Cell-penetrating porcine single-chain antibodies (transbodies) against nonstructural protein 1ß (NSP1ß) of porcine reproductive and respiratory syndrome virus inhibit virus replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS) worldwide, especially in domestic pigs, with an enormous economic impact, estimated at $664 million in losses every year to the pig industry. Current vaccines confer limited protection, and no direct-acting anti-PRRS treatment is available. Non-structural protein (NSP) 1ß, a cysteine-like protease (CLPro) of PRRSV plays an essential role in viral polyprotein processing, subgenomic RNA synthesis, and evasion of host innate immunity. Therefore, agents that interfere with the bioactivity of NSP1ß would be expected to inhibit virus replication. In this study, a porcine single-chain antibody (scFv)-phage display library was constructed and used as a tool for production of NSP1ß-specific porcine scFvs (pscFvs). The pscFvs to NSP1ß were linked to a cell-penetrating peptide to form cell-penetrating pscFvs (transbodies), which could be internalized and inhibit PRRSV replication in infected cells. A computer simulation indicated that the effective pscFvs used several residues in multiple complementarity determining regions (CDRs) to interact with multiple residues in the CLPro and C-terminal motifs, which might explain the mechanism of pscFv-mediated inhibition of virus replication. Although experiments are needed to determine the antiviral mechanism of the transbodies, the current data indicate that transbodies can potentially be applied for treatment and prevention of PRRSV infection.

Alteration of -656(G/T) and -607(C/A) polymorphisms in interleukin-18 (IL-18) gene in house dust mite-sensitive allergic rhinitis patients in Thailand.

Allergic rhinitis (AR) is an IgE-mediated inflammation of the nasal membranes, which is naturally triggered by aeroallergens. House dust mites (HDM) are the most common inhalant allergens. Interleukin-18 (IL-18) has been established as an essential cytokine that can activate the generation of IgE. This randomized controlled study aimed to identify the possible relationship of the genetic variations in the IL-18 gene with AR in mite-sensitive Thai patients. Study subjects consisted of 150 AR patients and 50 normal participants. Genomic DNA of 30 randomized AR patients and 30 randomized controls were screened by sequencing for the selection of candidate single nucleotide polymorphisms (SNPs), and further analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for all subjects. The following five SNPs were detected in the IL-18 gene: -656 G/T, -607 C/A, and -137 G/C in promoter 1 and -920 C/T and -373 C/G in promoter 2. The results showed that -656 G/T and -607 C/A SNPs were significantly correlated with IgE levels specific to Dermatophagoides pteronyssinus (Der p) allergen (P = 0.045 and P = 0.045, respectively), and significant differences were observed in the genotype distribution of AR patients when compared with controls [P = 0.044 and P = 0.044, respectively; odds ratios (ORs): 1.941 (95%CI, 1.014-3.715) and 1.941 (95%CI, 1.014-3.715), respectively]. Our findings indicate that the IL-18 alleles, -656T (rs1946519) and -607A (rs1946518), might be associated with the higher production of Der p allergen-specific IgE in mite-sensitive AR patients.

Human monoclonal single-chain antibodies specific to dengue virus envelope protein.

UNLABELLED: Dengue virus (DENV) infection is an arthropod-borne disease with increasing prevalence worldwide. Attempts have been made to develop therapeutic molecules for treatment for DENV infection. However, most of potentially therapeutic DENV monoclonal antibody was originated from mouse, which could cause undesirable effects in human recipients. Thus, fully human antibody is preferable for therapeutic development. Human single-chain variable fragments (HuScFv) with inhibitory effect to DENV infection were generated in this study. HuScFv molecules were screened and selected from the human antibody phage display library by using purified recombinant DENV full-length envelope (FL-E) and its domain III (EDIII) proteins as target antigens for biopanning. HuScFv molecules were then tested for their bindings to DENV particles by indirect ELISA and immunofluorescent microscopy. EDIII-specific HuScFv exhibited neutralizing effect to DENV infection in Vero cells in a dose-dependent manner as determined by plaque formation and cell ELISA. Epitope mapping and molecular docking results concordantly revealed interaction of HuScFv to functional loop structure in EDIII of the DENV E protein. The neutralizing HuScFv molecule warrants further development as a therapeutic biomolecule for DENV infection. SIGNIFICANCE AND IMPACT OF THE STUDY: No approved vaccine and specific drug for dengue virus (DENV) infection are available; thus, their developments are urgently required. The human single-chain variable antibody fragments (HuScFv) specific to DENV envelope (E) protein are potential to be developed as therapeutic biomolecules. HuScFv that bound specifically to recombinant full-length DENV E (FL-E) and its domain III (EDIII) were generated and testified for its inhibitory effect in DENV infection. EDIII-specific HuScFv inhibited DENV infection in a dose-dependent manner and has potential to be further developed as a therapeutic biomolecule for DENV infection.

Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand.

BACKGROUND & OBJECTIVES: El Tor Vibrio cholerae O1 carrying ctxB C trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand. METHODS: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpA C , tcpA E, hlyA C and hlyA E were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles. RESULTS: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid. INTERPRETATION & CONCLUSIONS: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxB C).

Up-regulation of Id1 in peripheral blood of psoriatic patients.

Although the precise causes of psoriasis are unclear, it is widely accepted that psoriasis is a disorder in which factors in the immune system, enzymes, and other biochemical substances that regulate skin cell division are impaired, leading to rapid proliferation of keratinocytes and incomplete keratinization. Expression of the helix-loop-helix transcription factor Id1 (inhibitor of differentiation/DNA binding), functioning as an inhibitor of differentiation, is known to increase in psoriatic skin. However, the molecular involvement of this particular biomarker in the psoriatic immune system remains to be elucidated. We measured Id1 mRNA expression in peripheral blood mononuclear cells (PBMCs) of psoriatic patients and healthy controls using semi-quantitative reverse transcriptase-PCR. The normalized level of Id1 transcripts in psoriatic patients was about 2-fold higher than that in controls (P < 0.05). When we examined the proliferation rate of PBMCs, the stimulation index obtained from the phytohemagglutinin stimulation assay was not significantly different in psoriatic patients. In patients with psoriasis, there was no correlation between the stimulation index and the psoriasis area severity index. We suggest that Id1 has a role in causing psoriatic immune cell symptoms.

Studies on immunodiagnosis of human paragonimiasis and specific antigen of Paragonimus heterotremus.

Adult Paragonimus heterotremus were recovered from the lungs and pleural cavity of cats orally infected with metacercariae. The worms were ground and extracted with distilled water. The soluble crude antigen (CA) contained about 40% proteins which could be fractionated by gel filtration on Sephadex G-200 into three profiles namely the F1, F2 and F3. The CA and its Sephadex profiles were used in an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to P. heterotremus in three groups of patients, i.e. patients whose sputum and/or faeces revealed P. heterotremus eggs (group 1), patients with other parasitic infections (group 2), bacterial proven tuberculosis patients (group 3) and healthy, parasite-free controls (group 4). The sensitivity and specificity of the assay when the F1 was used as the antigen were 100%. Western blot analysis revealed that specific antigen of P. heterotremus was a non-protein component of Mr35 kDa.

Specific antigen of Gnathostoma spinigerum for immunodiagnosis of human gnathostomiasis.

Sera from four patients with parasitologically confirmed gnathostomiasis, 15 patients with presumptive gnathostomiasis, 64 patients with various parasitic infections and 19 healthy adults were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis for their reactivities against somatic extract of Gnathostoma spinigerum third-stage larvae (L3). It was found that the L3 extract was highly complex consisting of more than 20 antigenic components, a few of which gave reactions with sera from the healthy controls. Extensive cross-reactions of the parasite's antigen with sera from patients with other parasitic infections occurred. A specific antigen of G. spinigerum with a mol. wt of 24,000 (24k) was found to react with all parasitologically proven patients, five of the presumptive patients, one of the patients with other parasitic infections and none of the healthy individuals. This 24k component of G. spinigerum is a potential diagnostic antigen for use in the immunodiagnosis of human gnathostomiasis.

Purification of Gnathostoma spinigerum specific antigen and immunodiagnosis of human gnathostomiasis.

