Prevalence of human T-cell lymphotropic virus infections in Germany.

The extent of human T-cell lymphotropic retrovirus HTLV-I and HTLV-II infections in the general population in central Europe has not been investigated fully. Two hundred forty-eight thousand blood donors from southern Germany were examined serologically for antibodies to the human lymphotropic retroviruses HTLV-I and HTLV-II: 0.021% were confirmed positive and 0.056% were "indeterminate". A limited number of seropositives and "indeterminate" samples were analyzed by polymerase chain reaction (PCR): the seropositives were confirmed as positive and 43% of the "indeterminate" samples were PCR-positive. The range of 0.021% HTLV-positives in 248,000 donors, i.e. about two in 10,000 individuals, mirrors closely the published data for the United States.

[A new variant of the simian T-lymphotropic retrovirus type I (STLV-IF) in the Sukhumi colony of hamadryas baboons]. / Novyi variant obez'ian'ego T-limfotropnogo retrovirusa pervogo tipa (STLV-1F) v Sukhumskoi kolonii pavianov gamadrilov.

Polymerase chain reaction (PCR) was developed for the detection of simian T-lymphotropic virus type 1 (STLV-1) infection of P. hamadryas and direct sequencing using oligo-nucleotide primer pairs specific for the tax and env regions of the related human T-lymphotropic virus type 1 (HTLV-1). Excellent specificity was shown in the detection of STLV-1 provirus in infected baboons by PCR using HTLV-1-derived primers. The nucleotide sequences of env 467bp and tax 159bp of the proviral genome (env position 5700-6137, tax position 7373-7498 HTLV-1, according to Seiki et al., 1983) derived from STLV-1-infected P. hamadryas were analysed using PCR and direct sequencing techniques. Two STLV-1 isolates from different sources (Sukhumi main-SuTLV-1 and forest stocks-STLV-1F) were compared. Two variants of STLV-1 among P. hamadryas with different level of homology to HTLV-1 were wound (83.8% and 95.2%, respectively). A possible role of nucleotide changes in env and tax sequenced fragments and oncogenicity of STLV-1 variants is discussed.

Production of human cloned antibodies specific for hepatitis D virus-encoded small and large protein.

Cloned antibodies to specific epitopes of hepatitis D virus were produced by transformation with Epstein-Barr virus and subsequent cloning of peripheral blood B lymphocytes from a patient with chronic hepatitis D virus infection. Several stable cloned B cell lines, derived from two parent cultures, produced hepatitis D-virus-specific IgG antibodies. Some cloned IgG antibodies detected hepatitis D virus-associated antigen in hepatitis D virus-infected woodchuck liver tissue sections by indirect immunofluorescence staining and some reacted in an inhibition ELISA test detecting hepatitis D virus antibodies; most cloned IgG lines detected hepatitis D antigen both in immunofluorescence tests and in inhibition ELISA. Cloned antibodies to hepatitis D antigen detected by ELISA and/or immunofluorescence staining recognized the two major specific native and denatured polypeptides, p27 and p29, in Western blot analysis. Such cloned antibodies for hepatitis D virus are potentially useful for clinical diagnosis and research.

The immune response to different doses of inactivated hepatitis A vaccine.

The immunogenicity and reactogenicity of different doses of hepatitis A vaccine was studied in healthy adult volunteers. Vaccinees (105) were immunized with 6.25, 12.5 or 25 ng of HAV antigen, each dose administered at 0, 1 and 6 months (groups B, C and D); one group (group A) obtained three 6.25 ng doses at 0, 1 and 2 months. After one single dose high seroconversion rates ranging between 63 and 85% were observed in all four groups. All participants had seroconverted after the third dose, irrespective of the antigen content per dose and the vaccination schedule. Geometric mean titers after three doses were 439 IU/l (group A, month 3) and 1492, 963 and 2772 IU/l in groups B, C and D at month 7. One year after the first injection all vaccinees tested still showed antibody levels well above 10 IU/l. The vaccine was very well tolerated. Minor localized symptoms were observed mainly such as slight pain at the injection site. These symptoms were not dose related; no serious side effects occurred.

