PCR for HBV, HCV and HIV-1 experiences and first results from a routine screening programme in a large blood transfusion service.

We adapted the PCR method to screen up to 3000 blood donations per day for hepatitis B, hepatitis C and HIV-1 virus contamination. Up to 600 aliquots (average 418 donations) are pooled by using an automatic sample processor with disposable tips (validated to avoid contamination) taken from blood donations which are serologically negative and free for clinical usage according to federal regulations. In the case of a positive PCR pool result the viraemic donation is identified by two additional PCR pools testing steps with smaller pool sizes. All of the steps are supported by electronic data processing. After virus concentration by ultracentrifugation, and in the case of HCV and HIV-1 an additional reverse transcription step, PCR amplifications are performed. PCRs are done for each virus in two genomic regions. Laser-induced detection after PAGE and computer-analysis are used to identify the amplification products. Using this validated methodology routine we have checked 428 896 donations up to the end of August 1996. During this survey we found at least 24 viruses-containing donations which were negative in corresponding serological tests and would have been transfused (2 HBV-, 22 HCV-, 0 HIV-1 -containing donations). It seems possible for large transfusion centres to shorten the diagnostic window periods with our PCR-methodology with acceptable costs (15 DM per donation for all three viruses including logistics, developments and investments).

[Routine PCR screening for HBV, HCV and HIV-I genome in a large blood donation services--experiences and initial results]. / PCR-Routine-Screening auf HBV-, HCV- und HIV-I-Genom in einem grossen Blutspendedienst--Erfahrungen und erste Ergebnisse.

We adapted the PCR method to screen up to 3,000 blood donations per day for HBV, HCV, and HIV-1 contamination. Concerning logistics: The first step is the generation of 3 identical microtiter plates (PT) by using the self-validated automatic sample processor with disposable tips. Using the first PT, we pooled up to 600 aliquots taken from blood donations which are serological negative and free for clinical usage according to actual federal regulations. In the case of a positive PCR pool result the viremic donation is identified by 2 additional PCR pool testing steps with smaller pool sizes using the second and third PT. All described steps are supported by electronic data processing. The PCR-method: After virus concentration by ultracentrifugation and--in case of HCV and HIV-1--additional reverse transcription PCR-amplifications were performed. PCR in two genomic regions are done for each virus. Laser-induced fluorescence detection after polyacrylamide gel electrophoresis and computer analysis were used to check the amplification products. Using this approach, a virus-containing donation can be detected in up to 599 negative samples with following sensitivity: HBV and HIV-1, 1,000-1,500 genome equivalents/ml; HCV, 2,000-2,500 genome equivalents/ml, thus sensitivity being in the range of commercial available PCR kits when testing nonpooled samples. The sensitivity was validated by using national and international available standards with known virus genome concentrations. All processing steps are checked using different controls such as, e.g., negative, positive, premix controls, reporter virus, inhibition controls. Routinely employing this validated methodology, we investigated 327,013 donations until the end of June 1996. During this survey, we found at least 16 virus-containing donations which are negative in corresponding serological tests and would have been transfused (4 HBV-, 13 HCV-, 0 HIV-1-containing donations), including one later seroconversion for HBV and one for HCV. Using our adapted PCR-methodology, it seems possible to shorten the diagnostic window periods with acceptable costs for large transfusion centers (15 DM per donation for all 3 viruses including all steps and investments). Therefore, PCR seems to become a new method of choice to prevent transfusion transmitted infections.

[Sensitivity studies on the demonstration of HIV-1 particles using RT HIV-1 PCR in pooled plasma before virus inactivation]. / Sensitivitätsstudien zum Nachweis von HIV-1-Partikeln mittels RT-HIV-1-PCR im Poolplasma vor Virusinaktivierung.

