Development and evaluation of a real-time RT-PCR assay for quantification of cell-free human immunodeficiency virus type 2 using a Brome Mosaic Virus internal control.

Quantification of cell-free virus in plasma is important for monitoring disease progression and for assessing the response to antiretroviral therapy in both human immunodeficiency type 1 and type 2 (HIV-1, HIV-2) infections. Although commercial assays suitable for HIV-1 quantification have been used for more than a decade, no commercial assays are yet available for the measurement of cell-free HIV-2. We have therefore developed a novel real-time RT-PCR assay which, unlike previously described 'in house' assays, incorporates a Brome Mosaic Virus (BMV) internal control to minimise the risk of generating false-negative or falsely low results due to unrecognised problems with viral RNA purification, cDNA synthesis or PCR amplification. The assay has a dynamic range of >5 log10, detects the clinically important HIV-2 subtypes A and B with high sensitivity and shows no cross reactivity with HIV-1. The 95% detection limit is approximately 100 HIV-2 RNA copies/ml and both the inter-assay and intra-assay variability are low (CV% at 1.8 x 10(5) copies/ml, 13.3% and 5.7%, respectively). Overall, plasma HIV-2 RNA was detected in 38% of 167 unselected HIV-2 antibody-positive samples analysed over a 2 year period. The assay described provides an ideal system for studying viral replication in HIV-2 infected patients and for monitoring antiretroviral therapy.

Evaluation of the role of real-time PCR in the diagnosis of invasive aspergillosis.

Recently, two developments relating to the diagnosis of invasive aspergillosis (IA) have occurred. First, the standardisation of criteria for determining the category of this disease according to the European Organisation for Research and Treatment of Cancer/Mycosis (EORTC) Study Group consensus definitions has allowed comparison of results from different studies to be undertaken. The second development is the generation of PCR assays based on real-time technologies that are able to quantify Aspergillus DNA. In this review the benefits and limitations of these new published assays are compared with nested-PCR assays and enzyme immuno assays. Results from studies where these real-time assays have been used and patient's infections were classified according the EORTC definitions are examined. The effect of anti-fungal treatment is noted. The requirement for both international standards and a consensus protocol that is sensitive enough for IA diagnosis, particularly in blood, is discussed.

Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control.

Quantitation of circulating hepatitis B virus (HBV) DNA is important for monitoring disease progression and for assessing the response to antiviral therapy. Several commercial and 'in house' assays for HBV DNA quantitation have been described but many of these have limitations of relatively low sensitivity and limited dynamic range. This study describes the development and evaluation of a FRET-based real-time PCR assay designed to overcome these limitations and to provide accurate quantitation of DNA from all eight genotypes of HBV (A-H). The assay employs a fully automated nucleic acid extraction system permitting high-sample throughput with minimal 'hands-on' time and incorporates a murine cytomegalovirus (mCMV) internal control to prevent false negative results and under-reporting due to unrecognised problems with viral lysis, DNA purification or PCR amplification. Sensitivity, assessed by Probit analysis at the 95% detection level, was 24.4 IU/ml, associated with an extremely wide dynamic range (approximately 9 log10). Coefficients of variation were low for both intra-assay and inter-assay variability (CV%, 7-11%) and quantitative data correlated well (R2 = 0.97) with the Digene hybrid capture assay. This assay provides an ideal system for therapeutic monitoring and for studying the relationship between HBV viral load and stage of disease.

HIV replicates in cultured human brain cells.

Adherent human embryo brain cells have been infected with HIV. Cells replicating HIV were maintained in culture for seven sequential passes over 7 months and continued to produce HIV during that time. Human embryo brain cells displayed glial-cell morphology and expressed glial fibrillary acidic protein. Electron microscopy showed clusters of virus particles around these cells as well as budding virus. Extracted, infected glial cells revealed bands for three major gag proteins, p18, p24 and p55, in Western blotting. It was not possible to detect CD4 antigen on the surface of these cells by indirect immunofluorescence or alkaline phosphatase staining with CD4 monoclonal antibodies. The results of these experiments indicate that HIV replicates in non-malignant brain cells. This observation strengthens the postulated aetiological link between HIV and the encephalopathy, dementia and other neurological symptoms observed in HIV-infected patients.

Epitope location of 13 anti-gag HIV-1 monoclonal antibodies using oligopeptides and their cross reactivity with HIV-2.

