Molecular typing of Vibrio cholerae O1 isolates from Thailand by pulsed-field gel electrophoresis.

The aim of the present study was to genotypically characterize Vibrio cholerae strains isolated from cholera patients in various provinces of Thailand. Two hundred and forty V. cholerae O1 strains, isolated from patients with cholera during two outbreaks, i.e. March 1999-April 2000 and December 2001-February 2002, in Thailand, were genotypically characterized by NotI digestion and pulsed-field gel electrophoresis (PFGE). In total, 17 PFGE banding patterns were found and grouped into four Dice-coefficient clusters (PF-I to PF-IV). The patterns of V. cholerae O1, El Tor reference strains from Australia, Peru, Romania, and the United States were different from the patterns of reference isolates from Asian countries, such as Bangladesh, India, and Thailand, indicating a close genetic relationship or clonal origin of the isolates in the same geographical region. The Asian reference strains, regardless of their biotypes and serogroups (classical O1, El Tor O1, O139, or O151), showed a genetic resemblance, but had different patterns from the strains collected during the two outbreaks in Thailand. Of 200 Ogawa strains collected during the first outbreak in Thailand, two patterns (clones)--PF-I and PF-II--predominated, while other isolates caused sporadic cases and were grouped together as pattern PF-III. PF-II also predominated during the second outbreak, but none of the 40 isolates (39 Inaba and 1 Ogawa) of the second outbreak had the pattern PF-I; a minority showed a new pattern--PF-IV, and others caused single cases, but were not groupable. In summary, this study documented the sustained appearance of the pathogenic V. cholerae O1 clone PF-II, the disappearance of clones PF-I and PF-III, and the emergence of new pathogenic clones during the two outbreaks of cholera. Data of the study on molecular characteristics of indigenous V. cholerae clinical isolates have public-health implications, not only for epidemic tracing of existing strains but also for the recognition of strains with new genotypes that may emerge in the future.

Murine monoclonal antibodies neutralizing the cytotoxic activity of diphtheria toxin.

In this study, murine monoclonal antibodies that specifically bound to the A and B subunits of diphtheria toxin (DT) were produced by conventional hybridoma technology using the spleens of BALB/c mice immunized with diphtheria DTP vaccine and CRM197. Monoclonal antibodies specific to the A subunit, i.e. clone AC5, as well as those specific to the B subunit, i.e. clone BB7, could neutralize the DT-mediated cytotoxicity to Vero cells in microcultures. The DT neutralizing mechanisms have yet to be determined. The MAbBB7 is hypothesized to either interfere with the DT receptor binding or with the pore forming function of the T domain of the B subunit. The MAbAC5 could neutralize the DT mediated cytotoxicity when mixed with the DT before adding to the Vero cell culture thus suggesting that the antibody interfered with the translocation of the A subunit. The A subunit-antibody complex might be too large to pass through the membrane channel formed by the T domain and thus prevent the accessibility of the A subunit to the cytosolic target. It is also possible that the MAb AC5 blocked the enzymatic active site of the enzyme catalytic subunit. While further experiments are needed to localize the epitopes of the two MAbs on the holo-DT in order to reveal the DT neutralizing mechanisms, both MAbs in their humanized forms have a high potential as human therapeutic antibodies for diphtheria.

Human monoclonal single chain antibodies (HuScFv) that bind to the polymerase proteins of influenza A virus.