Principal neutralizing domain of HIV-1 is highly immunogenic when expressed on the surface of hepatitis B core particles.

The development of subunit vaccines against HIV requires the identification of immunologically relevant antigens and a suitable method of antigen delivery. Ideally, defined epitopes with neutralizing activity should be included in a vaccine preparation. The carrier for such peptide sequences should enhance the immunogenicity of the selected epitopes. In this study hepatitis B virus core antigen (HBcAg) was used as a carrier moiety for the principal neutralizing domain (PND, V3-loop) of HIV-1. A 25 amino acid V3-loop sequence was fused to HBcAg at various positions by genetic engineering. The resulting hybrid HBcAg/HIV polypeptides were analysed for particle formation and immunogenicity. Fusion of the PND to an internal position replacing an immunodominant antibody-binding region of HBcAg or to a C-terminally truncated HBcAg resulted in the formation of hybrid particles with biochemical and biophysical properties similar to those of wild-type HBcAg particles. Both types of hybrids are recognized by monoclonal and polyclonal antisera raised against PND peptides of various HIV-1 isolates. Hybrid particles with a C-terminal fusion but not an internal fusion are also recognized by a polyvalent anti-HBcAg serum. In both cases the V3 domain is surface accessible. Immunization of mice with hybrid particles induces an enhanced antibody response against the V3 sequence. The internal fusion is more immunogenic than the C-terminal fusion.

The monoclonal CD4 antibody M-T413 inhibits cellular infection with human immunodeficiency virus after viral attachment to the cell membrane: an approach to postexposure prophylaxis.

Infectious cellular uptake of human immunodeficiency virus (HIV) is initiated by a complex sequence of interactions between the viral envelope gp120/gp41 complex and the cellular CD4 receptor resulting in the exposure of a hydrophobic region of gp41 that mediates the irreversible fusion of the virus with the cell membrane. Here we show that viral penetration into a susceptible cell can be inhibited by the high-affinity monoclonal CD4 antibody (CD4 mAb) M-T413 even when it is added as late as 30-120 min after the initial contact of virus with the cell membrane. Inhibition of infection was assessed by monitoring cultures for 34 days after exposure to virus using four different methods simultaneously, including detection of viral DNA by PCR. The interval during which HIV remains sensitive to postbinding neutralization by CD4 mAb depends on strain of virus and type of target cell. Preparations of recombinant soluble CD4 (and the immunoadhesin CD4-IgG1) were much less efficient when compared with mAb M-T413, particularly in blocking infection by fresh HIV-1 isolates. Also cellular transmission of HIV, as determined by syncytia formation within 24 hr, was prevented by mAb M-T413 when added within 45 min of contact of infected H9 cells with uninfected C8166 cells. Together with the favorable clinical experience obtained with CD4 mAbs as immunomodulatory drugs, these data suggest that infusion of CD4 mAb M-T413 may be a therapeutic modus for immediate prophylactic intervention after occupational exposure to HIV and for prevention of intrapartum mother-to-infant HIV transmission.

Heat stability of parvovirus B19: kinetics of inactivation.

Heat inactivation of parvovirus B19 (B19) was studied in a culture of hematopoietic progenitor cells generated in vitro from peripheral human blood. After inoculating cell cultures with identical volumes of plasma (MII) containing B19 (B19-MII) heat-treated (60 degrees C) for various periods of time, a time-dependent inactivation of the input virus was determined by a decrease of viral DNA replication. No B19 DNA was detected after infection with B19-MII heat-treated for 20 min or more by Southern blot. Viral B19 protein production decreased time-dependently and was not detected after infection with samples treated for 12 min at 60 degrees C or more determined by the enzyme immunoassay. This study indicates that infectivity of B19 virus in plasma can be reduced in vitro by heat-treatment (60 degrees C). However, this does not mean that the heat treatment completely inactivated B19 virus.