A series of 15-mer oligopeptides which overlapped by five amino acids (AA) across the p24 of HIV-1SF2 and a similar series across the p18 of HIV-1SF2 were used to identify the locations of 13 anti-gag monoclonal antibodies (MAbs). Three anti-p24 MAbs recognized sequences within the first 50 AA of the amino-terminal. Another anti-p24 recognized a conformational epitope in the centre of the protein and this MAb cross-reacted with two HIV-2 isolates suggesting conservation of this epitope between HIV-1 and HIV-2. One anti-p24 MAb recognized a linear sequence in the carboxy-terminal 100 AA and one p24 antibody was assumed to recognize a truly conformational epitope as it did not react with any of the linear peptides. Four anti-p18 MAbs were located at the carboxy-terminus of p18 with another MAb mapping slightly inwards from the carboxy-terminus and one anti-p18 MAb failed to bind to the p18 peptides. The carboxy-terminal distribution of the p18 MAbs indicated a highly immunogenic nature for this region in mice. None of the anti-p18 MAbs showed cross-reactivity with HIV-2 isolates, confirming the greater sequence variability of p18 over p24.

Monoclonal antibodies define linear and conformational epitopes of HIV-1 pol gene products.

Purified recombinant reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) was used to raise 21 monoclonal antibodies with anti-RT specificities. The antibodies were characterized using Western blotting against native virus and recognized either the p66 or p66, p51 components of RT. Further immunoblotting using either cyanogen bromide fragmented RT or truncated mutants of RT along with cross-competition studies enabled the location of various immunogenic regions of RT to be identified. Three antibodies recognized a linear epitope in the N-terminal region (amino acids 128-176). Also, a neutralizing RT antibody recognized a conformational epitope in this region. Three monoclonals had epitopes mapped to linear sequences in the RNase H region at the C-terminus of the RT. Another neutralizing antibody, also requiring folding of the RT protein had its epitope more centrally located (231-353). Of the remaining 13 monoclonals, 7 were roughly located in the C-terminal region and required folding of the protein for epitope recognition and only three of the remaining six could be mapped to conformational epitopes in N-terminal and central regions of the RT. None of the antibodies tested recognized HIV-2 RT products p68 and p55 in Western blot.

Detection of both hepatitis B e antigen and antibody in a single assay using monoclonal reagents.

Monoclonal antibodies raised against HBeAg were used to develop a HBeAg and anti-HBe detection assay. Monoclones containing anti-HBe were used both for the coating of the solid phase and for the fluid phase label in a sandwich type assay. The percentage binding of 125I-labelled anti-HBe to serum HBeAg was much greater than that seen in a similar assay using only polyclonal reagents. Therefore it was possible to add a small quantity of HBeAg for neutralising any anti-HBe present in a test serum without affecting HBeAg detection. This small amount of serum HBeAg was incorporated into each test sample thus allowing the determination of the e status of a patient using only one aliquot of test serum. This single test assay could be performed either as a radioimmunoassay or as an ELISA. The sensitivity of these assays was found to be greater than the conventional polyclonal assay particularly with regard to sera containing anti-HBe.

Comparison of a monoclonal anti-HIV 1 gag solid phase with a polyclonal anti-HIV solid phase for detecting anti-HIV 1 in a competition ELISA.

An anti-HIV 1 competitive ELISA was developed using a monoclonal anti-HIV 1 gag to capture viral antigen to the solid phase. This format of assay was compared with a competitive ELISA where a polyclonal human anti-HIV 1 was used, to capture the viral antigen. Several benefits were observed using the monoclonal-antibody form of the assay. Firstly, less viral antigen was needed on solid phase to give an equivalent test in terms of positive and negative optical density 450 nm values. Secondly, a slight increase in sensitivity was also gained without any loss of specificity and finally, as a result of the murine nature of the antibody on the solid phase, no cross-reaction with the labelled human antibody in the test conjugate was observed. Such cross-linking has been observed with the polyclonal form of the assay and can lead to false-negative reactions.

Quantitation of hepatitis delta virus using a single-step internally controlled real-time RT-qPCR and a full-length genomic RNA calibration standard.