Current anti-influenza drugs target the viral neuraminidase or inhibit the function of the ion channel M2 protein. Not only is the supply of these drugs unlikely to meet the demand during a large influenza epidemic/ pandemic, but also has an emergence of drug resistant influenza virus variants been documented. Thus a new effective drug or antiviral alternative is required. The influenza virus RNA polymerase complex consists of nucleoproteins (NP) that bind to three polymerase subunits: two basic polymerases, PB1 and PB2, and an acidic polymerase (PA). These proteins play a pivotal role in the virus life cycle; thus they are potential targets for the development of new anti-influenza agents. In this study, we produced human monoclonal antibodies that bound to the influenza A polymerase proteins by using a human antibody phage display library. Complementary DNA was prepared from the total RNA of a highly pathogenic avian influenza (HPAI) virus: A/duck/Thailand/144/2005(H5N1). The cDNA synthesized from the total virus RNA was used as template for the amplification of the gene segments encoding the N-terminal halves of the PB1, PB2 and PA polymerase proteins which encompassed the biologically active portions of the respective proteins. The cDNA amplicons were individually cloned into appropriate vectors and the recombinant vectors were introduced into Escherichia coli bacteria. Transformed E. coli clones were selected, and induced to express the recombinant proteins. Individually purified proteins were used as antigens in bio-panning to select the phage clones displaying specific human monoclonal single chain variable fragments (HuScFv) from a human antibody phage display library constructed from Thai blood donors in our laboratory. The purified HuScFv that bound specifically to the recombinant polymerase proteins were prepared. The inhibitory effects on the biological functions of the respective polymerase proteins should be tested. We envisage the use of the HuScFv in their cell penetrating version (transbodies) as an alternative influenza therapeutic to current anti-virus drugs.

Native troponin-T of the American cockroach (CR), Periplaneta americana, binds to IgE in sera of CR allergic Thais.

The American cockroach, Periplaneta americana, is the predominant cockroach (CR) species in Thailand and a major source of indoor allergens second only to the house dust mite. The incidence of CR allergy among allergic Thai patients is increasing but basic information on the allergenic components is scarce. In this study a recombinant troponin-T was produced by using cDNA prepared from RNA of the P. americana as a template and PCR primers designed from the P. americana troponin-T sequence deposited in the GenBank database. The recombinant protein (Mr approximately 50) did not bind to IgE in the sera of 18 skin prick test positive CR allergic patients. Rabbit polyclonal antiserum (PAb) against the recombinant troponin-T was produced and used in preparing an affinity column for the purification of native troponin-T from the crude P. americana extract (Mr approximately 47). IgE-immunoblotting revealed that the native protein bound to IgE in 3 of the 18 (16.7%) patients. Our results imply that native P. americana troponin-T, but not its recombinant counterpart, is a minor allergen among the CR allergic Thais.

Proteome and immunome of pathogenic Leptospira spp. revealed by 2DE and 2DE-immunoblotting with immune serum.

In this study, proteomes of two pathogenic Leptospira spp., namely L. interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni and L. borgpetersenii, serogroup Tarassovi, serovar Tarassovi, were revealed by using two dimensional gel electrophoresis (2DE)-based-proteomics. Bacterial cells were disrupted in a lysis buffer containing 30 mM Tris, 2 M thiourea, 7 M urea, 4% CHAPS, 2% IPG buffer pH 3-10 and protease inhibitors and then subjected to sonication in order to solubilize as much as possible the bacterial proteins. The 2DE-separated components of both Leptospira homogenates were blotted individually onto membranes and antigenic components (immunomes) were revealed by probing the blots with immune serum of a mouse readily immunized with the homogenate of L. interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni. The immunogenic proteins of the two pathogenic Leptospira spp. could be grouped into 10 groups. These are: 1) proteins involved in the bacterial transcription and translation including beta subunit transcription anti-termination protein of DNA polymerase III, elongation factors Tu and Ts, and tRNA (guanine-N1)-methyltransferase; 2) proteins functioning as enzymes for metabolisms and nutrient acquisition including acetyl-Co-A acetyltransferase, putative glutamine synthetase, glyceraldehyde-3-phospahte dehydrogenase, NifU-like protein, 3-oxoacyl-(acyl-carrier-protein) reductase, oxidoreductase, sphingomyelinase C precursor, spermidine synthase, beta subunit of succinyl-CoA synthetase, and succinate dehydrogenase iron-sulfur subunit; 3) proteins/enzymes necessary for energy and electron transfer, i.e. electron transfer flavoprotein, and proton-translocating transhydrogenase; 4) enzymes for degradation of misfolded proteins, i.e. ATP-dependent Clp protease; 5) molecular chaperone, i.e. 60 kDa chaperonin; 6) signal transduction system, i.e. response regulator; 7) protein involved in immune evasion in host, i.e. peroxiredoxin; 8) cell structure proteins including MreB (cytoskeletal) and flagellin/ periplasmic flagellin; 9) lipoproteins/outer membrane proteins: LipL32, LipL41, LipL45 and OmpL1; and 10) various hypothetical proteins. Many immunogenic proteins are common to both Leptospira spp. These proteins not only are the diagnostic targets but also have potential as candidates of a broad spectrum leptospirosis vaccine especially the surface exposed components which should be vulnerable to the host immune effector factors.