Virus infections: strategies of prevention.

Different approaches can be adopted in preventing virus infections. Public health measures can reduce the risk of infection but virus-specific prophylaxis, passive and most importantly active immunization are the most effective strategies for controlling and eradicating virus infections.

Replication of parvovirus B19 in hematopoietic progenitor cells generated in vitro from normal human peripheral blood.

Erythroid progenitor cells generated in vitro from peripheral human blood in the presence of interleukin-3 and erythropoietin were infected with human parvovirus B19. B19 virus DNA replication was highest 48 to 72 h after infection, and maximum levels of B19 virus proteins were detected in culture supernatants at 72 to 96 h after infection. B19 virus propagated in vitro was infectious. This cell culture system with peripheral blood cells facilitates studies in vitro of B19 virus replication.

Detection and characterization of hepatitis C virus sequence in the serum of a patient with chronic HCV infection.

A cDNA fragment corresponding to the nonstructural gene region of Hepatitis C virus was cloned and sequenced. cDNA was obtained by reverse transcription of viral RNA extracted from serum of a German patient with chronic post transfusion hepatitis. "Nested" PCR resulted in a cDNA fragment of 345 nt. The sequence showed a homology of 96% to the American prototype HCV.

Prevention of viral hepatitis A: past, present and future.

Before hepatitis A virus (HAV) was identified, spread of hepatitis A was prevented by public health measures. The first specific, preventive measure for hepatitis A was passive protection with standard, pooled human immune globulins. Human immune globulin contained sufficient HAV neutralizing antibodies for short-term, prophylactic passive protection and for control of the spread of local outbreaks. After many unsuccessful attempts, HAV was propagated in cell cultures and the development of vaccines for active immunization began. Formalin-inactivated, whole HAV induced protective immunity, and such formalin-inactivated hepatitis A vaccines are now being evaluated in large-scale clinical trials. HAV attenuated by serial propagation in cell culture has been used for several, live, attenuated hepatitis A vaccines and results of clinical trials are reassuring. Future approaches to protection against hepatitis A are likely to include vaccination with: hybrid viruses; hepatitis A antigen-expressing, genetically-engineered bacteria; purified hepatitis A antigens produced by molecular biological techniques and incorporated into slow or pulse-releasing systems; synthetic peptides or idiotypes.

Hepatitis A vaccination: schedule for accelerated immunization.

Hepatitis A vaccine, strain HM175, was investigated for immunogenicity and tolerability in a prospective multicentre trial. The following vaccination schedules and antigen contents were evaluated: days 0 and 14 with 720 ELISA units (El.U) of antigen, days 0 and 28 with 720 El.U and days 0 and 28 with 360 El.U. In all study groups, the seroconversion rates following two vaccinations were between 95 and 100%. Higher geometric mean concentrations of antibody to hepatitis A virus (anti-HAV) were reached by the vaccine containing 720 El.U of HAV antigen. The vaccine was equally well tolerated in all groups. In addition, an abbreviated schedule, in which 720 El.U of HAV antigen was given on days 0 and 14, resulted in 100% seroconversion by day 28 and a level of anti-HAV that was substantially higher than that observed after passive immunization. This implies that such a vaccine could replace immune globulin administration if time permits.

Vaccination against hepatitis A: comparison of different short-term immunization schedules.

