Function of Burkholderia pseudomallei RpoS and RpoN2 in bacterial invasion, intracellular survival, and multinucleated giant cell formation in mouse macrophage cell line.

Melioidosis, a human infectious disease with a high mortality rate in many tropical countries, is caused by the pathogen Burkholderia pseudomallei (B. pseudomallei). The function of the B. pseudomallei sigma S (RpoS) transcription factor in survival during the stationary growth phase and conditions of oxidative stress is well documented. Besides the rpoS, bioinformatics analysis of B. pseudomallei genome showed the existence of two rpoN genes, named rpoN1 and rpoN2. In this study, by using the mouse macrophage cell line RAW264.7 as a model of infection, the involvement of B. pseudomallei RpoS and RpoN2 in the invasion, intracellular survival leading to the reduction in multinucleated giant cell (MNGC) formation of RAW264.7 cell line were illustrated. We have demonstrated that the MNGC formation of RAW264.7 cell was dependent on a certain number of intracellular bacteria (at least 5 × 104). In addition, the same MNGC formation (15%) observed in RAW264.7 cells infected with either B. pseudomallei wild type with multiplicity of infection (MOI) 2 or RpoN2 mutant (∆rpoN2) with MOI 10 or RpoS mutant (∆rpoS) with MOI 100. The role of B. pseudomallei RpoS and RpoN2 in the regulation of type III secretion system on bipB-bipC gene expression was also illustrated in this study.

Correlation between biofilm production, antibiotic susceptibility and exopolysaccharide composition in Burkholderia pseudomallei bpsI, ppk, and rpoS mutant strains.

Burkholderia pseudomallei is the cause of melioidosis, a fatal tropical infectious disease, which has been reported to have a high rate of recurrence, even when an intensive dose of antibiotics is used. Biofilm formation is believed to be one of the possible causes of relapse because of its ability to increase drug resistance. EPS in biofilms have been reported to be related to the limitation of antibiotic penetration in B. pseudomallei. However, the mechanisms by which biofilms restrict the diffusion of antibiotics remain unclear. The present study presents a correlation between exopolysaccharide production in biofilm matrix and antibiotic resistance in B. pseudomallei using bpsI, ppk, and rpoS mutant strains. CLSM revealed a reduction in exopolysaccharide production and disabled micro-colony formation in B. pseudomallei mutants, which paralleled the antibiotic resistance. Different ratios of carbohydrate contents in the exopolysaccharides of the mutants were detected, although they have the same components, including glucose, galactose, mannose, and rhamnose, with the exception being that no detectable rhamnose peak was observed in the bpsI mutant. These results indicate that the correlation between these phenomena in the B. pseudomallei biofilm at least results from the exopolysaccharide, which may be under the regulation of bpsI, ppk, or rpoS genes.

Prediction of the functional effect of novel SLC25A13 variants using a S. cerevisiae model of AGC2 deficiency.

AGC2, a member of the mitochondrial carrier protein family, is as an aspartate-glutamate carrier and is important for urea synthesis and the maintenance of the malate-aspartate shuttle. Mutations in SLC25A13, the gene encoding AGC2, result in two age dependent disorders: neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and type II citrullinemia (CTLN2). The clinical features of CTLN2 are very similar to those of other urea cycle disorders making a clear diagnosis difficult. Analysis of the SLC25A13 gene sequence can provide a definitive diagnosis, however the predictive value of DNA sequencing requires that the disease association of variants be characterized. We utilized the yeast Saccharomyces cerevisiae lacking AGC1 as a model system to study the effect on the function of AGC2 variants and confirmed that this system is capable of distinguishing between AGC2 variants with normal (p.Pro632Leu) or impaired function (p.Gly437Glu, p.Gly531Asp, p.Thr546Met, p.Leu598Arg and p.Glu601Lys). Three novel AGC2 genetic variants, p.Met1? (c.2T>C), p.Pro502Leu (c.1505C>T), and p.Arg605Gln (c.1814G>A) were investigated and our analysis revealed that p.Pro502Leu and p.Arg605Gln substitutions in the AGC2 protein were without effect and these variants were fully functional. The p.Met1? mutant is capable of expressing a truncated p.Met1_Phe34del AGC2 variant, however this protein is not functional due to disruptions in a calcium binding EF hand as well as incorrect intracellular localization. Our study demonstrates that the characterization of AGC2 expressed in yeast cells is a powerful technique to investigate AGC2 variants, and this analysis should aid in establishing the disease association of novel variants.

