.04, P < 0.0001), and livers (1,550 ± 68.72 and 13,400 ± 1,066.67, P < 0.0001) than in the control group, but not in the lungs (15,300 ± 761.10 and 1,320 ± 41.63, P < 0.0001), and almost all were negative at 48 h postinfection. This study for the first time shows that early activation of iNKT cells by α-GalCer helps clearance of B. pseudomallei and prolongs mouse survival.

Decrease of spleen invariant natural killer T and natural killer cell numbers in Burkholderia thailandensis-infected C57BL/6 mice.

BACKGROUND: Burkholderia thailandensis is a non-pathogenic bacterium that is closely related to B. pseudomallei. Invariant natural killer T (iNKT) cells are innate lymphoid cells that play a role in bacterial infections; however the iNKT cells in B. thailandensis infections are still uncharacterized. OBJECTIVE: To study the cytokine production in α-galactosylceramide (α-GalCer)-stimulated lymphocytes from mouse organs. The numbers of spleen iNKT cells, natural killer (NK) cells, dendritic cells and macrophages in B. thailandensis- infected C57BL/6 (B6) mice were investigated. METHODS: Lymphocytes, obtained from mouse lungs, liver, and spleen, were cultured for 48 hours with α-GalCer, and their cytokine levels were determined. iNKT, dendritic, macrophage and NK cells in the spleen of B. thailandensis-infected B6 mice or iNKT knock out (KO) mice, stimulated with either phosphate-buffered saline (PBS) or α-GalCer, were analyzed by flow cytometry. This was also done in adoptive cell transfer experiments. RESULTS: Interferon gamma (IFN-γ) was predominantly produced in α-GalCer-stimulated mouse spleen and liver lymphocytes, while interleukin (IL)-13 was the main cytokine found in the lungs. B. thailandensis-infected mice had a significantly lower number of splenic iNKT, NK and dendritic cells, but not macrophages, compared to the control. Interestingly, the number of NK cells was significantly decreased in iNKT wild type and iNKT KO mice after B. thailandensis infection. The number of NK cells recovered by activation with α-GalCer or after adoptive transfer of iNKT cells into KO mice. The iNKT cell-mediated reduction of dendritic and NK cells might be related to infection by B. thailandensis. CONCLUSIONS: B. thailandensis decreased the number of iNKT and NK cells in the spleen of infected mice.

Development of an immunomagnetic separation-ELISA for the detection of Burkholderia pseudomallei in blood samples.

BACKGROUND: Septicemic melioidosis caused by Burkholderia pseudomallei is a serious cause of morbidity and mortality. An effective, rapid and simple diagnostic method is required for detection of B. pseudomallei infection. OBJECTIVE: To develop immunomagnetic beads (IMB) coupled with ELISA (IMB-ELISA) for detection of B. pseudomallei in blood samples of patients with suspected melioidosis. METHODS: For separation of B. pseudomallei from buffer, blood samples and hemoculture, 200 nm immunomagnetic beads (IMBs) coated with 4B11 monoclonal antibody (4B11-IMBs) against exopolysaccharide antigens were used. The detection was done by an ELISA based biotin-streptavidin system. The sensitivity and specificity were evaluated. RESULTS: 4B11-IMBs (100 µg) were successfully developed and used for detection of B. pseudomallei in 1 ml samples. Transmission electron microscopy (TEM) imaging demonstrated B. pseudomallei was captured by 4B11-IMBs. The IMBs showed high capture efficiency (98%) with B. pseudomallei in buffer. The IMB-ELISA assay was highly specific for B. pseudomallei. It showed no cross-reactions with other bacteria, except B. mallei. The limits of the B. pseudomallei assay detection for detecting B. pseudomallei in either buffer solution or blood was 102 CFU/ml. The IMB-ELISA detection sensitivity in blood samples was 44.5%. Although it did not give the highest sensitivity, it was useful for detection with hemoculture that was faster than conventional methods. CONCLUSION: This study suggests the IMB-ELISA assay offers a simple and highly specific method with a turnaround time of 6 h for detection of B. pseudomallei. The developed assay can be applied in hospitals for surveillance of B. pseudomallei.

