Recombinant Dense Granule Protein (NcGRA4) Is a Novel Serological Marker for Neospora caninum Infection in Goats.

Neospora caninum is widely recognised as one of the most significant causes of abortion in cattle, with infections also occurring in sheep and goats. To prevent and control animal neosporosis, it is crucial to develop sensitive and specific methods for detecting N. caninum infection. Recently, several recombinant proteins have been utilised in serological assays for the diagnosis of neosporosis. In this study, we used commercial gene synthesis to produce dense granular antigen 4 (NcGRA4) recombinant protein. NcGRA4 plasmids were expressed in the Escherichia coli system and then purified. The purified recombinant protein was analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. To evaluate the diagnostic potential of recombinant NcGRA4 protein, we tested 214 serum samples from goat farms via indirect enzyme-linked immunosorbent assay (iELISA) and compared the results to those from the indirect fluorescent antibody test (IFAT). Western blotting analysis revealed a single NcGRA4 band with an expected molecular weight of 32 kDa. The specific IgG against N. caninum was detected in 34.1% and 35% of samples evaluated by NcGRA4 iELISA and IFAT, respectively. The sensitivity and specificity of the NcGRA4 iELISA were 71.6% and 86.3%, respectively, when compared with the results from IFAT. Our results demonstrate that a recombinant protein that can be used to detect animal neosporosis can be produced using a synthetic NcGRA4 gene. Overall, recombinant NcGRA4 shows promise as a sensitive and specific serological marker for identifying target IgG in goat samples.

Development of an animal-derived component-free medium for Spodoptera frugiperda (Sf9) cells using response surface methodology.

OBJECTIVES: To develop an animal-derived component-free medium for Spodoptera frugiperda (Sf9) growth and green fluorescent protein (GFP) expression. RESULTS: OSF9-ADCFM contained optimum concentrations of CDLC, YE and ST at 0.5% (v/v), 11.0 g/L, and 3.0 g/L, respectively. A mean viable cell concentration of 1.71 ± 0.14 × 105 cells/mL was obtained from 5 passages (P1-P5). The use of both peptones after 10 kDa ultrafiltration had a significant effect on Sf9 cell growth. Grace's insect medium with 10% FBS gave higher un-infected cell number than SF-900II and OSF9-ADCFM for 4.29 and 5.38 times, respectively. The average cell number of un-infected cells and GFP-fluorescent cells of SF-900II were higher than OSF9-ADCFM 1.25 and 7 times, respectively. CONCLUSION: In-house OSF9-ADCFM could support growth and GFP expression in Sf9 less than commercial SF-900II. However, it could lower the production cost at least 50% comparing to commercial SF-900II. The development of in- house OSF9-ADCFM would be continued to increase both cell numbers and protein expression in the next step.

Peptide microarray-based identification of dormancy-associated Mycobacterium tuberculosis antigens inducing immune responses among latent tuberculosis infection individuals in Thailand.

Multi-stage tuberculosis (TB) vaccines composed of active- and dormancy-associated antigens are promising to trigger the immune protection against all TB stages. However, scientists are still in quest of the suitable vaccine candidates. In this study, we identified the potential targets for this vaccine in a high TB burden country, Thailand. Peptide microarray was applied to gauge IgA and IgG antibodies specific to 16,730 linear epitopes of 52 dormancy-associated Mycobacterium tuberculosis (M. tb) proteins in three study groups: active tuberculosis (ATB), latent tuberculosis infection (LTBI) and endemic healthy control (EHC). Preferential IgA recognition against epitopes of dormancy-associated proteins was identified in LTBI group. Validation of these findings revealed that LTBI subjects exhibited the greater levels of Rv2659c- and Rv1738-specific IgA than those of household contacts, but less than did ATB subjects. Frequencies of IFNγ-producing CD4+ and CD8+ T cells induced by proteins Rv2659c and Rv1738 were higher in LTBI than ATB individuals. The results indicated that LTBI group in a high TB burden country demonstrated cell-mediated immune response to proteins Rv2659c and Rv1738 stronger than those of ATB. These immune responses likely contribute to natural protection against dormant M. tb and might be potential targets for a multi-stage TB vaccine.

SARS-CoV-2 Seroprevalence in Unvaccinated Adults in Thailand in November 2021.

