Germline variable region gene segment derivation of human monoclonal anti-Rh(D) antibodies. Evidence for affinity maturation by somatic hypermutation and repertoire shift.

To date, there has been no systematic study of the process of affinity maturation of human antibodies. We therefore sequenced the variable region genes (V genes) of 14 human monoclonal antibodies specific for the erythrocyte Rh(D) alloantigen and determined the germline gene segments of origin and extent of somatic hypermutation. These data were correlated with determinations of antibody affinity. The four IgM antibodies (low affinity) appear to be derived from two germline heavy chain variable region gene segments and one or two germline light chain variable region gene segments and were not extensively mutated. The 10 IgG antibodies (higher affinity) appear to be derived from somatic hypermutation of these V gene segments and by use of new V gene segments or V gene segment combinations (repertoire shift). Affinity generally increased with increasing somatic hypermutation; on average, there were 8.9 point mutations in the V gene segments of the four IgM antibodies (Ka = 1-4 x 10(7)/M-1) compared with 19 point mutations in the V gene segments of the 10 IgG antibodies. The four highest affinity antibodies (Ka = 0.9-3 x 10(9)/M-1) averaged 25.5 point mutations. The use of repertoire shift and somatic hypermutation in affinity maturation of human alloantibodies is similar to data obtained in inbred mice immunized with haptens.

An improved method for the detection of cell surface antigens in samples of low viability using flow cytometry.

A high non-specific background fluorescence signal was observed when cell surface antigen analysis was carried out using flow cytometry on a cell sample which contained a high proportion of dead and dying cells. To overcome this problem it was necessary to analyse the cells in three stages. First the intact cells were identified by their forward (FWD) and 90 degree scatter profile. These cells were gated-on, then analysed on the basis of their FWD scatter and propidium iodide (PI) signal, allowing the dead PI positive cells to be gated out. The PI negative cells were then displayed using their 90 degree scatter and fluorescence signals following staining with the irrelevant antibody control. This revealed a population of dead cells, which despite being PI negative, were non-specifically binding antibody molecules. Such multiparameter analysis permitted the successful analysis of cell surface antigens in preparations of low viability by gating out the high background fluorescence associated with dead PI positive and negative cells.

Polymorphism in the promoter region of tumor necrosis factor-alpha gene is associated with severe meliodosis.

Melioidosis is an important infectious disease endemic in Southeast Asia and the Northern territories of Australia. Septicemic melioidosis, is the leading cause of fatality from community acquired septicemia in northeastern part of Thailand where death often occurs within a few days after hospitalization. The present study was carried out to investigate the polymorphisms of the position -308 promoter region of the TNF-alpha gene, as well as of the intron 1 of the TNF-beta gene in patients with melioidosis compared with normal uninfected controls in the same endemic area. The gene frequency of TNF2 allele was significantly higher in melioidosis patients compared with control subjects (p = 0.0097, relative risk 2.32). The increase in TNF2 allele in melioidosis patients was found in both heterozygous and homozygous forms. In addition, the increase in TNF2 allele was most apparent in patients who had fatal outcome from septicemic melioidosis (p = 0.017), but was also observed with lesser degree in other groups of melioidosis patients. However, no difference in the frequency of TNF-beta polymorphism the melioidosis patients was observed.

HLA-DR and -DQ associations with melioidosis.