The goal of this study was the establishment of a reverse-transcription polymerase chain reaction (PCR) test and the evaluation of its sensitivity to detect an HIV-1 contamination in pooled plasma samples prior to the solvent-detergent (SD) inactivation procedure. Pooled plasma samples were spiked with known concentrations (1,000-0.1 TCID50/ml) of HIV-1, originated from tissue culture supernatants. Unspiked plasma samples were used as negative controls. After reverse transcription, a PCR in 4 different HIV-gene regions (gag, pol, env, ltr) followed by a hybridisation with specific probes was done. The achieved sensitivity in 3 tests (pol. gag, ltr) was 0.1 TCID50/ml and 1.0 TCID50/ml in the env-PCR system. It was shown in previous studies that by inactivation of pooled plasma with the SD technique a reduction of HIV-1 infectivity greater than 10(6) is achieved. The combination of PCR testing and the SD inactivation procedure makes it possible for the first time to define the maximum amount of contaminating HIV-1 in case of a negative PCR result. According to this procedure, the residual HIV-1 load of virus-inactivated plasma would not exceed 1 TCID50 per 1,000 litres at worst.

Relevance of hepatitis B DNA detection in patient's serum.

Detection of HBV-DNA showed a satisfactory sensitivity (6 pg/ml) and high specificity: 15 patients with hepatitis A, 6 with CMV, 7 with EBV infection, 81 with hepatitis NANB as well as 174 persons with isolated HBc antibodies and 9 hepatitis B vaccinees gave negative results. However, out of 118 persons with a history of hepatitis B (HBs and HBc antibodies positive), one repeatedly had a positive result. We followed 58 patients with chronic hepatitis B, 25 healthy antigen carries, and 16 HBV-infected dialysis patients over an extended period with multiple blood sampling. Most of those patients showed changes of detectability of HBV-DNA. Typically, periods with positive DNA tests were followed by intervals with negative tests which could last for up to a few years. 75% of dialysis patients were positive in all sera available, while 48% of healthy antigen carriers were consistently negative. Additionally, 9 patients with a history of recent hepatitis B and a positive HBs antibody test as well as 4 patients with simultaneous infection by A and B were tested for HBV-DNA. In none of these two groups, a variation from the pattern usually found in hepatitis B was established. A case of intrauterine infection with hepatitis B virus is reported. Because the blood of the mother was free from HBV-DNA and HBe antigen at delivery and because the hepatitis B immunoglobulin prophylaxis failed in the child who developed HBs antigen very soon after delivery, it has been concluded that the child was infected in utero. The highest values of HBV-DNA determined quantitatively were found in dialysis patients which explains the high risk of acquiring hepatitis B in the dialysis unit. The results from earlier studies on the prognostic relevance of HBV-DNA and the risk of medical personnel as a source of infection with hepatitis B are discussed briefly.

Infectivity of medical staff for hepatitis B.

Prior to hepatitis B vaccination, 36,000 persons of the medical staff were tested for HBs antigen, HBc antibodies, and HBs antibodies. 210 sera were found positive for HBs antigen and HBc antibodies. Of these sera, 171 were available for testing for hepatitis B virus DNA as a marker of infectivity by spot hybridization. DNA was detected in only 15. One hundred and thirty-nine had HBe antibodies but no detectable HBe antigen, and only two of these were hepatitis B virus DNA positive. 12 had neither HBe antigen nor HBe antibodies and none had hepatitis B virus DNA. Hepatitis B virus DNA was, however, detected in 13 of 20 HBe antigen-positive but HBe antibody-negative sera. Our study confirms epidemiological observations that medical staff hardly plays any role as a source of HBV infection for patients.

[Viral nucleic acids in the serum of hepatitis B patients]. / Virale Nukleinsäure im Serum von Hepatitis-B-Patienten.