Between the first case of COVID-19 in January 2020 and the end of 2021, Thailand experienced four waves of the epidemic. The third and fourth waves were caused by the alpha and delta strains from April 2021 to November 2021. Serosurveillance studies provide snapshots of the true scale of the outbreak, including the asymptomatic infections that could not be fully captured by a hospital-based case detection system. We aimed to investigate the distribution of SARs-CoV-2 seroprevalence in unvaccinated adults after the delta wave outbreak. From November to December 2021, we conducted a cross-sectional survey study in 12 public health areas (PHAs) across Thailand. A total of 26,717 blood samples were collected and tested for SARs-CoV-2 antibodies (anti-S IgG) using a qualitative immunoassay. The results showed that seropositive prevalence in this cohort was 1.4% (95% CI: 1.24 to 1.52). The lowest prevalence was in the northern region (PHA 1) and in central Thailand (PHA 3) at 0.4% (95% CI: 0.15 to 0.95), while the highest was in the southern region of Thailand (PHA 12) at 5.8% (95% CI: 4.48 to 7.29). This seropositive prevalence was strikingly lower than the reports from other countries. Our serosurveillance results suggest that the vaccination of unvaccinated groups should be accelerated, especially in the public health areas with the lowest seroprevalence.

The Pilot Study of Immunogenicity and Adverse Events of a COVID-19 Vaccine Regimen: Priming with Inactivated Whole SARS-CoV-2 Vaccine (CoronaVac) and Boosting with the Adenoviral Vector (ChAdOx1 nCoV-19) Vaccine.

In response to the SARS-CoV-2 Delta variant, which partially escaped the vaccine-induced immunity provided by two doses of vaccination with CoronaVac (Sinovac), the National Vaccine Committee recommended the heterologous CoronaVac-ChAdOx1 (Oxford−AstraZeneca), a prime−boost vaccine regimen. This pilot study aimed to describe the immunogenicity and adverse events of the heterologous CoronaVac-ChAdOx1 regimen, in comparison with homologous CoronaVac, and homologous ChAdOx1. Between May and August 2021, we recruited a total of 354 participants from four vaccination groups: the CoronaVac-ChAdOx1 vaccinee (n = 155), the homologous CoronaVac vaccinee (n = 32), the homologous ChAdOx1 vaccinee (n = 47), and control group of COVID-19 patients (n = 120). Immunogenicity was evaluated by measuring the level of IgG antibodies against the receptor-binding domain (anti-SRBD) of the SARS-CoV-2 spike protein S1 subunit and the level of neutralizing antibodies (NAbs) against variants of concern (VOCs) using the plaque reduction neutralization test (PRNT) and pseudovirus neutralization test (pVNT). The safety profile was recorded by interviewing at the 1-month visit after vaccination. The anti-SRBD level after the second booster dose of the CoronaVac-ChAdOx1 group at 2 weeks was higher than 4 weeks. At 4 weeks after the second booster dose, the anti-SRBD level in the CoronaVac-ChAdOx1 group was significantly higher than either homologous CoronaVac, the homologous ChAdOx1 group, and Control group (p < 0.001). In the CoronaVac-ChAdOx1 group, the PRNT50 level against the wild-type (434.5 BAU/mL) was the highest; followed by Alpha variant (80.4), Delta variant (67.4), and Beta variant (19.8). The PVNT50 level was also found to be at its highest against the wild-type (432.1); followed by Delta variants (178.3), Alpha variants (163.9), and Beta variant (42.2), respectively. The AEs in the CoronaVac-ChAdOx1 group were well tolerated and generally unremarkable. The CoronaVac-ChAdOx1 heterologous regimen induced higher immunogenicity and a tolerable safety profile. In a situation when only CoronaVac-ChAdOx1 vaccines are available, they should be considered for use in responding to the Delta variant.

Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats.

BACKGROUND: The development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT). RESULTS: Western blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT. CONCLUSION: Our findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera.

Coordinated In Vitro Release of Granulysin, Perforin and IFN-γ in TB and HIV/TB Co-Infection Associated with Clinical Outcomes before and after Anti-TB Treatment.