Melioidosis is an important infectious disease of southeast Asia caused by an intracellular bacterium, Burkholderia pseudomallei. Cellular immunity is postulated to play important roles in immunity to melioidosis that may influence the severity and clinical outcome of the disease. The present study was undertaken to investigate possible associations of melioidosis with HLA class II alleles. HLA typing of HLA-DRB1, -DQA1, and -DQB1 was performed using polymerase chain reaction and sequence-specific oligonucleotide hybridization (PCR-SSO). Seventy-nine melioidosis patients and 105 healthy, ethnically and geographically matched controls were studied. Among 24 DRB1 alleles, 7 DQA1 alleles, and 13 DQB1 alleles identified in this population, an association with melioidosis was observed with DRB1*1602 which was increased in melioidosis patients (10.1%) compared to normal controls (4.8%), p = 0.047 (odds ratio (OR) = 2.25). In addition, significant increase of DRB1*1602 allele frequency and decrease of DQA1*03 were also observed in septicemic melioidosis patients, the most severe form of the disease (p = 0.01, OR = 3.10; and p = 0.047, respectively). Furthermore, a trend of association of DRB1*0701, DQA1*0201, and DQB1*0201 with relapse cases of melioidosis was also noted. In contrast, no HLA association was observed in localized melioidosis or melioidosis with diabetes mellitus. These findings provide the suggestive evidence of an immunogenetic basis of certain aspects of melioidosis.

HLA association with hepatitis C virus infection.

Hepatitis is one of the most important infectious diseases in Thailand. The knowledge of host factors that influence the course of the disease is still limited. In this study, the HLA class I and class II phenotypes were analyzed in the 2 groups of HCV-infected Thai populations. The first group included 43 individuals with transient HCV infection (HCV antibody positive, HCV RNA PCR negative), and the second included 57 individuals with persistent chronic HCV infection (HCV antibody positive, PCR positive). HLA class I typing was performed by 2-stage microlymphocytotoxicity test, and HLA class II typing, by PCR-SSO. No significant difference in the frequencies of HLA-A and -B antigens was observed between the 2 groups of HCV-infected individuals. The frequency of DRB1*0301 and DQB1*0201 was significantly higher in the persistent-infection group than in the transient-infection group (Pc = 0.03, Pc = 0.04, respectively). In addition, DRB1*0701 and DQA1*0201 were significantly decreased in all the HCV-infected patients compared with levels in the normal controls (Pc = 0.003, Pc = 0.001, respectively). This study demonstrated that DRB1*0301 and DQB1*0201 are associated with persistent HCV infection, whereas DRB1*0701 and DQA*0201 are associated with protection against HCV infection.

Speed of detection of Burkholderia pseudomallei in blood cultures and its correlation with the clinical outcome.

Burkholderia pseudomallei is a major cause of fatalities from nonhospital-acquired, gram-negative bacterial septicemia in northeastern part of Thailand. Rapid isolation of the bacterium is critical for diagnosis and treatment. Bacterial culture is currently the gold standard method for laboratory diagnosis of melioidosis. The present study describes the time to detection of B. pseudomallei in blood cultures using a BacT/Alert automated blood culture system, and the correlation between the speed of detection and the clinical outcome of the patients. Of 813 consecutive positive blood cultures, 75 blood cultures from 71 patients were positive for B. pseudomallei. The mean +/- SD time to detection of growth of B. pseudomallei was 23.9 +/- 14.9 hr (95% confidence interval = 20.4-27.5 hr). A total of 62.5% of the B. pseudomallei-positive cultures was detected within 24 hr of incubation, and 93.1% within 48 hr. Interestingly, fatalities occurred in 73.7% of those in which the bacterial growth was detected within the first 24 hr, as compared with only 40.9% in those with a time to detection of culture more than 24 hr (P = 0.012). The shorter time of detection of the bacterial growth in blood cultures may reflect a higher bacterial level in the patient at the time blood was taken, and may be responsible for the poor clinical outcome.

Differentiation between non-virulent and virulent Burkholderia pseudomallei with monoclonal antibodies to the Ara+ or Ara- biotypes.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal tropical infectious disease endemic in Southeast Asia. Environmental isolates of B. pseudomallei have two distinctive biotypes. Some soil isolates are arabinose-assimilators (Ara+ biotype) and are non-virulent in experimental animals. The others cannot assimilate arabinose (Ara- biotype) and are virulent in experimental animals. The Ara- biotype is found in almost all B. pseudomallei clinical isolates. In the present study, a panel of eight monoclonal antibodies that agglutinate the bacteria were produced and tested. The first group, Bps-D2, -D3, -D5, -L1, and -L2 agglutinated 100% of Ara+ clinical and soil isolates of B. pseudomallei. Another group Bps-A1, -A2, and -D1 agglutinated 92.9% and 90.9% of Ara- clinical and soil isolates, respectively. This panel of monoclonal antibodies may be useful for rapid differentiation between non-virulent Ara+ and virulent Ara- B. pseudomallei.