RpoS and oxidative stress conditions regulate succinyl-CoA: 3-ketoacid-coenzyme A transferase (SCOT) expression in Burkholderia pseudomallei.

Burkholderia pseudomallei, a pathogenic gram-negative bacterium, causes the severe human disease melioidosis. This organism can survive in eukaryotic host cells by escaping reactive oxygen species via the regulation of stress responsive sigma factors, including RpoS. In B. pseudomallei, RpoS has been reported to play a role in the oxidative stress response through enhanced activity of OxyR and catalase. In this study, the RpoS dependent oxidative stress responsive system was further characterized using comparative proteomic analysis. The proteomic profiles of wild-type B. pseudomallei following exposure to H2 O2 and between wild-type and the rpoS mutant strains were analyzed. Using stringent criteria, 13 oxidative responsive proteins, eight of which are regulated by RpoS, were identified with high confidence. It was observed that ScoA, a subunit of the SCOT enzyme not previously shown to be involved directly in the oxidative stress response, is significantly down-regulated after hydrogen peroxide treatment. ScoA and ScoB have been predicted to be organized in a single operon using computational methods: in this study it was confirmed by RT-PCR that these genes are indeed co-transcribed as a single mRNA. The present study is the first to report a role for RpoS in the down-regulation of SCOT expression in response to oxidative stress in B. pseudomallei.

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.

Feasibility of using 454 pyrosequencing for studying quasispecies of the whole dengue viral genome.

BACKGROUND: Dengue is the world's most common mosquito-borne viral disease. Poor proofreading by RNA polymerase during its replication results in the accumulation of mutations in its genome. This leads to a diversity of genotypes in the viral population termed quasispecies. Quasispecies play an important role in disease severity. The study of quasispecies in dengue has been hindered because of the requirement for large amounts of cloning and sequencing, which could be overcome by 454 pyrosequencing. In this study, we attempted to demonstrate the feasibility of using 454 pyrosequencing to study genome diversity of dengue virus quasispecies by sequencing a pool of known dengue viral strains. RESULTS: Two sets of dengue DNA templates were sequenced by 454/Roche GS FLX. The total number of reads for data 1 and data 2 were 54,440 and 134,441, with average lengths of 221 and 232 bp, respectively. Reads containing ambiguous base Ns were excluded (6.00% in data 1, 7.05% in data 2). More than 99% of reads could be aligned back to the correct serotypes by BLAST. The reads covered the whole genome without any gaps, and the minimum coverage depth was 50×. Frequencies of known strains detected from each data set were highly correlated with the input ratios. We also explored criteria for filtering error reads and artifacts from true variations. CONCLUSIONS: This study showed that 454 pyrosequencing, coupled with our analysis procedure, could sequence the whole genome of dengue virus with good coverage. The ratio of detected variants in the sequencing data reflected the starting ratio, proving that the proposed technique could be used to study the frequencies of variants in quasispecies.

Regulation of a quorum sensing system by stationary phase sigma factor RpoS and their co-regulation of target genes in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of a fatal disease, melioidosis. However, the mechanisms of pathogenesis and genes involved in its virulence are not understood. In the current study, whether stationary phase and stress response sigma factor RpoS and BpsI-mediated quorum sensing (QS) system co-regulate its target genes was investigated. Positive regulation of RpoS on bpsI expression and autoinducer production in stationary phase, but not on fliC and ppk expression, was observed. In contrast, expression of rpoS was not affected by deletion of bpsI. The present results also indicate that production of extracellular protease and siderophore, two QS-controlled exo products, is regulated via RpoS supporting the previously known inhibitory role of QS on those two factors. Proteomic analysis revealed that expression of 74 protein spots representing 60 genes is controlled by QS. Of those, 45 genes are co-regulated by both RpoS and QS, and regulation of genes involved in transcription and translation is favored by QS. Taken together, our findings indicate major target genes expression in stationary phase that is influenced by hierarchical control of RpoS over QS. We propose that this regulation may play an important role in the pathogenicity of B. pseudomallei.