Granule-associated killing molecules released from cytotoxic T lymphocytes participate as a crucial step in immunity against tuberculosis (TB), but the role of coordinated production remains controversial. Coordinated release of effector molecules in vitro after stimulating peripheral blood mononuclear cells (PBMCs) of active TB or HIV/TB coinfection patients with PPD, purified protein derivative of tuberculin and avirulent Mtb, H37Ra, an attenuated strain were investigated in association with clinical outcomes. Perforin, granzyme-B, granulysin and IFN-γ were measured using ELISA. Before anti-TB treatment, PBMCs of TB stimulated with PPD or H37Ra released higher perforin, granzyme-B, and granulysin levels than in HIV/TB and released significantly higher IFN-γ (p = 0.045, p = 0.022). Granulysin positively correlated with perforin in TB (p = 0.042, r = 0.385), HIV/TB coinfection (p = 0.003, r = 0.941) after PPD stimulation, and after H37Ra stimulation in TB (p = 0.005, r = 0.549), but negatively correlated with granzyme B in TB (p = 0.042, r = -0.386), HIV/TB coinfection (p = 0.042, r = 0.754) were noted. After anti-TB treatment, increased levels of perforin, granulysin and IFN-γ in TB or HIV/TB upon PPD or H37Ra stimulation, and decreased granzyme-B levels after PPD (p = 0.003) or H37Ra (p = 0.028) stimulation in TB were observed. These results suggest that granulysin may act synergistic with perforin and IFN-γ in TB, indicating its crucial function in host immunity to tuberculosis. Future studies with larger numbers of patients ought to be conducted in the future.

Potential function of granulysin, other related effector molecules and lymphocyte subsets in patients with TB and HIV/TB coinfection.

BACKGROUND: Host effector mechanism against Mycobacterium tuberculosis (Mtb) infection is dependent on innate immune response by macrophages and neutrophils and the alterations in balanced adaptive immunity. Coordinated release of cytolytic effector molecules from NK cells and effector T cells and the subsequent granule-associated killing of infected cells have been documented; however, their role in clinical tuberculosis (TB) is still controversy. OBJECTIVE: To investigate whether circulating granulysin and other effector molecules are associated with the number of NK cells, iNKT cells, Vγ9(+)Vδ2(+) T cells, CD4(+) T cells and CD8(+) T cells, and such association influences the clinical outcome of the disease in patients with pulmonary TB and HIV/TB coinfection. METHODS: Circulating granulysin, perforin, granzyme-B and IFN-γ levels were determined by ELISA. The isoforms of granulysin were analyzed by Western blot analysis. The effector cells were analyzed by flow cytometry. RESULTS: Circulating granulysin and perforin levels in TB patients were lower than healthy controls, whereas the granulysin levels in HIV/TB coinfection were much higher than in any other groups, TB and HIV with or without receiving HAART, which corresponded to the number of CD8(+) T cells which kept high, but not with NK cells and other possible cellular sources of granulysin. In addition, the 17kDa, 15kDa and 9kDa isoforms of granulysin were recognized in plasma of HIV/TB coinfection. Increased granulysin and decreased IFN-γ levels in HIV/TB coinfection and TB after completion of anti-TB therapy were observed. CONCLUSION: The results suggested that the alteration of circulating granulysin has potential function in host immune response against TB and HIV/TB coinfection. This is the first demonstration so far of granulysin in HIV/TB coinfection.

Decreased plasma granulysin and increased interferon-gamma concentrations in patients with newly diagnosed and relapsed tuberculosis.

Granulysin and interferon-gamma (IFN-γ) have broad antimicrobial activity which controls Mycobacterium tuberculosis (M. tuberculosis) infection. Circulating granulysin and IFN-γ concentrations were measured and correlated with clinical disease in Thai patients with newly diagnosed, relapsed and chronic tuberculosis (TB). Compared to controls, patients with newly diagnosed, relapsed and chronic TB had lower circulating granulysin concentrations, these differences being significant only in newly diagnosed and relapsed TB (P < 0.001 and 0.004, respectively). Granulysin concentrations in patients with newly diagnosed and relapsed TB were significantly lower than in those with chronic TB (P= 0.003 and P= 0.022, respectively). In contrast, significantly higher circulating IFN-γ concentrations were found in patients with newly diagnosed and relapsed TB compared to controls (P < 0.001). The IFN-γ concentrations in newly diagnosed and relapsed patients were not significantly different from those of patients with chronic TB. However, in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from patients with newly diagnosed, relapsed and chronic TB with purified protein derivative (PPD) or heat killed M. tuberculosis (H37Ra) enhanced production of granulysin by PBMCs. In vitro, stimulation of PBMCs of newly diagnosed TB patients with PPD produced greater amounts of IFN-γ than did controls, while those stimulated with H37Ra did not. The results demonstrate that patients with active pulmonary TB have low circulating granulysin but high IFN-γ concentrations, suggesting possible roles in host defense against M. tuberculosis for these agents.