High prevalence of hepatitis C infection among blood donors in northeastern Thailand.

Previous studies on the prevalence of hepatitis C virus (HCV) infection in Asian countries reported an average prevalence of less than 1.5%. In this study a combination of second- and third-generation enzyme immunoassays (EIAs), immunoblot analysis, and polymerase chain reaction was used to evaluate the prevalence of HCV infection in 3,255 volunteer blood donors in northeastern Thailand. Antibodies to HCV were detected in 6.5% of male blood donors and 0.9% of female blood donors, giving an overall prevalence of 5.6% in this population (gender-adjusted prevalence of 3.7%). The prevalence was higher in males than in females (P < 0.0001) and increased with age, reaching a peak at 31-40 years of age. More than 90% of the EIA-positive samples tested positive by immunoblot analysis, giving an estimated minimal prevalence of antibodies to HCV in the blood donors of 5.2%. Approximately 80% of the EIA-positive blood donors were viremic as determined by the presence of HCV RNA detected by the polymerase chain reaction, indicating that at least 4.5% of volunteer blood donors had detectable HCV RNA and were considered potentially infectious. The prevalence of HCV infection in this population was higher than that in previous reports for central and northern Thailand, while the prevalence of HBV infection was similar to that in other regions of the country. This study clearly demonstrated a very high prevalence of HCV infection in northeastern Thailand, especially in the male population.

Rapid identification of Burkholderia pseudomallei in blood cultures by latex agglutination using lipopolysaccharide-specific monoclonal antibody.

Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in Southeast Asia. The septicemic form of melioidosis is the leading cause of death due to community-acquired bacteremia in the northeastern part of Thailand. The delay in isolation and identification of the causative organism is a major contributing factor to the high mortality. The present study describes the evaluation of a latex agglutination test for rapid identification of the bacteria directly from blood cultures. The Bps-L1 monoclonal antibody recognized the lipopolysaccharide antigen of 96.8% of B. pseudomallei clinical isolates and was highly specific for B. pseudomallei. The diagnostic value of the latex agglutination test based on Bps-L1 monoclonal antibody was prospectively evaluated in an area endemic for melioidosis. The agglutination test kit was evaluated in 88 blood cultures with gram-negative bacteria identified with Gram staining. The sensitivity and specificity of the test kit were both 100%. These results indicated that the detection of B. pseudomallei lipopolysaccharide by specific monoclonal antibody in a latex agglutination format is clinically useful for the rapid identification of the bacteria in blood cultures in areas endemic for melioidosis.

Diagnostic value of an antibody enzyme-linked immunosorbent assay using affinity-purified antigen in an area endemic for melioidosis.

Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in southeast Asia. The septicemic form of melioidosis is the leading cause of death from nonhospital-acquired septicemia in the northeastern part of Thailand. A major factor that contributes to the high mortality is the delay in isolation and identification of the causative organism. The present study was undertaken to evaluate the use of enzyme-linked immunosorbent assays based on an immunoaffinity-purified antigen for detecting specific IgG and IgM antibodies to this organism as a rapid serodiagnostic method for melioidosis. The diagnostic value of these tests was evaluated in an actual clinical situation in an area endemic for melioidosis. The specificity of specific IgG test (82.5%) and the specific IgM test (81.8%) were significantly better than that of the indirect hemagglutination (IHA) test (74.7%). The sensitivity of the specific IgG assay (85.7%) was higher than that of the IHA test (71.0%) and the specific IgM test (63.5%). Specific IgG antibody was detected in a majority of septicemic melioidosis (87.8%), as well as in localized forms (82.6%). The specific IgG test was also better than the specific IgM test and the IHA test in identifying acute melioidosis cases in the first five days after admission. In addition, the IgG antibody level to this antigen remained high over a period of more than five years in those who had recovered from melioidosis and remained clinically free of the disease. These results indicate that the detection of specific IgG antibody is clinically useful for the diagnosis of acute melioidosis in an endemic area.