Monoclonal antibody that neutralizes pertussis toxin activities.

Pertussis or whooping cough is a disease with high mortality among infants and small children. The disease is caused by infection of the respiratory tract by a gram negative bacterium, Bordetella pertussis. The superficial colonized bacteria produce a myriad of toxins which enter the circulation causing various pathophysiologicalal changes in the host. Although antimicrobial therapy reduces the number of the coughed out bacteria and also the infectious time of the infected host, but it is not effective in amelioration of the clinical manifestations as the pertussis morbidity is due principally to the pertussis toxin (PT). Antibody based-therapy is frequently practiced in conjunction with other supportive measure to resuscitate the patient. Nevertheless, human derived antiserum against PT is of the limited supply and the ethical concern. Thus in this study a hybridoma clone, i.e. clone PT6-2G6, secreting monoclonal antibody (MAb) specific to the S1 subunit, the active enzyme of the PT that intracellularly ADP-ribosylates the host Gi-protein, was produced. The MAbPT6-2G6 inhibited the in vitro hemagglutination of chicken erythrocytes which is the activity of the B oligomer of PT; thus we hypothesize that the MAb bound to its epitope on the S1 subunit and stereologically hinders the binding sites of the B subunits. The MAb also inhibited ex vivo Chinese hamster ovarian cell clustering and neutralized the in vivo leucocytosis- promotion in mice which are usually mediated by intracellular S1 subunit. The large molecular nature of the intact MAb and its molecular hydrophilicity led us to speculate that the observed PT neutralizing activities of the MAb were due to interfering with the cellular entry of the S1 rather than the intracellular enzyme neutralizing activity per se. While further experiments are needed to pinpoint the MAb neutralizing activity and to identify the amino acid sequence and location of the MAbPT6-2G6 epitope, our findings indicate that this murine MAb, in its humanized-version, should have high therapeutic potential for pertussis.

OmpL1 DNA vaccine cross-protects against heterologous Leptospira spp. challenge.

Available leptospirosis vaccines made up of inactivated bacteria or their membrane components elicit immunity which is serovar specific and unsatisfactory immunological memory. A vaccine that protects across Leptospira serogroups/serovars, i.e. broad spectrum, and induces long-lasting memory is needed for both human and veterinary uses. In this study, a plasmid DNA vaccine was constructed from cloning gene encoding a transmembrane porin protein, OmpL1, of pathogenic Leptospira interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni into a mammalian expression vector pcDNA3.1(+). The protective efficacy of the ompL1-pcDNA3.1(+) plasmid DNA vaccine was studied by immunizing hamsters intramuscularly with three doses of the vaccine (100 microg per dose) at two week intervals. The empty pcDNA3.1(+) and PBS were used as mock as negative vaccine controls, respectively. All animals were challenged with the heterologous Leptospira interrogans, serogroup Pomona, serovar Pomona (10 LD50), at one week after the last vaccine booster. The ompL1-pcDNA3.1(+) plasmid DNA vaccine rescued some vaccinated animals from the lethal challenge and delayed death time, reduced morbidity, e.g. fever, and/or the numbers of Leptospira in the tissues of the vaccinated animals. While the results are encouraging, further studies are needed to optimize the immunization schedule, vaccine dosage and formulation in order to maximize the efficacy of the vaccine.

Traffic of antibody-secreting cells after immunization with a liposome-associated, CpG-ODN-adjuvanted oral cholera vaccine.