Association between circulating full-length osteopontin and IFN-gamma with disease status of tuberculosis and response to successful treatment.

The T helper type 1 (Th1) immune response plays an important role in protective immunity, pathophysiology and development of tuberculosis (TB). To investigate whether osteopontin (OPN) and other Th1 response-related molecules are associated withTB disease status, including co-infection with HIV, and response to anti-TB treatment, circulating levels of full-length OPN (F-OPN), thrombin-cleaved N-terminal fragment of OPN (N-half OPN), IFN-gamma, IP-10, IL-18, IL-12/ IL-23 (p40), IL-10, IL-15 and C-reactive protein (CRP) were measured before and after anti-TB treatment. Patients with newly active pulmonary TB had significantly higher plasma levels of F-OPN, IFN-gamma and CRP than healthy controls (HC). F-OPN, N-half OPN, IFN-gamma, IP-10, IL-18 and IL-10 levels were higher in patients with extensive TB/HIV co-infection than in patients with a single disease of TB or HIV. Plasma levels of F-OPN correlated well with those of IP-10, IL-18 and N-half OPN among patients with active TB. The F-OPN, IFN-gamma, IP-10 and CRP levels decreased significantly after effective anti-TB treatment. These data suggest that circulating OPN and Th1 response-related molecules, including IFN-gamma, may be regulated in response to expansion of active TB and could serve as markers of disease activity before and during treatment.

Study of anti-inflammatory activities of the pure compounds from Andrographis paniculata (burm.f.) Nees and their effects on gene expression.

In inflammation, the responses to noxious stimuli are controlled by the highly modulated interactions between various immune cells and chemical mediators. The purpose of this study is to evaluate and compare the anti-inflammatory effect of diterpenoids isolated from Andrographis paniculata, including dehydroandrographolide (AP1), andrographolide (AP2) and neoandrographolide (AP3), on the production of inflammatory cytokines and COX activities. Furthermore, the alteration of gene expression involved in this activity was investigated in the most potent compound to elucidate the other possible molecular mechanisms. AP1 (30.1 µM; 10 µg/ml) and AP2 (28.5 µM; 10 µg/ml) markedly inhibited COX-1 in ionophore A23187-induced human platelets. AP2 (28.5 µM) and AP3 (20.8 µM; 10 µg/ml) strongly suppressed the LPS-stimulated COX-2 activity in human blood. In addition, AP2 modulated the level of LPS-induced TNF-α, IL-6, IL-1ß and IL-10 secretion in human blood in a concentration-dependent manner. The results revealed that AP2 exhibited the highest efficacy. Therefore, changes in the levels of mRNA transcripts by AP2 were further measured using human cDNA microarrays. The molecular response to AP2 was complex and mediated by various processes. Among the altered gene expressions, the genes involved in immune and inflammation processes were selectively down-regulated, such as cytokines and cytokine receptors (TNFSF14, TNF, TNFRSF6, and IL1A), chemokines (CCL8 and CXCL11), JAK/STAT signaling (JAK3 and STAT5A), TLRs family (TLR4 and TLR8) and NF-κB (NFKB1). Expression of some genes was validated using RT-PCR. The results demonstrated that AP1, AP2 and AP3 exhibited the anti-inflammatory effect by interfering COX and inflammatory cytokines and the underlying mechanisms of AP2 may be related to down-expression of genes involved in inflammatory cascade.

Identification of the suppressive factors for human immunodeficiency virus type-1 replication using the siRNA mini-library directed against host cellular genes.

We performed the screening to find the novel host factors affecting human immunodeficiency virus type-1 (HIV-1) replication using the siRNA mini-library consisted with 257 siRNAs directed against cellular genes. J111 cells, a human acute monocytic leukemia cell line, were transfected with individual siRNA, followed by either infected or transfected with the HIV-1 molecular clone with luciferase reporter gene in 96-well plate format. The results showed that six siRNAs significantly enhanced the HIV-1 replication in J111 cells, indicating that the target cellular genes of those siRNAs may negatively regulate HIV-1 replication in normal cell culture condition. We also discuss the possible mechanisms by which those cellular proteins regulate viral replication.