Detection of hepatitis B viral DNA (HBV-DNA) is the most reliable test for infectivity of a patient's serum. HBV-DNA was detected by spot hybridization on nylon membrane. HBV-DNA was detected during the first or second week of jaundice in 22/133 (16.5%) sera from patients with acute hepatitis who did not develop chronic disease later on. However, in patients with acute hepatitis who later on developed the chronic form, HBV-DNA was found in 12/12 (100%) cases in the first or second week after onset. In other patients who had chronic hepatitis for longer than one year HBV-DNA was detected in 92/113 (81.4%) sera when HBe antigen was also detectable and in 11/104 (10.6%) when HBe antibodies were found. HBV-DNA was present in 32/38 (84.2%) sera from healthy antigen carriers when HBe antigen was detected and only in 2/173 (1.2%) sera of HBe antibody positive HBs antigen carriers. The highest levels of HBV-DNA were found in patients with chronic hepatitis B infection who were found in patients with chronic hepatitis B infection who were on haemodialysis therapy. Almost all had HBe antigen and HBV-DNA was also detectable in 82/84 (97%) sera. 11/28 (39.3%) sera from patients with chronic hepatitis B as well as HIV infection showed HBV-DNA. Detection of HBV-DNA has significantly improved the accuracy of diagnosis of hepatitis B viral infection.

Detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay, indirect immunofluorescence, and virus isolation: a comparative study.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of respiratory syncytial virus (RSV) antigens in nasopharyngeal secretions (NPS) from children with acute respiratory disease. Antisera against RSV nucleocapsids were used as immunoreagents for this test system. The results obtained by RSV antigen ELISA were compared to those of indirect immunofluorescence (IF) and tissue culture virus isolation (TC). Of the 404 NPS obtained, 278 were tested in parallel by ELISA and IF and 205 by ELISA and TC, and 89 were screened in parallel by all three methods. The sensitivity of ELISA in relation to IF was 86.7%, the specificity 95.7%. Sensitivity and specificity obtained by ELISA were 89.9% and 94.4%, respectively, compared to TC. False-negative results were obtained with all three test systems used.

A model study of the use of monoclonal antibodies in capture enzyme immunoassays for antigen quantification exploiting the epitope map of tick-borne encephalitis virus.

On the basis of an epitope model, capture enzyme immunoassay systems using monoclonal antibodies have been devised for the detection and quantification of Tick-borne encephalitis virus and compared with a reference system employing polyclonal sera. Monoclonal antibodies were used both as capture and detector antibodies, their suitability depending primarily on their avidity and intrinsic background activity. A considerable increase in sensitivity was achieved by combining antibodies to different non-overlapping epitopes. Biotinylation of the detector antibodies allowed the construction of multiple site simultaneous binding assays. Furthermore the use of monoclonal antibodies of defined serological specificity made virus type identification possible. This assay can therefore be used as a rapid 'test of identity' as required during the manufacture of viral vaccines.

Immunogenicity of tick-borne encephalitis virus glycoprotein fragments: epitope-specific analysis of the antibody response.

After digestion with trypsin, alpha-chymotrypsin, or chemical cleavage using CNBr, fragments of the tick-borne encephalitis (TBE) virus glycoprotein were isolated which retained their reactivity with neutralizing monoclonal antibodies defining a denaturation-resistant antigenic domain. Upon immunization of mice, these fragments induced antibodies reactive with the immunizing peptide, the denatured glycoprotein and the native glycoprotein as a constituent of the whole virus. The immune sera revealed the same properties as the monoclonal antibodies that were used to select the fragments for immunization: neutralizing activity; haemagglutination-inhibiting activity; blocking of the binding of antibodies used for selection; enhancement of the binding of other monoclonal antibodies defining a denaturation-sensitive antigenic domain. It was shown that the natural immune response against certain functionally important, denaturation-resistant immunogenic domains on the native protein can be closely mimicked by immunization with defined protein fragments. Antigenic sites present on these fragments may therefore represent essential constituents of a synthetic vaccine. The fine specificities of antibody populations in anti-peptide or anti-protein immune sera were analysed on the basis of single antigenic determinants by blocking assays using radiolabelled monoclonal antibodies that define eight distinct epitopes on the TBE virus glycoprotein. Quantitative differences in the blocking of certain monoclonal antibodies were also observed between human convalescent sera. The establishment of such blocking profiles using a panel of well-characterized monoclonal antibodies may represent a general method for dissecting the specificities of antibody populations present in polyclonal immune sera and could allow investigations on determinant-restricted differences of immune responses and its possible implications for the course of the disease.