Specific antigen of G. spinigerum which has been shown to be a protein with a relative mol. wt of 24,000 (24K) was prepared from the advanced third-stage larvae (L3) obtained from the livers of naturally infected eels. The L3 were ground and extracted with water. Purification procedures involved gel filtration, chromatofocussing and anion exchange column chromatographies, while characterization of the specific antigen was performed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining, Western blot analysis and isoelectric focussing. The specific antigen which has a pI of 8.5 was used as antigen in the indirect enzyme-linked immunosorbent assay (ELISA) to detect specific antibody in four groups of individuals, namely five parasitologically diagnosed gnathostomiasis patients (group 1); 15 clinically diagnosed gnathostomiasis patients (group 2); 136 patients with other parasitic infections (group 3); and 25 normal healthy parasite-free controls. Sensitivity, specificity and predictive values (positive and negative) of the assay were 100%.

Immunodiagnosis of human trichinellosis and identification of specific antigen for Trichinella spiralis.

Crude antigens obtained from the infective stage larvae of Trichinella spiralis were used in an ELISA for detecting IgG antibodies to T. spiralis in serum samples collected from three groups of individuals. The individuals of the first group were parasitologically confirmed trichinellosis patients, while those of group 2 were patients with other helminthiasis and group 3 were healthy, parasite-free individuals. The specificity of the assay was 96.8% when performed on sera of groups 2 and 3. Cross-reaction was observed with the sera of patients with capillariasis, gnathostomiasis, opisthorchiasis, and strongyloidiasis and opisthorchiasis with hookworm infection. The sensitivity of the test was 100% when performed on sera of group 1, which were collected 57 days after infection. Western blot analysis revealed that a specific antigen for T. spiralis was a component of M(r) 109.

Towards a suitable antigen for diagnosis of Gnathostoma spinigerum infection.

Advanced third-stage larvae of G. spinigerum were obtained from two separate sources, namely from cysts in the livers of naturally infected eels (L3E) and from experimentally infected mice (L3M). Morphology of the L3E was studied microscopically. The larvae were homogenized in distilled water, 1% Triton X-100 or 1% sodium deoxycholate containing protease inhibitors. Protein compositions of the three crude extracts were compared, on the same weight basis, by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining while their antigenicities were studied by Western blot analysis using serum of a patient with parasitologically confirmed gnathostomiasis. Distilled water was found to be the best extraction solution in solubilizing proteins especially the diagnostic antigen, namely the 24,000 (24 kDa) mol. wt component from the larvae. The L3E and L3M contained relatively equal amounts of the 24 kDa antigen. This diagnostic component was anatomically located in the body fluid, oesophagus and intestine of the larva.

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody.

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody. International Journal for Parasitology 22: 527-531. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting Opisthorchis viverrini antigen in faecal extracts of four groups of individuals. These were 24 patients with O. viverrini infection only (group 1), 31 patients with O. viverrini and other parasitic infections (group 2), 141 patients with other parasitic infections (group 3) and 21 normal, parasite-free individuals (group 4). The first antibody used in the ELISA was polyclonal immunoglobulin G prepared from the serum of a rabbit previously immunized with crude extract of O. viverrini. The second antibody was monoclonal antibody specific to an antigen located in the worm tegument and muscular tissue. Sensitivity of the assay was 31% while specificity was 100%. Considerations for improving the sensitivity are discussed.

Serum antibody responses in opisthorchiasis.

We evaluated an enzyme-linked immunosorbent assay using crude parasite homogenates as a diagnostic test for Opisthorchis viverrini infection in humans. Serum antibody (Ab) responses to O. viverrini adult worm homogenate (AWH) and metacercaria homogenate (MH) were studied in 83 infected residents of an opisthorchiasis-endemic area in Thailand. Elevated levels of Ab persisted for over 1 year following curative treatment with praziquantel, and cross-reactivity to O. viverrini AWH and MH antigens was observed in sera from individuals with other parasitic infections. Serum Ab to crude AWH and MH are therefore unsuitable for immunodiagnosis since they may be non-specific and would not differentiate between ongoing and past infection.

Monoclonal antibody to a diagnostic Mr 24,000 antigen of Gnathostoma spinigerum.