Phosphoproteomic analysis of apoptotic hematopoietic stem cells from hemoglobin E/ß-thalassemia.

BACKGROUND: Hemoglobin E/ß-thalassemia is particularly common in Southeast Asia and has variable symptoms ranging from mild to severe anemia. Previous investigations demonstrated the remarkable symptoms of ß-thalassemia in terms of the acceleration of apoptotic cell death. Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear. METHODS: The phosphoproteome of bone marrow HSCs/CD34⁺ cells from HbE/ß-thalassemic patients was analyzed using IMAC phosphoprotein isolation followed by LC-MS/MS detection. Decyder MS software was used to quantitate differentially expressed proteins in 3 patients and 2 normal donors. The differentially expressed proteins from HSCs/CD34⁺ cells were compared with HbE/ß-thalassemia and normal HSCs. RESULTS: A significant change in abundance of 229 phosphoproteins was demonstrated. Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors. Moreover, in the HSCs patients a significant increase was observed in a specific type of phosphoserine/threonine binding protein, which is known to act as an important signal mediator for the regulation of cell survival and apoptosis in HbE/ß-thalassemia. CONCLUSIONS: Our study used a novel method to investigate proteins that influence a particular pathway in a given disease or physiological condition. Ultimately, phosphoproteome profiling in HbE/ß-thalassemic stem cells is an effective method to further investigate the cell death mechanism of ineffective erythropoiesis in ß-thalassemia. Our report provides a comprehensive phosphoproteome, an important resource for the study of ineffective erythropoiesis and developing therapies for HbE/ß-thalassemia.

A proteome reference map of the causative agent of melioidosis Burkholderia pseudomallei.

Burkholderia pseudomallei is the etiologic agent of melioidosis. Using 2DE and MALDI-TOF MS, we report here a proteome reference map constructed from early stationary phase, a bacterial adaptation process. We identified 282 protein spots representing 220 ORFs; many of them have been implicated in bacterial pathogenesis. Up to 20% of identified ORFs belong to post-translational modification and stress responses. The proteome reference map will support future analysis of the bacterial gene and environmental regulation and facilitate comparative proteomics with its sibling species.

Biomarker discovery in the biofilm-forming process of Burkholderia pseudomallei by mass-spectrometry.

A serious human infectious disease called Melioidosis is a result of Burkholderia pseudomallei infection. Treatment for infected individuals is difficult due to a wide range of ineffective antibiotics including a high level of antibiotic tolerance which has been known to be caused by biofilm production. However, biofilm forming processes of this bacterium are not well documented despite multiple-methodologies being applied. In this study, we utilized a proteomics strategy called whole cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (whole cell MALDI-TOF MS) to discover a potential biomarker relating biofilm forming in B. pseudomallei. The results presented a novel specific type of enzyme amylo-alpha-1, 6-glucosidase, which was demonstrated by a higher level of gene expression during the biofilm development. Our results also suggested a list of candidate markers that might be involved in this scenario. Eventually, this knowledge may expand valuable data to the biofilm study that may increase effective treatments for people infected with B. pseudomallei and possibly other antibiotic tolerant bacteria.

Comparative Proteome-Wide Analysis of Bone Marrow Microenvironment of ß-Thalassemia/Hemoglobin E.

ß-thalassemia/Hb E is a global health issue, which is characterized by a range of clinical symptoms from a mild and asymptomatic anemia to severe disorders that require transfusions from infancy. Pathological mechanisms of the disease involve the excess of unmatched alpha globin and iron overload, leading to ineffective erythropoiesis and ultimately to the premature death of erythroid precursors in bone marrow (BM) and peripheral organs. However, it is unclear as to how BM microenvironment factors contribute to the defective erythropoiesis in ß-thalassemia/Hb E patients. Here, we employed mass spectrometry-based comparative proteomics to analyze BM plasma that was collected from six ß-thalassemia/Hb E patients and four healthy donors. We identified that the differentially expressed proteins are enriched in secretory or exosome-associated proteins, many of which have putative functions in the oxidative stress response. Using Western blot assay, we confirmed that atypical lipoprotein, Apolipoprotein D (APOD), belonging to the Lipocalin transporter superfamily, was significantly decreased in BM plasma of the tested pediatric ß-thalassemia/Hb E patients. Our results highlight that the disease condition of ineffective erythropoiesis and oxidative stress found in BM microenvironment of ß-thalassemia/Hb E patients is associated with the impaired expression of APOD protein.

