Patrice O. Yarbough

Solid-phase enzyme-linked immunosorbent assay for hepatitis E virus IgG and IgM antibodies utilizing recombinant antigens and synthetic peptides

Four recombinant antigens representing two distinct antigenic domains from two different strains of hepatitis E virus (HEV), were used individually to develop four ELISAs designed to detect antibodies to HEV. Both IgG and IgM class antibodies to HEV were detected in 7 of 8 pedigreed serum/plasma from known outbreaks of HEV in Mexico, Burma, Somalia and Pakistan. In addition, specific HEV-antibodies were detected in cynomolgus macaques following inoculation with various HEV strains. Anti-HEV was also detected in 8 of 386 (2.1%) randomly selected American blood donors. Supplemental tests utilizing both synthetic peptides and specific blocking assays provided additional serologic data confirming the presence of anti-HEV. Similar prevalence studies on a limited number of available sera from other geographical regions (Alaska, Japan, Germany, New Zealand, Thailand and Mexico) confirmed the presence of anti-HEV in at least 1.1 to 7.6% of the specimens.

Antiretroviral Activity of the Anti-CD4 Monoclonal Antibody TNX-355 in Patients Infected with HIV Type 1

BACKGROUND We wished to determine the safety and anti-human immunodeficiency virus (HIV) type 1 activity of single doses of TNX-355, a humanized IgG4 anti-CD4 monoclonal antibody with potent activity against HIV-1 in vitro, in HIV-infected subjects. METHODS Sequential cohorts of 6 HIV-1-infected subjects each received infusions of TNX-355. Data included plasma HIV-1 RNA level, CD4+ T cell count, TNX-355 coating of CD4+ T cells, and serum TNX-355 levels. RESULTS Dose-related reductions in plasma HIV-1 RNA loads correlated with complete CD4+ T cell coating by TNX-355. Peak median decreases in plasma HIV-1 RNA loads were 0.56, 1.33, and 1.11 log10 copies/mL and occurred on days 4-7, 14, and 21 for the 3.0, 10, and 25 mg/kg doses, respectively. Dose-dependent increases in CD4+ T cell count occurred within 24 h of dosing. CONCLUSIONS Single doses of TNX-355 reduced plasma HIV-1 RNA loads and increased CD4+ T cell counts in HIV-infected subjects. The further assessment of therapeutic potential awaits data from longer-duration trials.

Fulminant or subfulminant non-A, non-B viral hepatitis: The role of hepatitis C and E viruses

BACKGROUND Although non-A, non-B (NANB) viral hepatitis has been implicated as an etiology of fulminant hepatitis, hepatitis C virus (HCV) has not been shown to result in acute hepatic failure and hepatitis E virus (HEV) has predominantly been associated with fulminant hepatitis among pregnant women. METHODS Using polymerase chain reaction to detect HCV and HEV genomes, four-antigen radioimmunoblot assay (4-RIBA) to measure anti-HCV antibodies, and enzyme-linked immunosorbent assay (ELISA) to detect anti-HEV immunoglobulin M (IgM) antibodies, 17 patients with sporadic fulminant or subfulminant hepatitis of presumed NANB viral etiology were studied. RESULTS The diagnosis of acute NANB viral hepatitis was made based on clinical information, serological tests, biochemical profiles, and pathological features. All 17 patients were negative for anti-HEV IgM antibodies and HEV RNA in either serum and/or liver. HCV RNAs were detected in 2 patients although anti-HCV antibodies were negative in all of them. CONCLUSIONS It is shown that HCV is infrequently associated with and HEV is not an identifiable cause of presumed NANB fulminant or subfulminant hepatitis in this patient population. Although further studies will be required for identification of the causative agent, it is possible that another agent is responsible for the occurrence of sporadic NANB fulminant or subfulminant hepatitis.

Comparison of tests for antibody to hepatitis E virus

The results of serologic tests for hepatitis E virus have varied widely from laboratory to laboratory, making interpretation of seroepidemiologic studies difficult. The present study compares serologic results with different antigens and tests developed in two laboratories for their ability to diagnose hepatitis E and measure antibody prevalence in a high risk population in Saudi Arabia. The results confirm that tests based upon open reading frame (ORF) 3 of HEV are of limited value for seroepidemiologic studies, whereas ORF2‐based antigens have broad utility and yield data that are reproducible in more than one laboratory. J. Med. Virol. 55:134–137, 1998.