Invariant Natural Killer T (iNKT) cells response in human melioidosis.

BACKGROUND: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. In infected mice, IFN-γ can provide protection against B. pseudomallei infection. Invariant Natural Killer T (iNKT) cells are a subpopulation of T lymphocytes, activated by recognition of glycolipid ligands such as α-Galactosylceramide presented by CD1d, produce and secrete several cytokines, including IFN-γ and IL-4. The response of iNKT cells in human melioidosis was then investigated. OBJECTIVE: To determine the iNKT cells response in human melioidosis. METHODS: The number of human iNKT cells and its activation states were investigated in sepsis melioidosis patients compared with healthy controls using flow cytometry. The iNKT cells activation was confirmed in vitro using heatkilled B. pseudomallei with normal peripheral blood mononuclear cells. The components induced iNKT cell were also determined using different concentration of B. pseudomallei lipopolysaccharide (LPS), heat-killed B. pseudomallei treated with or without DNase, RNase, or proteinase. RESULTS: The number of human iNKT cells was significantly lower while the percentage of activated iNKT cells was higher in sepsis melioidosis when compared to control. In addition, B. pseudomallei can stimulate human iNKT cells in vitro. Heat-killed B. pseudomallei could activate iNKT cells but not relate to nucleic acid, proteins, or LPS. CONCLUSIONS: We found for the first time that the iNKT cells were activated during B. pseudomallei infection in human. However, the roles and the mechanism of iNKT cells during early state of infection needed to be further investigated.

Characterization and functional analysis of novel circulating NK cell sub-populations.

Natural killer (NK) cells are innate lymphoid cells having potent cytolytic function that provide host defense against microbial infections and tumors. Using our generated monoclonal antibody (mAb), named FE-1H10, new NK cell sub-populations in peripheral blood were identified. The molecules recognized by mAb FE-1H10 were expressed on a sub-population of CD3-CD56dim NK cells. The epitope recognized by mAb FE-1H10 was demonstrated to be N-glycan and proven to be different from CD57. Upon K562 stimulation, the CD56dimFE-1H10+ NK cell sub-population exhibited significantly lower cytolytic function with low ability to degranulate and release cytolytic granules compared to the CD56dimFE-1H10- NK cell sub-population. Moreover, the CD56dimFE-1H10+ NK cells produced less IFN-γ and TNF-α than the CD56dimFE-1H10- NK cells. We demonstrated here that mAb FE-1H10 could identify two sub-populations of circulating CD56dim NK cells with different functions. Our discovery of new sub-populations of NK cells improves our understanding of NK cell biology and may lead to the development of new approaches for NK cell therapy.

D-LL-31 in combination with ceftazidime synergistically enhances bactericidal activity and biofilm destruction in Burkholderia pseudomallei.

Melioidosis is a severe disease caused by Burkholderia pseudomallei. The biofilm of B. pseudomallei acquires resistance to several antibiotics and may be related to relapse in melioidosis patients. Here, the killing activity of antimicrobial peptides (LL-37, LL-31) and the D-enantiomers (D-LL-37, D-LL-31) in combination with ceftazidime (CAZ) against B. pseudomallei 1026b, H777 and a biofilm mutant M10, derived from H777 grown under biofilm-stimulating conditions was observed. Using static conditions, D-LL-31 exhibited the strongest killing activity against the three isolates in a dose-dependent manner. IC50 values for D-LL-31 ranged from 1 to 6 µM, for isolates M10, H777, and 1026b, respectively. Moreover, D-LL-31 combined with CAZ synergistically decreased the IC50 values of the peptide and antibiotic and caused also disruption of biofilms of B. pseudomallei 1026b under flow conditions. Thus a combination of D-LL-31 and CAZ may enhance the efficacy of the currently used antibiotic treatments against B. pseudomallei.