Hepatitis C virus genotypes in patients with hepatocellular carcinoma and cholangiocarcinoma in Thailand.

The prevalences of infections with hepatitis C virus (HCV) and hepatitis B virus (HBV) were determined in 110 Thai patients with liver cancer, of whom 80 and 30 had histological diagnoses of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), respectively. Hepatitis B surface antigen was detected in 63.8% of HCC patients and 16.7% of those with CCA. Antibodies to HCV, detected by a third-generation enzyme immunoassay, were found in 11.3% of HCC patients and in no CCA patient. HCV ribonucleic acid (RNA) was detected by polymerase chain reaction in 6 anti-HCV positive patients, and also in 2 patients who had no detectable anti-HCV antibody. A total of 11 patients had evidence of HCV infection, 8 of whom were infected with HCV alone. HCV genotypes were determined in all 8 patients who had HCV RNA; genotype 3a was the most common (62.5%). These results demonstrate that, in Thailand where both HBV and HCV are endemic, HBV infection is still the most important risk factor for HCC, but HCV also has an important role in those without HBV infection. In addition, the genotypic distribution of HCV in HCC in Thailand is similar to that in the general population. No specific association between genotype 1b and HCC was observed.

Multiple replicons constitute the 6.5-megabase genome of Burkholderia pseudomallei.

Burkholderia pseudomallei is a causative agent of melioidosis, a fatal tropical infectious disease endemic in Southeast Asia and Northern Australia. In order to determine the size and characteristics of the bacterial genome, the B. pseudomallei genome and genes were analyzed by pulsed field gel electrophoresis of the undigested, intact megabase DNA, and by computational analysis of nucleotide sequences of B. pseudomallei genes which have been sequenced by several investigators and already deposited in a public database. The results showed that the B. pseudomallei genome consists of two large replicons, and that both contain ribosomal RNA gene sequences, indicating the presence of two chromosomes. The classical arabinose-negative B. pseudomallei isolate K96243 has chromosomes of approximately 3563 +/- 73 and 2974 +/- 40 kilobase-pairs in size, giving a total genome size of about 6.5 million base-pairs. The arabinose-positive nonvirulent biotype of B. pseudomallei also has two replicons which are smaller than those of the arabinose-negative biotype. Analysis of the publicly-available nucleotide sequences showed that the average B. pseudomallei gene is approximately 1031 base-pairs in size, with an average G + C content of 65.7%. The genome is gene-rich and about 89% of the coding capacity is used as coding sequences. It can therefore be estimated that the entire B. pseudomallei genome encodes about 5600 genes.

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.

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.

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.

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.

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.

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.

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.

Fusion protein of Salmonella typhi flagellin as antigen for diagnosis of typhoid fever.

We previously established the specific 52 kDa antigen of Salmonella typhi, detected by our monoclonal antibodies, which was a flagellin protein. Comparison of the nucleotide sequences of phase-1 flagellin of Salmonella species available through GenBank database showed high homology at both ends of the genes with lower degree of homology in the middle portion which contained the antigenically variable regions. Thus, proteins from the central regions of flagellin genes should be species specific and could be used as specific antigens for the immunodiagnostic tests. In this report, recombinant protein derived from the central region of S. typhi flagellin was produced as a fusion protein with glutathione-S-transferase. This fusion protein was used as specific S. typhi antigen for the immunodiagnostic test to detect IgM antibodies in sera using enzyme-linked immunosorbent assay. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of this test were 53.5, 98.0, 91.5, 82.1 and 92.4%, respectively.