Crude water extract (CA) was prepared from the advanced third-stage larvae of Gnathostoma spinigerum collected from livers of naturally infected eels. The extract was partially purified by chromatofocussing column chromatography and the fraction which contained specific antigen of G. spinigerum which was an Mr 24,000 glycoprotein was used to immunize five Balb/c mice for preparing immune splenocytes. Spleen cells were collected from one mouse which showed high serum titre by indirect enzyme-linked immunosorbent assay and contained specific antibody to the Mr 24,000 antigen as checked by Western blot analysis. The spleen cells were fused with myeloma Sp2/0 cells at a ratio of 10 spleen cells per one myeloma cell using polyethylene glycol 3350 as a fusogen. Thirteen out of 174 growing polyclones (7.5%) produced antibodies to the partially purified CA fraction. Among them, two polyclones produced antibody directed to the Mr 24,000 protein. These two polyclones were subjected to monocloning by limiting dilution and a monoclone GN6/24 which produced monoclonal antibody to the specific Mr 24,000 protein of G. spinigerum was obtained.

Specific monoclonal antibodies to Opisthorchis viverrini.

A Balb/c mouse was immunized with a crude soluble antigen of Opisthorchis viverrini adult worms (OVAA) over a period of 7 months. Spleen cells from the immune mouse were fused with Sp2/0 myeloma cells. Among the 264 tissue culture wells containing the fused cells, cells of 96 wells (36%) produced antibodies to the immunizing agent. Antibodies produced by cells in several wells reacted with antigens from other species of parasite. Cells of 17 wells produced antibodies specific only to OVAA, thus cells from three representative wells were cloned by limiting dilution. Hybrids obtained produced antibodies which could be classified according to their tissue specificities into three groups. The first group of antibodies reacted strongly to the worm integument and weakly with the muscles while those belonging to the second group reacted only to muscles of the worms. The monoclonal antibodies of the third group gave a positive reaction to both muscles and tegument.

Monoclonal antibodies to Paragonimus heterotremus and their potential for diagnosis of paragonimiasis.

Monoclonal antibodies (MAbs) specific to the lung fluke (Paragonimus heterotremus) were produced against the soluble metabolic products (excretory-secretory antigen). Three hybrids secreting MAbs specific for P. heterotremus antigens were identified by an indirect enzyme-linked immunosorbent assay (ELISA) against a panel of homologous and 24 heterologous parasite antigens and Mycobacterium tuberculosis. Of the three specific clones, clone 10F2, which was IgG1 producing and which gave immune complex bands with 31.5-kD and 22-kD polypeptides by gel electrophoresis and immunoblotting, was selected for further characterization and evaluation of its possible diagnostic potential. The result obtained from an indirect immunofluorescent antibody test suggested that MAb 10F2 reacted with mucosa and contents of the worm's intestine. The antibody could be readily used to prepare an affinity-purified antigen for use in an indirect ELISA that was highly sensitive and specific for the detection of circulating antibody in sera of paragonimiasis patients.

Serum antibody response to Opisthorchis viverrini antigen as a marker for opisthorchiasis-associated cholangiocarcinoma.

The liver flukes Opisthorchis viverrini and Clonorchis sinensis chronically infect over 30 million people in south-eastern Asia, resulting in significant morbidity and a predisposition to cholangiocarcinoma (CCA). Liver fluke-associated CCA carries a poor prognosis, partly because it is often detected at a late and advanced stage. The development of improved diagnostic methods, particularly for early CCA, may improve chances of survival and cure. Accordingly, we explored the use of immunological responses to liver fluke antigens as a potential means of identifying individuals at high risk for liver fluke-associated CCA. Serum antibody responses to O. viverrini adult worm homogenate and metacercaria homogenate (MH) were studied using enzyme-linked immunosorbent and immunoblot assays in 65 infected residents of an opisthorchiasis-endemic area in Thailand. Antibody levels correlated with liver ultrasonography (U/S) findings, and immunoblot analysis revealed a 91/93 kDa MH doublet recognized only by sera of individuals with severe liver U/S findings, including CCA. These results suggest that serum antibody responses to liver fluke antigens may be useful in the identification of infected individuals who are at high risk for liver fluke-associated CCA.