Hamster IFN-γ+CD4+ and IL-4+CD4+ T cell responses against leptospires are significantly higher than those of mice.

BACKGROUND: Leptospirosis is a bacterial disease caused by the Leptospira interrogans. The hamster is considered a susceptible host while the mouse is resistant. The knowledge of hamster T cell immunity is limited compared to the mouse. The reason why the hamster and the mouse give different responses to leptospires remains unclear. OBJECTIVE: To determine the differential responses of CD4+ T cells between hamsters and mice using Leptospira interrogans as an infectious model. METHODS: The CD4+ T-cell reactivity and their intracellular cytokine responses after infection with live L.interrogans serovar Autumnalis or leptospiral antigens, or injection with recombinant LipL32 protein (rLipL32) were elucidated. For secondary immune responses, mononuclear cells were re-stimulated with leptospiral crude antigens (LAg) or rLipL32. Intracellular cytokines and CD4+ T cells were determined using flow cytometry. RESULTS: There were no significant differences between the percentages of hamster and mouse CD4+ and CD25+CD4+ T cell responses to live bacteria. Mouse CD4+ (24.50±1.98%) and CD25+CD4+ T cells (3.83±0.88) responded significantly higher than those of hamster (15.07±2.82% and 2.00±0.37%) when infected and re-stimulated with LAg. The numbers of IFN-γ and IL-4 producing cells in hamsters at 1.76±0.10% and 0.82±0.25% for IFN-γ+CD4+ and IL-4+CD4+ T cells were significantly higher than those in resistant mice at 0.10±0.02% and 0.23±0.03% for IFN-γ+CD4+ and IL-4+CD4+ T cells. CONCLUSION: Hamsters responded significantly higher in secondary stimulation especially in the levels of the IFN-γ+ and IL-4+CD4+ T cells. The mechanisms of this dissimilarity remain to be elucidated.

BURKHOLDERIA PSEUDOMALLEI BIOFILM PLAYS A KEY ROLE IN CHRONIC INFLAMMATION IN C57BL/6 MICE.

Burkholderia pseudomallei is a causative agent of melioidosis. Clinical signs of melioidosis vary from acute septicemia to chronic inflammation or subclinical infection. This study investigated the role of B. pseudomallei biofilm in chronic inflammation in lungs of infected C57BL/6 mice. Low doses of B. pseudomallei H777 and its biofilm defective M10 mutant were fed intra-gastrically to C57BL/6 mice and inflammatory responses were investigated by histopathological techniques. Two hundred colony forming units (CFUs) of B. pseudomallei H777 induced chronic inflammatory responses in mice on day 20 post-infection, with discrete interstitial infiltration by mononuclear inflammatory cells. On day 40 postinfection, there were marked thickening of alveolar septa and congested capillaries, which increased in severity by day 60. On the other hand, mice infected with B. pseudomallei M10 showed less mononuclear infiltration. The results indicate that B. pseudomallei defective in biofilm production gave rise to less severe pathology, resulting a higher rate of survival in infected mice; and pulmonary melioidosis could be developed in C57BL/6 mice by intra-gastric feeding makes it a possible animal model of chronic human melioidosis.

Relapsed Melioidosis Model in C57BL/6 Mice.

BACKGROUND: Burkholderia pseudomallei are the causative agents of melioidosis, a disease that has a high relapse rate in endemic areas. The mechanism of relapse is unclear OBJECTIVE: This study aimed to establish relapsed melioidosis in C57BL/6 mice by induction with B. pseudomallei. MATERIAL AND METHOD: Low doses of B. pseudomallei H777 and its biofilm defective mutant (M10) were intra-gastric fed to C57BL/6 mice. All the infected mice had suppressed immune status by intra-peritoneal injection of hydrocortisone at 2.5 mg per mouse at day 60 post-infection. Inflammatory response to the infection was investigated by histo-pathological studies and monitoring bacterial counts in the blood and organs. RESULTS: All the infected mice were found to have a high infiltration of mononuclear cells at day 60 post-infection. The results showed high bacterial counts in the blood in both strains post-suppressed immune status after two days. The biofilm mutant and wild type strains produced relapse in C57BL/6 mice but the latter was responsible for significantly more severe inflammation than the biofilm mutant. CONCLUSION: Low immune status may cause relapsed melioidosis in hosts with chronic inflammation.