Antibody-induced conformational changes result in enhanced avidity of antibodies to different antigenic sites on the tick-borne encephalitis virus glycoprotein.

By the use of monoclonal antibodies we have recently defined eight distinct epitopes on the structural glycoprotein of tick-borne encephalitis (TBE) virus which differ with respect to location, function, or serological specificity (Heinz et al, Virology 126, 525-537, 1983). The present investigation reveals a complex network of interactions between antibodies directed against distinct nonoverlapping epitopes leading to enhanced binding of certain antibodies in the presence of bound second antibodies. The enhancement between antibody pairs can be either unidirectional or bidirectional. In addition, there are domains of predilection, which bind enhanceable antibodies (domain A) whereas others bind antibodies which preferentially induce enhancement (domain B). These domains represent structurally unrelated entities, domain A being sensitive to denaturation and fragmentation and domain B being resistant. Quantitative evaluation of binding data by Scatchard analysis revealed that the observed enhancement phenomenon is due to a two- to sixfold increase of antibody avidity. In the system described, enhancement of antibody binding is not dependent on antibody bivalency since it could also be demonstrated with purified Fab fragments acting either as enhanced or as enhancing antibody. It is therefore concluded that binding of antibodies to certain epitopes on the TBE virus glycoprotein induces conformational changes in distant parts of the molecule which can result in increased avidity of antibodies directed to conformationally changed epitopes. A possible explanation for the origin of this enhancement phenomenon is presented.

Location of immunodominant antigenic determinants on fragments of the tick-borne encephalitis virus glycoprotein: evidence for two different mechanisms by which antibodies mediate neutralization and hemagglutination inhibition.

A model showing the topological distribution, functions, and serological specificities of eight distinct, monoclonal antibody-defined epitopes on the tick-borne encephalitis (TBE) virus glycoprotein has been presented in a previous publication (F. X. Heinz, R. Berger, W. Tuma, and Ch. Kunz (1983). Virology 126, 525-537.) In the present report the influence of conformational change, chemical modification, and fragmentation on the antigenic reactivity of each epitope has been analyzed by the use of blocking enzyme immunoassays and "Western blotting." One of the two major antigenic domains (A), composed of three different epitopes, completely lost its antigenicity upon incubation at pH 5.0 or by treatment with guanidine-HCl/urea, SDS, reduction and carboxymethylation, as well as by proteolytic (trypsin, alpha-chymotrypsin, thermolysin) and chemical (CNBr) fragmentation. The second major antigenic domain (B), however, defined by four distinct monoclonal antibodies, three of which are hemagglutination (HA)-inhibiting, neutralizing, and protective, was shown to be resistant to low pH, guanidine-HCl/urea treatment, and proteolytic cleavage of the native protein. Also, polyclonal immune sera from mice and rabbits contained antibody populations reactive with antigenic determinants which are resistant and others which are sensitive to conformational change and fragmentation. Glycoprotein fragments with molecular weights of about 9000, generated by proteolysis of the native protein, were immunoreactive with neutralizing and protective monoclonal antibodies (defining domain B) as well as with a polyclonal mouse immune serum. Thus, these fragments appear to contain antigenic determinants which are immunodominant on the native protein and play an important role in the induction of a protective immune response against TBE virus. In addition, these results show that antibody binding to antigenic domains which are topologically and structurally completely unrelated may result in neutralization and/or HA inhibition. As the presence of two receptor-binding sites is unlikely, different effector mechanisms may account for the effects of these antibodies. The antigenic reactivity of domain A is sensitive to the same treatments which also inactivate HA activity of TBE virus, whereas domain B is resistant. These treatments include a change of domain A induced by incubation at slightly acidic pH which also results in inactivation of virus infectivity. Antibodies to domain A therefore presumably block viral activities by direct binding at or near the putative receptor-binding site whereas antibodies to domain B may cause loss of biological activities by inducing a conformational change of the receptor-binding site.