Genotypes and phenotypes of Shiga toxin producing-Escherichia coli isolated from healthy cattle in Thailand.

Shiga toxin producing-Escherichia coli (STEC) has not yet been identified as an important aetiologic agent of human disease in Thailand. To evaluate the potential for STEC to contribute to human disease in Thailand, 139 fecal samples were collected from healthy cattle from five different provinces and analysed by genotypic and phenotypic methods for STEC. Of 139 samples, 27 (19.4%) were positive for stx1 and/or stx2 by multiplex polymerase chain reaction, or for O157 lipopolysaccharide (LPS) by immunoassay. Isolates positive for stx and/or O157 were subdivided into 49 strains that varied in the presence of the virulence determinants stx1+/stx2+ (22 strains), stx2+ (22 strains), stx1+ (4 strains), and O157 LPS (1 strain). Within these 49 distinguishable strains, other virulence determinants varied as follows: hlyA+ (77.6%), eae+ and tir+ (4.1%), and katP+ (6.12%). The most predominant profile (22 isolates) was stx1+/stx2+, eae-, tir-, etpD-, hlyA+, katP-. For further characterization of the isolated strains by two molecular typing assays, plasmid profiles and ERIC PCR were performed. The results suggest that the genetic and phenotypic profiles of STEC associated with human disease are not prevalent at this time in cattle in Thailand.

Differential diagnosis of schistosomiasis mekongi and trichinellosis in human.

An indirect (plate) ELISA and, a more convenient version, a dot-blot (membrane) ELISA have been developed using haemocyanin of a mollusk, Megathura crenulata, i.e. keyhole limpet haemocyanin (KLH) and purified, specific antigen of Trichinella spiralis (APTsAg) obtained from a monoclonal antibody-affinity column chromatography, for differential diagnosis of schistosomiasis mekongi and trichinellosis. Serum samples of patients with parasitologically confirmed trichinellosis were reactive to both antigens in both versions of ELISA while sera of patients with schistosomiasis mekongi were positive only to the KLH. Both ELISA were negative when used to test sera of normal controls and patients with gnathostomiasis, paragonimiasis and opisthorchiasis.

Separation and characterization of adult worm proteins and glycoproteins from the liver fluke Opisthorchis viverrini.

Detailed studies of liver fluke proteins and antigens are necessary to facilitate further investigation of the human immune responses to these parasites. Accordingly, Opisthorchis viverrini antigens were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. We initially encountered excessive background smearing, vertical streaking, and indistinct bands that were similar to problems previously described by investigators studying this and other trematodes including Schistosoma mansoni. These problems were especially evident with silver staining of proteins and occurred despite the extensive use of protease inhibitors. They were minimized by using mini (vs. large) SDS-PAGE and Coomassie blue protein staining. With the latter 2 techniques, adult worm somatic proteins and excretory-secretory products were separated and characterized. Immunoblots using rabbit anti-adult worm sera demonstrated that some of these proteins were antigens common to both the adult and metacercarial stages. Several of these antigens also corresponded (according to molecular weight) to glycoproteins, detected by concanavalin A blotting. These findings form a base for subsequent studies of the human immune response to liver fluke infection.

Detection of Paragonimus heterotremus in experimentally infected cat feces by antigen capture-ELISA and by DNA hybridization.

An antigen capture enzyme-linked immunosorbent assay (antigen capture-ELISA) and DNA hybridization technique were developed and evaluated for their application in the detection of Paragonimus heterotremus infection in experimentally infected cats. An IgG fraction prepared from serum of a rabbit immunized with P. heterotremus excretory-secretory (ES) products was used as the capture antibody. An IgG1 monoclonal antibody specific to the 22- and 31.5-kDa ES products of P. heterotremus was used as the antigen probe. As little as 0.24 ng of the ES products could be detected by this technique. A specific P. heterotremus DNA probe derived from the P. heterotremus genomic DNA library containing 1,500 base pairs was used in a dot-blot hybridization assay for the detection of parasite DNA. The radioactively labeled probe could detect DNA released from as few as 2 P. heterotremus eggs. Both ELISA and DNA hybridization were found to have 100% specificity, with sensitivities of 73.7% and 100%, respectively.