A total of 114 healthy young adults were immunized with hepatitis A vaccine using different vaccination schedules. Individuals received either a single dose (group 1), two doses given simultaneously (group 2), two doses at days 0 and 14 (group 3) or at days 0 and 28 (group 4), or three doses at days 0, 7 and 21 (group 5). Two weeks after a single dose, seroconversion rates between 77 and 85% were achieved (groups 1, 3, 4). All individuals immunized with two doses within two weeks (groups 2, 3, 5) had antibodies to hepatitis A vaccine (anti-HAV positive) by week 3; these participants also showed clearly higher mean anti-HAV values (geometric mean titres, GMTs) at this time than those individuals vaccinated only once. GMTs at week 8 were 560 IU/l in group 5, 236, 339 and 428 IU/l in groups 2-4 and 102 IU/l in group 1. Of participants with anti-HAV at week 8, 82 were again tested 4 months later; all were still seropositive. Ten individuals were tested during the first three weeks at 3-4 day intervals for anti-HAV immunoglobulin M (IgM); specific IgM responses were not detectable before day 10 but were present in eight of 10 vaccinees by day 14.

Detection and characterization of T-cell leukemia virus-like proviral sequences in PBL and tissues of baboons by PCR.

The "high lymphoma-prone" baboon stock (Papio hamadryas) of the Sukhumi Primate Center colony is characterized by a high prevalence of antibodies to the STLV-I/HTLV-I type of retrovirus and a high manifestation of human ATL-type (adult T-cell leukemia/lymphoma) malignancies (Yakovleva et al., this symposium). This is in contrast to other primate colonies and wild monkeys, which have low seroprevalence and very few if any ATL-type T-cell malignancies. To characterize the type of T-cell lymphoma retrovirus involved in the Sukhumi disease, a PCR (polymerase chain reaction) DNA analysis of peripheral blood lymphocytes (PBL) and of various tissues of healthy "at-risk", or ill baboons was performed. Proviral STLV/HTLV sequences were detected in all monkeys with symptoms of T-cell malignancy and/or antibodies to STLV-I/HTLV-I. For precise identification and characterization of the Sukhumi T-cell lymphoma virus, parts of the virus genome were mapped and sequenced from PCR derived fragments. A 420 nucleotide fragment of the env (gp 46) gene (analysed from 3 different DNA's) revealed 16.2% nucleotide divergence to the Japanese strain of HTLV-I and 14.8% to the Japanese strain of STLV-I including one deletion of a triplet. On the level of amino acid (a.a) sequence this revealed an exchange of 6 a.a. to STLV-I (4.3%), but only of 4 a.a. to HTLV-I (2.8%). The analysis of 120 nucleotides of the tax sequence (identical in 6 different DNAs) resulted in 5% nucleotide divergence to the HTLV-I (2.4% on the a.a. level) and 10% (7.3% a.a.) to the STLV-1. These results indicate that the Sukhumi T-cell lymphoma virus is a representative of the T-cell leukemia/lymphoma virus family, apparently more closely related to HTLV-I than to STLV-I genome. Furthermore, the infected monkeys from Sukhumi develop at a high rate a T-cell malignancy not observed among other baboons carrying STLV.

Prevalence of hepatitis C virus infections in dialysis patients and their contacts using a second generation enzymed-linked immunosorbent assay.

The prevalence of antibodies to the hepatitis C virus (HCV) was determined in 498 hemodialysis patients from three german dialysis units, 121 staff members and 42 family members using an enzyme-linked immunosorbent assay (ELISA) of the second generation which detects antibodies to a structural (C22) and to non-structural (C33c, C100, 5-1-1) recombinant antigens to HCV. Using the second generation ELISA 115 patients (23.1%) were anti-HCV positive versus 77 (15.5%) when sera were tested by an ELISA of the first generation containing only a non-structural antigen (C100). In 34 of these 40 discordant sera antibodies against at least one viral protein was found by a recombinant immunoblot assay. Of 5 sera containing antibodies to only one viral protein (C22) 3 were HCV RNA positive by polymerase chain reaction. Epidemiological evaluation of the patients revealed that the prevalence of anti-HCV was correlated to the duration of dialysis but not to the number of blood transfusions. Of 121 staff members 2 (1.6%) and 2 of 42 family members (4.7%) were positive indicating a low risk of the patients' contacts of acquiring HCV infection.