An oral cholera vaccine made up of heat-treated recombinant cholera toxin (rCT), V. cholerae lipopolysaccharide (LPS), and recombinant toxin-co-regulated pili subunit A (rTcpA), entrapped in liposomes in the presence of unmethylated bacterial CpG-DNA (ODN#1826) was used to orally immunize a group of eight week old rats. A booster dose was given 14 days later. Control rats received placebo (vaccine diluent). The kinetics of the immune response were investigated by enumerating the antigen specific-antibody secreting cells (ASC) in the blood circulation and intestinal lamina propria using the ELISPOT assay and a histo-immunofluorescence assay (IFA), respectively. ASC of all antigenic specificities were detected in the blood of the vaccinated rats as early as two days after the booster dose. The numbers of LPS-ASC and TcpA-ASC in the blood were at their peak at day 3 post booster while the number of CT-ASC was highest at day 4 after the booster immunization. At day 13 post immunization, no ASC were detected in the blood. A several fold increase in the number of ASC of all antigenic specificities in the lamina propria above the background numbers of the control animals were found in all vaccinated rats at days 6 and 13 post booster (earlier and later time points were not studied). Vibriocidal antibody and specific antibodies to CT, LPS and TcpA were detected in 57.1% and 52.4%, 14.3%, and 19.0% of the orally vaccinated rats, respectively. The data indicated that rats orally primed with the vaccine could produce a rapid anamnestic response after re-exposure to the V. cholerae antigens. Thus, a single dose of the vaccine is expected to elicit a similar anamnestic immune response in people from cholera endemic areas who have been naturally primed to V. cholerae antigens, while two doses at a 14 day interval should be adequate for a traveler to a disease endemicarea.

Prognostic improvement of patients with advanced liver cancer after active hexose correlated compound (AHCC) treatment.

Most patients with liver cancer are diagnosed when they are not suitable for resection. Although some palliative approaches can be applied to these patients, the overall survival rate remains unsatisfactory. Active hexose correlated compound (AHCC), a newly developed functional food, has been shown to act as a potent biological response modifier in in vitro experiments. Recently, AHCC was found to improve the prognosis of hepatocellular carcinoma patients following surgical treatment. We investigated whether AHCC could prolong survival and improve the prognosis of patients with advanced liver cancer. A prospective cohort study was performed with 44 patients with histologically confirmed liver cancer. All of the patients underwent supportive care. Survival time, quality of life, clinical and immunological parameters related to liver function, cellular immunity, and patient status were determined. Of the 44 patients, 34 and 10 received AHCC and placebo (control) orally, respectively. Patients in the AHCC treated-group had a significantly prolonged survival when compared to the control group by Mann-Whitney test (95% CI, p = 0.000). Quality of life in terms of mental stability, general physical health status, and ability to have normal activities were significantly improved after 3 months of AHCC treatment when tested using the Wilcoxon signed-rank test (on one-sided test, p = 0.028, 0.037, and 0.040, respectively). The apparent different clinical parameters between the two groups were the levels of albumin and percentage of lymphocytes with p-values of 0.000 and 0.026 at 1 and 2 months after treatment, respectively. Unlike the control patients, AHCC treated-patients with longer survival time had the tendency of better outcomes since the levels of AST and ALT had not increased rapidly from their baselines at follow-up. In addition, the levels of total IL-12 and neopterin were slightly increased in AHCC treated-patients. This study suggests that AHCC intake could prolong the survival and improve the prognosis of patients with advanced liver cancer and delay the gradual decline of their physiological status.

Preferences for treatment of diarrhoea and dysentery in Kaengkhoi district, Saraburi province, Thailand.

To estimate the proportion of cases missed in a passive surveillance study of diarrhoea and dysentery at health centres and hospitals in Kaengkhoi district, Saraburi province, Thailand, a community-based cluster survey of treatment-seeking behaviours was conducted during 21-23 June 2002. Interviews were conducted at 224 households among a study population of 78,744. The respondents reported where they sought care for diarrhoea and dysentery in children aged less than five years and adults aged over 15 years. Health centres or hospitals were the first treatment choice for 78% of children with dysentery (95% confidence interval [CI] 63-94%), 64% of children with diarrhoea (95% CI 54-74%), 61% of adults with dysentery (95% CI 40-82%), and 35% of adults with diarrhoea (95% CI 17-54%). A high degree of heterogeneity in responses resulted in a relatively large design effect (D=3.9) and poor intra-cluster correlation (rho=0.3). The community survey suggests that passive surveillance estimates of disease incidence will need to be interpreted with caution, since this method will miss nearly a quarter of dysentery cases in children and nearly two-thirds of diarrhoea cases in adults.

An antigen detection assay for diagnosing filariasis.