A PCR-BASED DETECTION OF BURKHOLDERIA PSEUDOMALLEI DIVERSITY USING MYOVIRIDAE PROPHAGE TYPING.

PCR-based detection of Myoviridae lysogenic phages in Burkholderia pseudomallei was developed using primers targeting K96243 prophage GI2, phiE12-2 and phi52237/phiX216. Investigation of 50 clinical and 50 environmental (soil) isolates revealed that K96243 prophage GI2 was the most common (48%) among the isolates, followed by phiE12-2 (38%) and phi52237/phiX216 (35%), with K96243 prophage GI2 being significantly more frequent in soil (64%) than clinical (32%) samples. Twenty-four percent of soil isolates contained all three prophage types, while clinical isolates harbored no more than two types. Although B. pseudomallei isolates from soil were found to be more diverse based on prophage typing, all isolates were equally susceptible to a battery of lytic phages (although to different extents), suggesting the possibility of using lytic phages to control environmental B. pseudomallei.

THE CONTRIBUTION OF SOIL PHYSICOCHEMICAL PROPERTIES TO THE PRESENCE AND GENETIC DIVERSITY OF BURKHOLDERIA PSEUDOMALLEI.

Burkholderia pseudomallei (Bp), the causative agent of melioidosis, is unevenly distributed in the complex soil environment. Physicochemical factors in the soil have been reported to affect microbial communities in the soil. The effect of physicochemical factors on the number and diversity of organisms in the soil has not been reported. Twenty-five each B. pseudomallei-positive and -negative soil samples were collected from a melioidosis-endemic area. The amount of Bp in each soil sample was measured by culture and quantitative PCR (qPCR). The following physicochemical properties from each soil sample were measured: pH, total organic carbon (TOC), total nitrogen (TN), carbon to nitrogen ratio (C:N ratio), exchangeable calcium (EC) and extractable iron (EI). All the physico- chemical properties measured were significantly different between the Bp-positive and -negative soil samples. The Bp-positive soil samples had lower C:N ratios and lower EC and a higher EI (p < 0.05) than the Bp-negative samples. The average pH was lower (3.7-5.0) in the Bp-negative samples. Among the Bp-positive soil samples, the EC was negatively correlated with the PCR copy number. The amount of bacteria detected with the qPCR method was higher than with the culture method, suggesting the presence of unculturable forms of bacteria that might re-grow when the environmental conditions was suitable. A total of 117 Bp isolates obtained from the soil samples were classified into 25 groups using BOX-PCR. The genetic diversity of Bp, did not correlate with the physicochemical factors investigated. A suitable pH range and C:N ratio may be important for the presence of Bp. The EI supports the needs and EC probably alters the growth of Bp. The genetic diversity of the bacteria was not influenced by the soil factors investigated in this study. This information shows the environment conducive to the growth of Bp. This gives us information about how to potentially control or decrease Bp in the soil in the future.

Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis.

LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265-284 and lactoferricin17-30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.

Characterization of a novel two-component system response regulator involved in biofilm formation and a low-iron response of Burkholderia pseudomallei.