Polyphosphate kinase 1 of Burkholderia pseudomallei controls quorum sensing, RpoS and host cell invasion.

Burkholderia pseudomallei is a Gram negative bacterium and the causative agent of melioidosis. Nonetheless, how virulence factors and pathogenic mechanisms are regulated have been elusive. In this study, we determined a role of polyphosphate kinase 1 (Ppk1) in regulation of quorum sensing (QS) and the sigma factor RpoS, and identified genes co-regulated by Ppk1, QS and RpoS. We find that Ppk1 positively controls autoinducer production and expression of rpoS transcript. Proteomic analysis identified 70 protein spots that are differentially expressed between B. pseudomallei wildtype and its ppk1-deficient strain. Within Ppk1regulated proteins, expression of 31 proteins are co-regulated by both RpoS and QS, whose functions of the majority of these proteins are associated with energy production and stress response. Moreover, expression of proteins involved in type III secretion system (T3SS) is also controlled by Ppk1. Quantitative PCR analysis confirmed that the T3SS genes bipB, bsaR and hrpK are down-regulated in ppk1 mutant. In addition, the ppk1-deficient strain exhibits defects in adhesion and invasion into human lung epithelial cells. Our work therefore reveals regulation of virulence factors and a regulatory mechanism of RpoS and QS by Ppk1, which altogether participate in gene expression control, and might be crucial for pathogenicity of B. pseudomallei. SIGNIFICANCE: Polyphosphate kinase1 (Ppk1), which is a key enzyme in polyphosphate biosynthesis, is pivotal for virulence of the melioidosis pathogen B. pseudomallei. This enzyme is not present in human. Therefore, it has been proposed to be a key target for anti-bacterial drugs. An important step toward development of novel antibiotics and therapeutic strategies is an analysis of proteins that are controlled by Ppk1. By using proteomics, we find that Ppk1 co-regulates virulence-associated genes together with quorum sensing (QS) and the sigma factor RpoS. Moreover, we reveal that Ppk1 is critical for bacterial adhesion and host cell invasion, supporting the finding from our proteome analysis.

Investigation of host-pathogen interaction between Burkholderia pseudomallei and autophagy-related protein LC3 using hydrophobic chromatography-based technique.

BACKGROUND: Burkholderia pseudomallei is an intracellular bacteria causing Melioidosis, the disease widely disseminates in Southeast Asia and Northern Australia. B. pseudomallei has ability to invade various types of host cell and to interfere with host defense mechanisms, such as nitric oxide (NO). Due to the cross-talk among alternative killing mechanisms in host immune response against invading microbes, autophagy is the molecular mechanism belonging to intracellular elimination of eukaryotic cells that has been widely discussed. However, bacterial evasion strategy of B. pseudomallei and host-bacterial protein-protein interaction within autophagic machinery remain unknown. METHODS: Here, we demonstrated the protein-protein interaction study between different strains of B. pseudomallei, including wild type PP844 and rpoS mutant, with autophagy-related protein LC3 that has been constructed, using the modified immunoaffinity hydrophobic chromatography based-technique. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis was utilized for identifying the eluted proteins obtained from the established column. In addition, the expression level of gene encoding candidate protein was predicted prior to verification using real-time quantitative reverse transcription PCR assay (RT-qPCR). RESULTS: LC3 recombinant proteins could be entrapped inside the column before encountering their bacterial interacting partners. Based on affinity interaction, the binding capacity of LC3 with antibody displayed over 50% readily for hydrophobically binding with bacterial proteins. Following protein identification, bacterial ATP-binding cassette (ABC) transporter periplasmic substrate-binding protein (BPSL2203) was identified as a candidate LC3-interacting protein, which was found only in B. pseudomallei wild type. Gene expression analysis and bioinformatics of BPSL2203 were validated the proteomic result which are suggesting the role of RpoS-dependent gene regulation. CONCLUSIONS: Remarkably, utilization of the modified immunoaffinity hydrophobic chromatography with LC-MS/MS is a convenient and reliable approach to a study in B. pseudomallei-LC3 protein-protein interaction.

