Burkholderia pseudomallei-specific recombinant protein and its potential in the diagnosis of melioidosis.

Melioidosis is an important public health problem in Southeast Asia and Northern Australia. This disease is caused by the gram-negative bacilli, Burkholderia pseudomallei. Wide spectra of clinical manifestations are observed in melioidosis ranging from asymptomatic to septicemic infection. Although serodiagnostic methods of melioidosis have been improved significantly in recent years, a highly specific diagnostic test that can differentiate asymptomatic seropositive individuals and melioidosis patients remains to be the subject of current investigations. In this study, a B. pseudomallei-specific gene, pBps-1, expressing a novel 18.7 kDa recombinant protein was selected from genomic libraries of two B. pseudomallei virulent isolates by using pooled sera from septicemic melioidosis patients. Nucleotide sequence analysis demonstrated that this gene is unique and does not show substantial similarity with any known genes in the Genbank database. The Bps-1 recombinant protein was evaluated for its potential in serodiagnosis of melioidosis by Western blot analysis. A high degree of specificity was demonstrated using sera from healthy individuals in the endemic (98.5%) and non-endemic areas (100%), with moderate sensitivity (69.7%) in melioidosis patients. The study demonstrated that this approach can be used to obtain highly specific recombinant antigens such as that described in the present report. A combination of such antigens should provide materials for successful serodiagnosis of melioidosis in the endemic areas.

Dengue virus specific T cell responses to live attenuated monovalent dengue-2 and tetravalent dengue vaccines.

The proliferative T cell responses to dengue vaccines were studied using the parental strains of dengue vaccines as antigens in 26 dengue immune individuals who resided in Bangkok which is the endemic area of dengue infection. The magnitude of the T cell responses in subjects with flavivirus cross-reactive neutralizing antibody was much higher and the cross-reactivity was broader than in those with dengue serotype-specific neutralizing antibodies, Japanese encephalitis (JE) specific antibodies or dengue cross-reactive antibodies. The T cell response in those with neutralizing antibody against a single serotype or in those who had dengue cross-reactive neutralizing antibody was relatively low, independent of the level or degree of cross-reactivity of the antibody. Evaluation of the proliferative T cell responses in 8 recipients of the monovalent dengue-2 (16681-PDK53) or the tetravalent dengue vaccines demonstrated that both vaccines induced high levels of neutralizing antibody as well as high levels of T cell responses to all serotypes of dengue virus. These results indicate that the evaluated dengue vaccines efficiently induced humoral and cell mediated immunity comparable to natural infection with dengue virus.

Genetically engineered single-chain Fvs of human immunoglobulin against hepatitis C virus nucleocapsid protein derived from universal phage display library.

Specific single-chain Fvs (scFvs) of human immunoglobulin that specifically recognized the recombinant hepatitis C virus (HCV) nucleocapsid protein were isolated from a large phage display antibody library. This universal library of genetically engineered filamentous phagemids displayed random pairings of the variable regions of both human heavy and light chain immunoglobulin in the scFv format. Specific clones were isolated by affinity selection with purified recombinant HCV protein fused to glutathione-S-transferase (GST). The GST-specific clones were excluded by blocking the phagemid library with GST prior to the selection. After 4 rounds of selection, the HCV-reactive clones were enriched by a factor of 100,000. About 4% and 9% of the clones from rounds 4 and 5, respectively, specifically reacted to the HCV portion of the fusion protein in an enzyme immunoassay. The specificity was confirmed by specific binding inhibition with plasma from an HCV-infected individual. Nucleotide sequence analysis of 3 HCV-specific clones indicated that all 3 clones contained an almost identical VH gene sequence which was derived from the VH3 germline gene family. These clones had different VL gene sequences of the lambda type. There were some differences between nucleotide and amino acid sequences of the HCV-specific scFv genes and those of the closest matched germline genes, indicating the presence of somatic mutation. This study illustrated the feasibility of using antibody engineering technology with the universal phage display library to isolate human antibodies with predefined specificity to important microbial pathogen which may be useful for future therapeutic purpose.

The immunoreactivity profile of different HCV genotypes on immunoblot assay and its implications in the development of diagnostic assays.

The immunoreactivity profiles of plasma samples obtained from patients infected with different hepatitis C virus (HCV) genotypes were studied using immunoblot assay containing multiple HCV antigens. The immunoblot assay was found to be positive in 81.5% of 195 blood donors who had anti-HCV antibodies as detected by second generation enzyme immunoassays. The samples reacted preferentially with the viral core, NS3-1 and NS5 antigens, and these reactivities were not influenced by HCV genotype. However, the reactivities with NS3-2 and NS4 antigens varied depending on HCV genotypes. The samples from patients infected with HCV genotype 1 reacted well with NS3-2 and NS4 antigens whereas those with other genotypes did not. In addition, samples with the unclassified HCV genotype reacted poorly with all antigens, except NS3-1. This study demonstrates the importance of the core, NS3-1 and NS5 antigens in the detection of antibodies against HCV, especially in areas where more than one genotypes of HCV are present. It also demonstrates that there is a need for further improvement of the currently used assays as new HCV genotypes are recently discovered.

Improved amplification system for detection of hepatitis C virus genome that simultaneously differentiates viral genotypes.