Two-component systems (TCSs) regulate an adaptive response to environmental conditions, leading to changes in bacterial cellular processes. In this study, we identified a novel TCS response regulator gene, designated as bfmR (biofilm formation-associated regulator) that regulates biofilm formation by Burkholderia pseudomallei (Bp). An insertion mutant of the Bp bfmR gene resulted in a significant decrease in expression of fimbriae chaperone-usher assembly genes (BPSL2O28 and BPSL22 7), leading to suppression of assembly of fimbriae on the cell surface and reduced biofilm formation. The defective phenotypes of the mutant strain were restored by introducing a complementing plasmid having an intact bfmR gene. Using RT-PCR analyses, we found that bfmR gene expression was upregulated under low-iron growth conditions. In addition, the bfmR mutant strain showed retarded growth in low-iron medium and in phagocytic cells compared to the wild-type strain. These results indicate that bfmR is a novel positive regulator for controlling assembly of fimbriae and biofilm formation, and is upregulated under low-iron conditions.

A heterodimer comprised of two bovine lactoferrin antimicrobial peptides exhibits powerful bactericidal activity against Burkholderia pseudomallei.

Melioidosis is a severe infectious disease that is endemic in Southeast Asia and Northern Australia. Burkholderia pseudomallei, the causative agent of this disease, has developed resistance to an increasing list of antibiotics, demanding a search for novel agents. Lactoferricin and lactoferrampin are two antimicrobial domains of lactoferrin with a broad spectrum of antimicrobial activity. A hybrid peptide (LFchimera) containing lactoferrampin (LFampin265-284) and a part of lactoferricin (LFcin17-30) has strikingly higher antimicrobial activities compared to the individual peptides. In this study, the antimicrobial activities of this chimeric construct (LFchimera1), as well as of another one containing LFcin17-30 and LFampin268-284, a shorter fragment of LFampin265-284 (LFchimera2), and the constituent peptides were tested against 7 isolates of B. pseudomallei and compared to the preferential antibiotic ceftazidime (CAZ). All isolates including B. pseudomallei 979b shown to be resistant to CAZ, at a density of 10(5) CFU/ml, could be killed by 5-10 µM of LFchimera1 within 2 h, while the other peptides as well as the antibiotic CAZ only inhibited the B. pseudomallei strains resulting in an overgrowth in 24 h. These data indicate that LFchimera1 could be considered for development of therapeutic agents against B. pseudomallei.

Environmental management procedures following fatal melioidosis in a captive chimpanzee (Pan troglodytes).

A 40-yr-old male captive chimpanzee (Pan troglodytes) presented with depression and anorexia for 7 days. The tentative diagnosis, following a physical examination under anesthesia, was pneumonia with sepsis. Despite antibiotic treatment and supportive care the chimpanzee died a week following presentation. Gross pathology confirmed severe purulent pneumonia and diffuse hepatosplenic abscesses. Detected in serum at the time of the initial examination, the melioidosis serum antibody titer was elevated (> 1:512). Soil samples were collected from three sites in the exhibit at three depths of 5, 15, and 30 cm. By direct and enrichment culture, positive cultures for Burkholderia pseudomallei were found at 5 and 15 cm in one site. The other two sites were positive by enrichment culture at the depth of 5 cm. To prevent disease in the remaining seven troop members, they were relocated to permit a soil treatment with calcium oxide. The exhibit remained empty for approximately 1 yr before the chimpanzees were returned. During that period, the soil in the exhibit area was again cultured as before and all samples were negative for B. pseudomallei. Following the soil treatment in the exhibit, all chimpanzees have remained free of clinical signs consistent with melioidosis.

A two-arm analysis of the immune response to heterologous boosting of inactivated SARS-CoV-2 vaccines.

Several vaccine programs were introduced during the COVID-19 pandemic, which included inactivated virus, DNA viral vectors and mRNA vaccines. Booster programs are recommended, especially for those in high-risk groups. However, many of these booster programs involve heterologous vaccines. This study enrolled volunteers who first received two full-dose CoronaVac vaccinations before receiving heterologous boosters with DNA- and/or mRNA-vaccines for an additional 2 doses (n = 40) or an additional 3 doses (n = 16). Our results showed no difference in side effects, neutralizing antibodies, or T-cell responses for any of the heterologous vaccination programs. However, the neutralizing capacity and IFN-γ responses against the Omicron variant in volunteers who received 4 or 5 doses were improved. Polarization of peripheral memory T cells after stimulation in all booster groups with Omicron peptide showed an increased trend of naïve and central memory phenotypes of both CD4+ and CD8+ T cells, suggesting that exposure to Omicron antigens will drive T cells into a lymphoid resident T cell phenotype. Our data support a continuous vaccination program to maximize the effectiveness of immunity, especially in people at high risk. Furthermore, the number of boosting doses is important for maintaining immunity.