Quantitation of circulating hepatitis delta virus (HDV) RNA is important for assessing the response to antiviral therapy and for understanding the complex dynamic interactions between hepatitis B virus (HBV) and HDV replication. Although several PCR assays for HDV RNA have been described none of them incorporate an internal control or use a full-length RNA calibration standard for absolute quantitation. This study describes the development and evaluation of a novel single-step real-time RT-qPCR assay for HDV RNA quantitation which incorporates a Brome Mosaic virus internal control to prevent false negatives and under-reporting due to inhibitors or due to inefficient RNA purification, reverse transcription or PCR amplification. The assay has a dynamic range of ≥7log(10) and is designed to detect all HDV genotypes. The 95% detection limit is ∼3800 HDV RNA copies/ml, 700 copies/ml being detectable in 20% of repeats. Both intra-assay and inter-assay variability are low (CV 8% and 17%, respectively). Plasma HDV RNA was detected in 75% of 59 HDV antibody-positive samples with titres ranging from 8.4×10(4) to 4.4×10(8) copies/ml. The assay described provides a reliable and sensitive quantitative system for therapeutic monitoring and for studying the dynamic interplay between hepatitis B virus replication and HDV viral load.

Monoclonal antibodies to hepatitis Be antigen (HBeAg) derived from hepatitis B core antigen (HBcAg): their use in characterization and detection of HBeAg.

A panel of mouse hybridomas secreting monoclonal antibody to serum hepatitis Be antigen (HBeAg) was produced from mice immunized with denatured hepatitis B core antigen (HBcAg). This panel could be divided broadly into two groups. Within each group, the monoclonal antibodies recognized a single antigenic site, designated either e-alpha or e-beta, and generally exhibited a high degree of cross-inhibition. In contrast, between the two groups of antibodies there was little or no cross-inhibition. The antigens of serum HBeAg and denatured HBcAg appeared to be very similar. Both behaved as molecules carrying only a single e-alpha or e-beta site, in spite of native serum HBeAg having an apparent molecular weight of 300 000. It is inferred that e-alpha and e-beta sites may be involved in the polymerization of HBeAg into HBcAg and that during this process mutual masking of antigenic sites may occur.

Human and monoclonal antibodies to hepatitis B core antigen recognise a single immunodominant epitope.

Monoclonal antibodies were raised against hepatitis B core antigen (HBcAg) synthesized in Escherichia coli. They identified a single immunodominant determinant on both liver-derived and E. coli-derived HBcAg. Cross-inhibition studies demonstrated that HBsAg-containing human sera which contained antibodies to HBcAg (anti-HBc) together with either hepatitis B e antigen (HBeAg) or its antibody (anti-HBe) essentially only recognised the same single determinant as the murine antibodies. The identification of a single dominant HBcAg determinant may be important if future hepatitis B virus vaccines contain HBcAg.

Characterization of monoclonal antibodies against the human immunodeficiency virus (HIV) gag products and their use in monitoring HIV isolate variation.

Monoclonal antibodies were raised against the gag proteins of a British isolate of the human immunodeficiency virus (HIV), CBL-1. Seven of the monoclonal antibodies recognized HIV gag-coded proteins of 55,000 mol. wt. (p55) and 24,000 mol. wt. (p24), three recognized p55/p18 and three p18 alone. Cross-competition assays suggested that at least two independent epitopes exist on the p24 cleaved from the p55 precursor, whereas a cluster of closely related epitopes was recognized by the p55/p18 and p18 antibodies. The panel of monoclonal antibodies was then used to compare by immunofluorescence the expression of gag-encoded antigenic determinants on HIV isolates from different geographical locations. Minor changes in epitope expression were observed in isolates from the U.S.A. and Haiti with the most notable changes being detected in isolates from Zaire, Tanzania and Uganda.

Characterisation of a panel of monoclonal antibodies raised against recombinant HCV core protein.

Hepatitis C virus (HCV) has as yet no practical culture system so any antigen or antibody studies must be carried out using recombinant antigens. In this study, HCV core sequence was amplified by PCR, inserted into pRSET, and expressed in E. coli. The resultant core protein was purified using nickel affinity chromatography which bound the six histidine tag attached to the N-terminus of the protein. After elution in imidazole buffer, the core protein was used to immunise Balb/c mice and monoclonal antibodies against HCV core were raised. Six monoclonals were examined in a variety of assays. All of them recognised the p27 kDa protein which they were raised against and 2D2 and 3D7 recognised the core component of an HCV Recombinant Immunoblot Assay (RIBA). None of the antibodies recognised the linear peptides in an Innolia HCV assay. 2D2 showed cytoplasmic granular staining in 1-5% of cells in frozen section of HCV infected livers. Cross-competition assays between themselves and human anti-HCV core positive sera divided the antibodies into two main groups (I and II), with a sub-division of group I into a and b. Group I antibodies were unable to be inhibited by human anti-HCV sera whereas group II antibodies were inhibited by these sera (up to 62%). Epitopes recognised by all the monoclonals were probably conformational with the group I epitope being located within the first 105 amino acids of the core sequence and the group II epitope between amino acids 105 and 160.