Induction of specific immune responses against the Plasmodium vivax liver-stage via in vitro activation by dendritic cells.

Due to chronic morbidity, the risk of increasing drug resistance and the existence of the hypnozoite stage in Plasmodium vivax malaria, there is a need to find out how hosts develop immunity to compromise the malaria parasites. Here we focused on an in vitro model for immunotherapy and vaccine development. Immunosuppressive mechanisms in malaria include inhibition of T cell response and suppression of dendritic cell function. Using in vitro activation of lymphocytes by malaria antigen-pulsed dendritic cells could overcome the limitation of antigen presentation during acute infections. Here we showed that the sporozoite-pulsed dendritic cell could elicit cytotoxicity against liver stage of P. vivax. Analysis using immunophenotypic markers showed maturation of the dendritic cells and stimulation of cytotoxic T cells. Functional assay of the in vitro-activated cytotoxic T cells showed enhancement of specific killing of the P. vivax exoerythrocytic stages within infected hepatocytes. This model may be useful for vaccine development against human malaria.

Construction of scFv derived from a tumor-associated monoclonal antibody having tumoricidal activity on human hepatocellular carcinoma.

A mouse monoclonal antibody (Mab-HepTAA43), classified as an anti-tumor-associated antigen, was raised by immunizing BALB/c mice with the Thai human hepatocellular carcinoma S102 (HCC-S102) cell line cells using hybridoma techniques. The Mab-HepTAA43 reacted with and markedly inhibited the growth of human hepatocellular carcinoma cell lines as well as a tumor mass in an animal model. Human hepatoma transplanted into nude mice did not show metastasis after 20 injections amounting to a total of about 4 mg of the Mab over 1-month period. A single-chain variable fragment (scFv) molecule derived from the Mab was constructed by phage display method. DNA sequence analysis of the active variable regions of both heavy- and light-chains of the cDNA clone was subsequently performed. The scFv43 molecule contains a V(L) kappa type and a unique V(H) sequence having 88% amino acid homology to that of Mab-MAK B raised against tumor-associated antigen. Immunohistochemical staining on frozen sections of paired hepatoma (NCI-I) and normal liver tissue from the same individual showed that both scFv43 and Mab-HepTAA43 antibodies reacted with hepatoma but not with normal liver tissue. The results suggest that scFv43 may be useful in the immunotherapy of hepatocellular carcinoma.

Burkholderia pseudomallei rpoS mediates iNOS suppression in human hepatocyte (HC04) cells.

Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen and the causative agent of melioidosis, a widespread disease in Southeast Asia. Reactive nitrogen, in an intermediate form of nitric oxide (NO), is one of the first lines of defense used by host cells to eliminate intracellular pathogens, through the stimulation of inducible nitric oxide synthase (iNOS). Studies in phagocytotic cells have shown that the iNOS response is muted in B. pseudomallei infection, and implicated the rpoS sigma factor as a key regulatory factor mediating suppression. The liver is a main visceral organ affected by B. pseudomallei, and there is little knowledge about the interaction of liver cells and B. pseudomallei This study investigated the induction of iNOS, as well as autophagic flux and light-chain 3 (LC3) localization in human liver (HC04) cells in response to infection with B. pseudomallei and its rpoS deficient mutant. Results showed that the rpoS mutant was unable to suppress iNOS induction and that the mutant showed less induction of autophagy and lower co-localization with LC3, and this was coupled with a lower intracellular growth rate. Combining these results suggest that B. pseudomallei rpoS is an important factor in establishing infection in liver cells.

Utilization of Whole-Cell MALDI-TOF Mass Spectrometry to Differentiate Burkholderia pseudomallei Wild-Type and Constructed Mutants.

Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has been widely adopted as a useful technology in the identification and typing of microorganisms. This study employed the whole-cell MALDI-TOF MS to identify and differentiate wild-type and mutants containing constructed single gene mutations of Burkholderia pseudomallei, a pathogenic bacterium causing melioidosis disease in both humans and animals. Candidate biomarkers for the B. pseudomallei mutants, including rpoS, ppk, and bpsI isolates, were determined. Taxon-specific and clinical isolate-specific biomarkers of B. pseudomallei were consistently found and conserved across all average mass spectra. Cluster analysis of MALDI spectra of all isolates exhibited separate distribution. A total of twelve potential mass peaks discriminating between wild-type and mutant isolates were identified using ClinProTools analysis. Two peaks (m/z 2721 and 2748 Da) were specific for the rpoS isolate, three (m/z 3150, 3378, and 7994 Da) for ppk, and seven (m/z 3420, 3520, 3587, 3688, 4623, 4708, and 5450 Da) for bpsI. Our findings demonstrated that the rapid, accurate, and reproducible mass profiling technology could have new implications in laboratory-based rapid differentiation of extensive libraries of genetically altered bacteria.

Role of Burkholderia pseudomallei Sigma N2 in Amino Acids Utilization and in Regulation of Catalase E Expression at the Transcriptional Level.

Burkholderia pseudomallei is the causative agent of melioidosis. The complete genome sequences of this pathogen have been revealed, which explain some pathogenic mechanisms. In various hostile conditions, for example, during nitrogen and amino acid starvation, bacteria can utilize alternative sigma factors such as RpoS and RpoN to modulate genes expression for their adaptation and survival. In this study, we demonstrate that mutagenesis of rpoN2, which lies on chromosome 2 of B. pseudomallei and encodes a homologue of the sigma factor RpoN, did not alter nitrogen and amino acid utilization of the bacterium. However, introduction of B. pseudomallei rpoN2 into E. coli strain deficient for rpoN restored the ability to utilize amino acids. Moreover, comparative partial proteomic analysis of the B. pseudomallei wild type and its rpoN2 isogenic mutant was performed to elucidate its amino acids utilization property which was comparable to its function found in the complementation assay. By contrast, the rpoN2 mutant exhibited decreased katE expression at the transcriptional and translational levels. Our finding indicates that B. pseudomallei RpoN2 is involved in a specific function in the regulation of catalase E expression.

Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma.

BACKGROUND: Hep27 monoclonal (Hep27 Mab) is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102). We attempted to produce a single-chain fragment (scFv), a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques. RESULTS: The sequences encoding the variable regions of heavy (VH) and light (VL) chains of a murine Hep27 Mab were linked together by a linker peptide (Gly4Ser)3 and tagged with a hexa-histidine at the C-terminal; the resultant DNA construct was expressed in E. coli as an insoluble protein. The denatured scFv was refolded and purified by immobilized metal ion affinity chromatography (12 mg/l with a molecular weight of 27 kDa). Hep27scFv exhibited a tumoricidal activity against the HCC-S102 cell as its parental antibody (Hep27 Mab). CONCLUSION: This scFv may be a potential candidate for a targeting agent in HCC immunodiagnosis or immunotherapy.

Tumor suppressive monoclonal antibody belonging to the V(H) 7183 family directed to the oncodevelopmental carbohydrate antigen on human hepatocellular carcinoma.

Hep27 monoclonal antibody (Hep27 Mab) was raised by immunizing BALB/c mice with cells of the Thai human hepatocellular carcinoma (HCC) cell line HCC-S102 using hybridoma technology. The Hep27 Mab recognizes oncofetal development antigens by reacting with many HCC, other cancers, fetal and newborn liver but not adult liver. The Hep27 Mab alone markedly inhibits the growth of hepatocellular carcinoma cell lines (65% viability on the third day), suggesting its clinical usefulness. Moreover, complementary DNA (cDNA) for active variable regions of both heavy and light chains of the antibody has been cloned. Sequence analysis of the variable region of the Hep27 Mab revealed that the V(H) and V(L) genes belong to the V(H) 7183 and V(K) families, respectively. We have also characterized the reactivity of the Hep27 Mab to synthetic carbohydrate epitopes and 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP)-treated HCC-S102 cells. The results showed that the Hep27 Mab recognizes a neoglycolipid containing a mucin core unit and PDMP treatment reduced Hep27 Mab binding activity to HCC-S102 cells, indicating that the Hep27 Mab recognizes a glycolipid antigen on HCC-S102 cells. This Mab may be potentially useful for studying antigenic expression in hepatocellular carcinoma and as a targeting agent for radioimmunodetection and immunoconjugated therapy.