Melioidosis in animals, Thailand, 2006-2010.

We retrospectively estimated the incidence of culture-proven melioidosis in animals in Thailand during 2006-2010. The highest incidence was in goats (1.63/100,000/year), followed by incidence in pigs and cattle. The estimated incidence of melioidosis in humans in a given region paralleled that of melioidosis in goats.

Phenotypic and functional changes of T cell subsets after CoronaVac vaccination.

BACKGROUND: The pandemic coronavirus disease 2019 (COVID-19) is a major global public health concern and several protective vaccines, or preventive/therapeutic approaches have been developed. Sinovac-CoronaVac, an inactivated whole virus vaccine, can protect against severe COVID-19 disease and hospitalization, but less is known whether it elicits long-term T cell responses and provides prolonged protection. METHODS: This is a longitudinal surveillance study of SARS-CoV-2 receptor binding domain (RBD)-specific IgG levels, neutralizing antibody levels (NAb), T cell subsets and activation, and memory B cells of 335 participants who received two doses of CoronaVac. SARS-CoV-2 RBD-specific IgG levels were measured by enzyme-linked immunosorbent assay (ELISA), while NAb were measured against two strains of SARS-CoV-2, the Wuhan and Delta variants. Activated T cells and subsets were identified by flow cytometry. Memory B and T cells were evaluated by enzyme-linked immune absorbent spot (ELISpot). FINDINGS: Two doses of CoronaVac elicited serum anti-RBD antibody response, elevated B cells with NAb capacity and CD4+ T cell-, but not CD8+ T cell-responses. Among the CD4+ T cells, CoronaVac activated mainly Th2 (CD4+ T) cells. Serum antibody levels significantly declined three months after the second dose. INTERPRETATION: CoronaVac mainly activated B cells but T cells, especially Th1 cells, were poorly activated. Activated T cells were mainly Th2 biased, demonstrating development of effector B cells but not long-lasting memory plasma cells. Taken together, these results suggest that protection with CoronaVac is short-lived and that a third booster dose of vaccine may improve protection.

Opisthorchis viverrini antigens up-regulates the expression of CD80 and MHC class II in JAWSII mouse dendritic cells and promotes IL-10 and TGF-ß secretions.

Dendritic cells (DCs) are antigen-presenting cells (APC) involved in the initiation of immune responses. Maturation of DCs is characterized by the high expression of major histocompatibility complex (MHC) class II and co-stimulatory clusters of differentiation (CD) 40, CD80, and CD86 molecules. Matured DCs are required for T cell differentiation and proliferation. However, the response of DCs to Opisthorchis viverrini antigens has not yet been understood. Therefore, this study sought to determine the expression of surface molecules of JAWSII mouse DCs stimulated by crude somatic (CS) and excretory-secretory (ES) antigens of O. viverrini. ES antigen significantly induced only mRNA expression of CD80 and MHC class II in JAWSII mouse DCs, while CS antigen promoted up-regulation of both mRNA and protein levels of CD80 and MHC class II, indicating relative maturation of JAWII mouse DCs. Moreover, the secreted cytokines from the co-cultures of O. viverrini antigens stimulated JAWSII DC with naïve CD4+ T cells was determined. Significantly increased levels of immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-ß) were found. The up-regulation of these cytokines may indicate the response of regulatory T cells (Treg) to CS antigen-stimulated JAWSII DC. These findings may lead to a better understanding of the role that DCs play in O. viverrini infection.