In this study we examined the diagnostic potential of monoclonal antibodies (MAb) reactive to antigens of adult Brugia malayi, their microfilariae and antigen of Dirofilaria immitis. The MAb of clone 17E10, which were of IgM isotype, reacted to the inner cuticles and internal content of both male and female worms and also to the sheath and internal content of microfilariae in utero. However, these MAb did not react to the sheath of blood circulating microfilariae. The MAb 17E10 produced a smear pattern between 37 to > 200 kDa in the Western blot analysis against a SDS-PAGE separated extract of B. malayi. The epitopes were non-protein in nature as indicated by their resistance to proteinase-K treatment. The MAb 17E10 were applied in a sandwich ELISA to detect filarial antigen in the buffy coat and plasma of patients. We tested patients with different clinical manifestations of brugian filariasis, i.e. microfilaremia (M), lymphangitis (L) and elephantiasis (E), as well as non-symptomatic inhabitants of a filariasis endemic area (NE), and compared them to samples from non-symptomatic inhabitants of disease non-endemic areas (NNE). It was found that 22 of 31 (70.9%) of M, 7 of 13 (53.8%) of L, 2 of 14 (14.2%) of E, 10 of 100 (10.0%) of NE and none (0%) of the NNE were positive for antigenaemia. The assay was also positive in 14 of 15 (93.3%) blood samples from B. malayi microfilaremic cats and in 7 of 7 (100%) blood samples of Dirofilaria immitis microfilaremic dogs. The so-developed test has a high potential for routine diagnosis of active filariasis, for epidemiological studies in both humans and reservoir animals and for monitoring treatment efficacy.

CpG DNA, liposome and refined antigen oral cholera vaccine.

An oral cholera vaccine made up of three Vibrio cholerae antigens, i.e. lipopolysaccharide (LPS), recombinant toxin co-regulated pili (rTcpA) and heat-treated cholera toxin (H-CT) has been developed in six different formulations. Eight-week-old Wistar rats were divided into nine groups and immunized as follows: the first group received the oral vaccine 1 consisting of the three antigens (LPS, rTcpA and H-CT) associated with a liposome (L) and bacterial CpG-DNA (ODN#1826). The rats of groups 2 and 3 received oral vaccines 2 and 3 consisting of the liposome-associated three antigens with and without non-bacterial CpG-DNA (ODN#1982), respectively. Rats of groups 4 received oral vaccine 4 consisting of the three antigens mixed with the ODN#1826, similar to vaccine 1, but without liposome. Rats of groups 5 and 6 received oral vaccines 5 and 6 consisting of the three antigens with and without ODN#1982, respectively, similar to vaccines 2 and 3, but without liposome. Rats of groups 7, 8 and 9 received oral placebos, namely liposomes (L), ODN#1826 (CpG), and vaccine diluent, i.e. 5% NaHCO3 solution, respectively. All vaccines were given in three doses at 14-day intervals. It was found that the combination of liposome and ODN#1826 in vaccine 1 evoked the highest immune response to V. cholerae antigen compared to other vaccine formulations and placebos, as measured by the appearance of antigen-specific antibody-producing cells in the intestinal lamina propria. The immunogenicity according to the magnitude of the immune response was: V1>V2=V3>V4>V5=V6>V7=V8=V9. The results of this study indicate that CpG-DNA and liposome are effective mucosal adjuvants for an oral cholera vaccine prepared from refined V. cholerae antigens and their combination seems to be synergistic. The potential role of liposome as a vaccine delivery vehicle has been confirmed.

Cockroach allergen detection and cockroach allergens of allergic Thai patients.