Quantitation of Immunoglobulin to Hepatitis E Virus by Enzyme Immunoassay

ABSTRACT We developed a quantitative enzyme immunoassay (EIA) for antibody to hepatitis E virus (HEV) by using truncated HEV capsid protein expressed in the baculovirus system to improve seroepidemiology, to contribute to hepatitis E diagnosis, and to enable vaccine evaluations. Five antigen lots were characterized; we used a reference antiserum to standardize antigen potency. We defined Walter Reed antibody units (WR U) with a reference antiserum by using the four-parameter logistic model, established other reference pools as assay standards, and determined the conversion factor: 1 WR U/ml = 0.125 World Health Organization unit (WHO U) per ml. The EIA performed consistently; median intra- and intertest coefficients of variation were 9 and 12%, respectively. The accurate minimum detection limit with serum diluted 1:1,000 was 5.6 WR U/ml; the test could detect reliably a fourfold antibody change. In six people followed from health to onset of hepatitis E, the geometric mean antibody level rose from 7.1 WR U/ml to 1,924.6 WR U/ml. We used the presence of 56- and 180-kDa bands by Western blotting as a confirmatory test and to define true-negative and -positive serum specimens. A receiver-operating characteristics plot identified 30 WR U/ml as an optimum cut-point (sensitivity, 86%; specificity, 89%). The EIA detected antibody more sensitively than a commercially available test. The EIA was transferred to another laboratory, where four operators matched reference laboratory results for a panel of unknowns. Quantitation of antibody to HEV and confirmation of its specificity by Western blotting make HEV serology more meaningful.

Hepatitis E virus (HEV): The novel agent responsible for enterically transmitted non-A, non-B hepatitis

SummaryA normally endemic form of viral hepatitis is the cause of major epidemic outbreaks in developing countries. This disease has a global distribution and has been referred to as water-borne, epidemic or enterically transmitted non-A, non-B hepatitis (ET-NANBH). Although the fecal-oral route of transmission predominates, person-toperson routes of exposure were also suggested in some epidemiologic studies. The disease has been documented as having an extremely high mortality in pregnant women (~20%). Sporadic cases of ET-NANBH, as well as imported travel exposures, have been reported in developed countries. Molecular cloning was hampered by the lack of a tissue culture system for virus propagation, however, an available animal model and a newly developed non-specific amplification procedure were used to clone and identify an exogenous cDNA (ET1.1) from a Burma-isolate infected animal. Molecular clones were also identified by immunoscreening of a cDNA library made from a fecal specimen collected from a Mexican outbreak of ET-NANBH. The isolation and sequencing of a set of overlapping cDNA clones had led to the recognition that this form of hepatitis is caused by a virus unlike any of the other viral hepatitis agents. The molecular characterization of HEV will lead to important pathobiologic insights and hasten the development of potentially useful diagnostic and therapeutic products for ET-NANBH.

Detection of hepatitis E virus in genome and gene products in two patients with fulminant hepatitis E

Non-isotopic in situ hybridization (digoxigenin-labeled probe directed towards hepatitis E virus ORF1) and immunohistochemistry (against hepatitis E virus ORF2 and ORF3) were applied to detect hepatitis E virus genome and gene product in the liver tissue of two patients with fulminant hepatitis E seropositive for hepatitis E virus RNA. Both hepatitis E virus RNA and hepatitis E virus antigens were detected exclusively in the cytoplasm of hepatocytes and not detected in other cell types. In both patients, more than 50% of the hepatocytes were positive for both hepatitis E virus RNA and hepatitis E virus antigens, most of which showed degenerative changes. This is consistent with the histological appearance of marked loss of hepatocytes with acinar collapse. Interestingly, denaturation of the RNA before in situ hybridization was found to enhance hepatitis E virus RNA detection. We conclude that: (1) hepatitis E virus RNA and hepatitis E virus antigens can be demonstrated in the liver in hepatitis E virus-related fulminant hepatitic failure, (2) hepatitis E virus is hepatocyte-tropic within the liver, (3) cytoplasmic localization of hepatitis E virus RNA and hepatitis E virus antigens is consistent with cytoplasmic replication, and (4) the presence of degenerative changes in hepatitis E virus positive cells, together with the histological appearance of hepatocyte loss in the absence of significant inflammatory infiltrate, suggests that hepatitis E virus-related fulminant hepatitic failure is mediated by a cytopathic mechanism.

Assay Development of Diagnostic Tests for Hepatitis E

The development of convenient, sensitive, and specific diagnostic tests to investigate the serology of hepatitis E virus (HEV) infection will be crucial to the understanding of the immune response to this virus. Previously, we reported the identification of two cDNA clones from the 3′ end of open reading frame (ORF) 2 and 3 encoding specific HEV epitopes. We report here the development of an ELISA test to detect IgM and IgG antibodies to HEV based on these epitopes. Also contained within ORF2, is a broadly reactive epitope region (SG3) that has been useful in identifying previously undetected antibodies to HEV. In developing countries, serologic tests suggest that l%–2% of the population have been exposed to the virus. In countries where HEV is endemic, 7%–17% of the population have measurable antibodies to HEV. In cases of acute hepatitis E, IgM and IgG antibody responses have been confirmed by PCR amplification of the virus in the sera.