The cloning and expression in Escherichia coli of sequences coding for p24, the core protein of human immunodeficiency virus, and the use of the recombinant protein in characterizing a panel of monoclonal antibodies against the viral p24 protein.

The sequences encoding the p24 core protein of human immunodeficiency virus type 1 were identified in a cDNA library made from infected CEM cells. The nucleotide sequence of the DNA coding for p24 was shown to be very similar but not identical to the sequences of lymphadenopathy virus and human T-cell leukaemia virus type IIIb. These sequences were expressed in Escherichia coli at the amino terminus of beta-galactosidase and used to screen a panel of monoclonal antibodies raised against virus-expressed p24. Regions containing the epitopes of five of the monoclonal antibodies were located using a series of amino- and carboxy-terminal deletion mutants of the recombinant p24 protein.

Antibody assays for varicella-zoster virus: comparison of competitive enzyme-linked immunosorbent assay (ELISA), competitive radioimmunoassay (RIA), complement fixation, and indirect immunofluorescence assays.

The competitive ELISA test was compared with competitive RIA, complement fixation, and indirect fluorescent antibody test for the detection of antibody to varicella-zoster virus (VZV). ELISA and RIA were the most sensitive tests, being tenfold as sensitive as indirect immunofluorescence and 20 times as sensitive as complement fixation, which was surprisingly inadequate for detecting anti-VZV in sera from regular haemodialysis patients. The ELISA test was found to be the most suitable assay, employing a routine complement fixation test (CFT) grade viral antigen and not requiring radiolabelling and counting facilities, which are unavailable in most virology laboratories.

Rising prevalence of human T-lymphotropic virus type III (HTLV-III) infection in homosexual men in London.

The prevalence of antibody to HTLV-III has increased from 3.7% (4/107) amongst unselected British homosexual men attending a London sexually transmitted disease (STD) clinic during one week in March, 1982, to 21% (26/124) in those attending during one week in July, 1984. Seropositive men had a significantly higher prevalence of infection with hepatitis B virus than did seronegative men. 82% (27/33) of the seropositive men in 1984 were symptomless or had only local genito-urinary symptoms referable to the STD for which they were attending. The evidence suggests that HTLV-III was initially an imported but is now an endemic sexually transmitted agent. As of July, 1984, at least 2600 homosexual men in London would probably have been infected.

The expression in Escherichia coli of sequences coding for the p18 protein of human immunodeficiency virus and the use of the recombinant protein in characterizing a panel of monoclonal antibodies against the viral p18 protein.

The sequences coding for the p18 protein of CBL-1, a British human immunodeficiency virus (HIV) type 1 isolate, were expressed in Escherichia coli as beta-galactosidase fusion proteins. The recombinant proteins were used to screen a panel of five monoclonal antibodies (MAbs) raised against p18 expressed in CBL-1-infected cells. The regions containing the epitopes for four of the MAbs were located using carboxy deletion mutants and synthetic peptides. The epitope of one of the MAbs (1D9) was reconstructed as part of an unfused, E. coli-expressed p18 protein using the polymerase chain reaction technique. Four different HIV strains and one lymphadenopathy virus type 2 strain were analysed by fluorescence-activated cell sorting of live infected cells using the p18-reactive MAbs.

Prevalence of antibody to human T-lymphotropic virus type III in AIDS and AIDS-risk patients in Britain.

2000 persons in the UK were examined serologically for antibodies to human T-lymphotropic virus type III (HTLV-III). Sera reacting in a membrane immunofluorescence assay (IFA) to HTLV-III were also positive when tested against cells infected with lymphadenopathy virus (LAV-1), and cross-adsorption tests indicated that these retroviruses are probably identical. A competitive radioimmunoassay (RIA), which was wholly concordant with IFA, was used to screen the sera. 30/31 patients with the acquired immunodeficiency syndrome (AIDS) were seropositive, as were 89% patients with persistent generalised lymphadenopathy (PGL), 17% symptomless homosexual men, 34% haemophiliacs receiving pooled clotting factors, and 1.5% intravenous drug abusers. None of more than 1000 unselected blood donors was seropositive. These data confirm the close association between HTLV-III and AIDS and PGL and show that infection with HTLV-III is also prevalent in the populations in whom these syndromes are most likely to develop. However, it would be unwise to presume that AIDS will necessarily develop in seropositive subjects.