Hybridomas secreting monoclonal antibodies (MAb) specific to American cockroach (Periplaneta americana) were produced through a fusion of immune splenocytes of a BALB/c mouse immunized with crude cockroach (CR) extract and mouse myeloma cells. Two hybridomas namely 38G6 and 3C2 were established. These specific hybridomas secreted IgG1 monoclonal immunoglobulins with antigenic specificities to CR protein components of over 207 to 72 kDa and 45 to 40 kDa, respectively. The monoclonal antibodies were applied to select their specific epitopes out of the crude CR extract using affinity chromatography. A Prausnitz-Kustner test revealed that these epitopes were allergens which caused wheals and flares of the skin of a guinea-pig previously sensitized with a pool of serum samples from CR allergic patients. The monoclonal antibodies were also used in a capture ELISA to detect specific IgE in serum samples of allergic Thai patients. It was found that 72% and 76% of the patients had IgE antibodies to the epitopes of MAb 38G6 and MAb 3C2, respectively, indicating that the two epitopes are major CR allergens among the CR allergic Thai patients. An antibody-sandwich ELISA was developed for quantitative detection of CR allergens using the two monoclonal antibodies as a capture reagent and rabbit polyclonal antibodies to crude CR extract as a detection reagent. The assay could detect allergenic epitopes contained in as little as 122 pg of crude cockroach extract, and has high potential for direct measurement of the marker allergens in extracts of environmental samples.

Recombinant American cockroach component, Per a 1, reactive to IgE of allergic Thai patients.

Twelve similar recombinant Per a 1 clones were produced from an American cockroach (CR) cDNA library. The nucleotide sequence of a representative cline, i.e. clone A6, contained 579 base pairs (bp) and a 372 bp open reading frame (2-373) encoding 124 amino acids. A stop codon was found at position 374-376 followed by a 3' end untranslated region with an AATAAA polyadenylation signal and a poly (A) tail. The estimated molecular mass of the 24 amino acid residue protein was 13.8 kDa, with a predicted isoelectric point value of 4.74. Cysteine or N-linked glycosylation was not found. The deduced amino acid sequence of the A6 revealed 84.68-95.97% identity to other previously reported Per a 1 clones and 65.87-69.60% homology to the previously reported Bla g 1 clones. However, while previously reported Per a 1 clones showed homology to ANG12, a precursor protein in the midgut of the female Anopheles gambiae secreted after the blood meal, the A6 DNA sequence was found to have homology (37.1%) to DNA of G2, a putative protein in the midgut of Aedes aegypti (AY 050565). The deduced amino acid sequence of A6 contained a mitochondrial energy transfer protein signature, phosphorylation sites for the cAMP-and cGMP-dependent protein kinase C and casein kinase II. Hydrophobic and hydrophilic characteristics of the A6 deduced peptide indicated that it was a transmembrane protein. This is the first report that Per a 1 is a transmembrane protein. The deduced amino acid sequence of the A6, which contained the sequence LIRSLFGLP, differed in one amino acid from two previously reported epitopes, i.e. LIRALFGL and IRSWFGLP, of Per a 1.0104 which bound 80% and 100%, respectively, to IgE of the allergic patients tested. The A6 DNA sequence was deposited in the GenBank (Accession number AY 259514) and has been designated Per a 1.0105. The A6 expressed protein bound to monoclonal antibodies (MAb 3C2) specific to American cockroach and also bound to IgE of all (100%) of the 20 allergic Thai patients.

Specific monoclonal antibodies to Strongyloides stercoralis: a potential diagnostic reagent for strongyloidiasis.

In this study, specific hybridomas secreting monoclonal antibodies (MAb) to antigen of Strongyloides stercoralis filariform larvae were produced. Specific epitopes targeted by the MAb were protein in nature and located in situ in the internal content of the filariform larvae of the parasite but not in the esophagus. The MAb reacted to the homologous antigen in an indirect ELISA but did not reveal any reaction to the SDS-PAGE separated-homologous antigen in a Western blot analysis (WB) suggesting a conformational epitope specificity. The MAb were of IgG1 isotype which is the isotype known to have high affinity to this epitope so they were used in a dot-ELISA to detect the antigen of the parasite. The assay could detect the epitopes in 78 ng or more of the crude filariform larval extract but did not reveal any positive result when applied to detect antigen in stool samples of parasitologically confirmed strongyloidiasis patients. The negative antigen test results can be explained as follows. Either the MAb were filariform stage-specific and thus did not recognize the rhabditiform larval antigen mainly contained in the patient's stool or the amounts of antigen in the stool samples were too small and/or unevenly dispersed. In the latter instance, the MAb developed in this study would have a diagnostic potential if used in an immunological test design where more volume of fresh stool sample could be accommodated in the test, e.g. a sandwich plate ELISA.