Prevalence of cytomegalovirus, human herpesvirus-6, and Epstein-Barr virus in periodontitis patients and healthy subjects in the Thai population.

Fifty periodontitis patients and 30 healthy patients with oral cavities were selected from the Faculty of Dentistry, Mahidol University, Bangkok, Thailand, from March 2001 to November 2002. Their ages varied between 15 and 70 years. Among the periodontitis patients, specimens were collected from both disease and healthy sites. All samples were evaluated for the presence of CMV, HHV-6, and EBV-1 by nested PCR. Among the periodontitis patients, CMV was found in 34%, of which 8% were at the disease sites, 10% were at the healthy sites, and 16% were from both sites. EBV was not found in this group of the patients, while HHV-6 was found in 4%, at the disease sites only. CMV was found in one (3.3%) healthy control while HHV-6 and EBV-1 were not found. The depth of sample sites, various demographic and baseline characteristics eg sex, age, occupation and root planning were not associated with the presence of these viruses.

Preliminary clinical evaluation of a protein chip for tumor marker serodiagnosis of various cancers.

This preliminary study aimed to investigate sensitivity and specificity of a protein chip system for multi-tumor marker serodiagnosis of ten types of cancers, and to understand the possible clinical applications of this protein chip for the Thai population. The specific cancers diagnosed by this protein chip are lung, breast, liver, cervix, colo-rectal, stomach, ovary, esophagus, prostate and pancreas cancers. We analyzed 215 serum samples of which 165 were obtained from clinically confirmed cancer patients and 50 from healthy people with no evidence of cancer. The sensitivity and specificity of the protein chip were 82.4% and 94.0%, respectively. The success rate of the protein chip for detecting all 10 types of cancers varied from 57% to 100%. The value of the simultaneous measurement of multiple tumor markers using the protein chip for cancer screening lied in the higher sensitivity compared to using single tumor markers for each type of cancer. In short, protein chips may be useful in mass screening for cancer during health checkups as well as for metastasis follow-up of cancer patients.

Human herpesvirus 6 variant A but not variant B induces fusion from without in a variety of human cells through a human herpesvirus 6 entry receptor, CD46.

Human herpesvirus 6 (HHV-6) is a lymphotropic betaherpesvirus that productively infects T cells and monocytes. HHV-6 isolates can be differentiated into two groups, variants A and B (HHV-6A and HHV-6B). Here, we show a functional difference between HHV-6A and -6B in that HHV-6A induced syncytium formation of diverse human cells but HHV-6B did not. The syncytium formation induced by HHV-6A was observed 2 h after infection; moreover, it was found in the presence of cycloheximide, indicating that HHV-6A induced fusion from without (FFWO) in the target cells. Furthermore, the fusion event was dependent on the expression of the HHV-6 entry receptor, CD46, on the target cell membrane. In addition, we determined that short consensus repeat 2 (SCR2), -3, and -4 of the CD46 ectodomain were essential for the formation of the virus-induced syncytia. Monoclonal antibodies against glycoproteins B and H of HHV-6A inhibited the fusion event, indicating that the syncytium formation induced by HHV-6A required glycoproteins H and B. These findings suggest that FFWO, which HHV-6A induced in a variety of cell lines, may play an important role in the pathogenesis of HHV-6A, not only in lymphocytes but also in various tissues, because CD46 is expressed ubiquitously in human tissues.

Human herpesvirus-6 rep/U94 gene product has single-stranded DNA-binding activity.

The characterization is reported of the human herpesvirus-6B (HHV-6B) rep/U94 gene, which is a homologue of the adeno-associated virus type 2 rep. In this study, a monoclonal antibody was produced against HHV-6B REP (anti-REP mAb). Immunofluorescence staining using the anti-REP mAb showed that REP was localized to the nucleus in HHV-6-infected MT4 cells. It was first detected at 24 h post-infection (p.i.) and accumulated to higher levels by 72 h p.i. REP may be expressed only at very low levels in HHV-6-infected cells: even when the late protein glycoprotein H was detected in nearly 90% of HHV-6-infected cells, REP was detected in only a small percentage of them. Western blot analysis showed that the anti-REP mAb recognized a 56-kDa polypeptide in HHV-6B-infected MT4 cells. Furthermore, the REP protein was shown to bind single-stranded DNA.