An improved system for amplification of hepatitis C virus genome (HCV) was developed based on a multiplex nested polymerase chain reaction format. Two sets of oligonucleotide primers were used simultaneously. One was derived from the conserved sequences in the 5' non-coding region of the viral genome which can bind to the viral genome of all genotypes. The other set of primers was designed from a sequence in the nonstructural-5 region of HCV. HCV genotypes 1 and 3 can be differentiated by the banding patterns of amplified DNA products. All of 39 samples containing the HCV genotype 1 could be amplified with primers in the 5' non-coding region only, whereas 92% of those with genotype 3 could be amplified by both primer sets. In addition, HCV RNA can be detected in 81% of 84 anti-HCV-positive blood donors and in 0% of 34 anti-HCV-negative cases. Of the HCV RNA-positive specimens, 69% showed genotype 1-like patterns while 31% showed genotype 3-like patterns. The detection rate of HCV RNA in this study was much higher than that in our previous report due to the improvement of new primers which can detect all genotypes of the virus. In conclusion, this improved amplification system is a sensitive method for rapid identification of HCV RNA in clinical specimens that can simultaneously differentiate the two most common genotypes of HCV found in Thailand.

Molecular cloning and expression of hepatitis C virus core protein and production of monoclonal antibodies to the recombinant protein.

The gene encoding nucleocapsid (core) protein of hepatitis C virus (HCV) was isolated from a Thai blood donor infected with HCV genotype 1b. The nucleotide sequence of this clone showed a high degree of homology to that of 4 HCV strains isolated from other Thai blood donors as well as that of the HCV prototypes of genotypes 1a, 1b, 2a and 3a. The entire region of the core gene was cloned into an expression plasmid pGEX-3X to be expressed as a fusion protein with glutathione-S-transferase (GST). E. coli transformants containing this plasmid did not express the fusion protein. However, GST-HCV core fusion protein could be produced when the core gene was truncated at the 3' end resulting in a gene encoding only the first 123 amino acid residues of the core protein. This fusion protein was insoluble in standard buffers, but could be solubilized by sarkosyl and thus subsequently purified using glutathione-Sepharose 4B. The purified fusion protein was immunogenic and could react with antibodies from blood donors infected with all genotypes of HCV found in Thailand. In addition, two murine hybridoma clones secreting monoclonal antibodies specific to the recombinant HCV core protein were produced. The purified HCV core protein and the monoclonal antibodies to the recombinant protein will be useful for developing assay systems for detecting anti-HCV antibodies and HCV antigen, respectively.

Production of specific monoclonal antibodies to Burkholderia pseudomallei and their diagnostic application.

Hybridomas secreting monoclonal antibodies (MAbs) specific to Burkholderia pseudomallei were produced by immunizing BALB/cJ mice with crude culture filtrate of B. pseudomallei. Two monoclonal antibodies were found to be highly specific to B.pseudomallei as tested by indirect enzyme-linked immunosorbent assay and immunoblotting against a panel of crude whole cell extracts from B. pseudomallei, B. cepacia, Pseudomonas aeruginosa, P.putida, and Escherichia coli. One of the specific MAbs, clone SP-M, IgM subclass, could directly agglutinate all 42 B. pseudomallei, isolates. The study has shown that the agglutinating MAb has potential for rapid identification of B. pseudomallei in primary bacterial culture from clinical specimens. The antibody can be used in bacteriology laboratories to reduce time of biochemical methods, which require a few days.

The newly discovered non A-E hepatitis viruses.

Two biotechnology companies have recently announced the discovery of 4 new hepatitis viruses, provisionally named HGV and GBV agents (GBV-A, GBV-B, and GBV-C). Using a molecular biological approach, the genomes of these viruses were identified from non-A-E hepatients patients who had no markers to any previously known hepatitis viruses. The new viruses are members of family Flaviviridae, and are closely related to hepatitis C virus (HCV). Preliminary studies show that the prevalence of GBV agents and HGV are alarmingly high in blood donors in the United States, Europe, Africa and Japan. The viruses are transmitted parenterally, similar to HCV and hepatitis B virus (HBV), Chronic infection is common and can lead to cirrhosis. Some chronic hepatitis cases caused by these viruses respond to interferon treatment. The viruses can coinfect with HCV and/or HBV. A number of questions about these new viruses remain to be answered, including the magnitude of the problems, clinical significance, mode of transmission and populations at risk, as well as the appropriate treatment.

Hepatitis B- and hepatitis C-associated hepatocellular carcinoma: evaluation of alpha-fetoprotein as a diagnostic marker.

Hepatocellular carcinoma (HCC) is one of the most common cancers, especially in Asia and Africa. The prognosis of HCC is very poor because of the high malignancy and the failure of early diagnosis which is mainly dependent on the late onset of clinical symptoms. Chronic infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) is the most commonly known risk factor for developing HCC. Mass screening and monitoring of general population or of high-risk population, by measurement of serum alpha-fetoprotein (AFP), have been implemented in several countries. However, the use of AFP as a diagnostic marker for HCC is questionable due to its limited sensitivity and specificity. This article analyzed the serum level of AFP in 72 histopathologically confirmed hepatocellular carcinoma cases in Thailand. Elevation of serum AFP was detected in 75.6%, 88.9%, 79.2% and 80.0% of patients with HBsAg, anti-HCV antibody, HBV DNA, and HCV RNA, respectively. However, only 58.8% of HCC patients without any of the four markers had elevation of serum AFP. AFP is thus not a sensitive screening marker for HCC in general population, especially in those not associated with HBV or HCV. However, since elevated serum AFP was found in most patients with evidence of HBV or HCV infection, the monitoring of serum AFP level in those high-risk patients can be valuable for screening and monitoring of hepatocellular carcinoma.