A topological and functional model of epitopes on the structural glycoprotein of tick-borne encephalitis virus defined by monoclonal antibodies.

A topological and functional model of eight distinct epitopes on the structural glycoprotein of tick-borne encephalitis (TBE) virus was established by the use of monoclonal antibodies. The unique specificities and spatial relationships of these antibodies were determined by variant analysis, haemagglutination inhibition (HI), neutralization, passive mouse protection, and antibody blocking assays. Seven out of the eight distinct epitopes were shown to be partially linked and to cluster in two antigenically reactive domains (A, B). Each of these domains is inhomogeneous and contains constituents with different serological specificities and functions. Domain A is defined by three HA-inhibiting antibodies, two of which are flavivirus group-reactive, whereas the third is TBE virus subtype specific. Within this domain only the subtype-specific antibody is involved in virus neutralization, thus explaining the observation that neutralization tests with flaviviruses show higher serological specificities than HI tests and that HI tests can be made type and subtype specific by antibody absorption. Domain B is composed of three TBE-complex reactive epitopes, and the corresponding antibodies inhibit HA and neutralize the virus. A fourth epitope linked to this domain is neither involved in HA nor in neutralization and the same holds true for a subtype-specific epitope which is topologically independent of domains A and B. Each of two different nonneutralizing antibodies was capable of blocking the binding of distinct neutralizing antibodies. All eight epitopes are indistinguishably present on strains of the western subtype of TBE virus isolated all over Europe in different years from different hosts, thus again confirming the great stability of this virus.

Antigenic and immunogenic properties of defined physical forms of tick-borne encephalitis virus structural proteins.

Polymeric, delipidated glycoprotein complexes of defined size and composition were prepared from tick-borne encephalitis virus by solubilization with Triton X-100 or cetyltrimethylammonium bromide, followed by centrifugation into detergent-free sucrose density gradients. The antigenic reactivities and immunogenicities of these complexes were compared with those of complete inactivated virus. These glycoprotein preparations induced hemagglutination-inhibiting and neutralizing antibodies which proved to be protective in passive mouse protection tests and monospecifically reacted only with the viral envelope and not with the internal core. In a competitive radioimmunoassay the glycoprotein complexes revealed about 10-fold higher antigenicity than whole virus when tested at equal protein concentrations. The important implications of these results with respect to antigen quantification in vaccines are discussed. As shown in the mouse challenge potency test, glycoprotein complexes prepared after Triton X-100 solubilization actively protected mice almost as well as did complete inactivated virus at the same protein concentration, whereas those prepared after cetyltrimethylammonium bromide solubilization had a somewhat lower protective activity per microgram of protein.

[Detection of hepatitis A virus by means of electron microscopy (author's transl)]. / Elektronenoptischer Nachweis von Hepatitis-A-Virus.

During an outbreak of hepatitis A in an institution, 34 stools of children were investigated by means of electron microscopy. Hepatitis A virus was found in four samples. All four children had previously received gammaglobulin injections and did not develop any clinical symptoms.

Helium-neon laser stabilized on iodine: design and performance.

A helium-neon laser operating at 633 nm was frequency stabilized on a hyperfine component of (127)I(2) with saturated absorption. The stability achieved was 2.5 x 10(-12) at an averaging time of 400 sec. The frequency was reproducible to 5 x 10(-11).