Long-term persistence of hepatitis C virus antibodies in a single source outbreak.

The occurrence of antibodies to hepatitis C virus (HCV) was investigated in 81 patients who developed hepatitis non-A, non-B (HNANB) after parenteral administration of contaminated immunoglobulin to prevent Rh sensitization. Sera from 74 of the 81 patients (89.9%) were anti-HCV positive at either 6-12 months or 9-10 years after administration of immunoglobulin. Sera were not available from any patients at either of the times: however, 52 of 56 sera (92.9%) were anti-HCV positive 6-12 months after use of immunoglobulin, and anti-HCV was present in 45 of 65 sera (69.2%) 9-10 years after immunoglobulin treatment. Of the latter, only two of 13 (15.4%) sera from patients who recovered from hepatitis were anti-HCV positive, whereas 43 of 52 patients (82.7%) with chronic disease were anti-HCV positive. The ELISA using a recombinant antigen was found a good detector as marker for a HCV infection because 90% of patients infected by a common source became anti-HCV positive. However, 10 years after infection most patients who did not develop chronic disease no longer had detectable antibodies.

[Blood picture findings in children with Parvovirus B19 infections (fifth disease/erythema infectiosum)]. / Blutbildbefunde bei Kindern mit Parvovirus B19-Infektion (Ringelröteln/Erythema infectiosum).

The human parvovirus B19 provokes erythema infectiosum ("e.i."); moreover there is a wide range of diseases due to parvovirus B19 without exanthema/rash. The erythropoietic blast cells of the bone marrow seems to be the main target cells for this virus. Therefore in cases of prenatal infection the consequences are extremely similar to fetal erythroblastosis ("non-immunological" fetal hydrops). In postnatal life the parvovirus B19 infection causes hyporegenerative phases of the erythropoiesis with anaemia after 3-4 weeks. We studied the white blood cell count (WBC), erythrocytes and thrombocytes in children suffering from (serologically well documented) parvovirus B19 infection with exanthem/"e.i." (group 1; n = 23), without exanthem (group 2; n = 46) and with unknown febrile exanthematous rashes (group 3; n = 76). We did not find any characteristic data in the WBC for a diagnosis of parvovirus B19 infection. However we have for the first time documented a significant thrombocytopenia in "e.i." (group 1) not found in group 2. The thrombocytopenia appears earlier than the anaemia, because the lifespan of thrombocytes is considerably shorter than that of erythrocytes. These data suggest that parvovirus B19 attacks not only "erythropoietic" blast cells but also immature bone marrow cells, which are later responsible for the thrombocytopoiesis.

[Obstructive respiratory tract diseases in children and Parvovirus B19 infections]. / Obstruktive Respirationstrakterkrankungen bei Kindern und Parvovirus B19-Infektion.

Acute respiratory diseases (ARD) due to parvovirus B 19 infection can be observed relative frequently in children. In 21 children (infants, toddlers and school children) we have seen acute or prolonged obstructive bronchitis/bronchiolitis (15 infants), acute subglottic laryngitis (3 toddlers) and acute asthmatic attacks (3 children of school age) in connection with parvovirus B 19 infection. Other respiratory viruses (adeno-, influenza, parainfluenza and RS-virus) could be excluded as agents causing the ARD. We suggest that parvovirus B 19 can provoke ARD with obstructive ventilatory disturbances of the upper or lower airways in children with a specific endogenous predisposition (small or unstable bronchial walls, or bronchial or tracheal mucosal hyperreactivity).

Productive infection of in vitro generated haemopoietic progenitor cells from normal human adult peripheral blood with parvovirus B19: studies by morphology, immunocytochemistry, flow-cytometry and DNA-hybridization.

Parvovirus B19 exerts a highly selective cytopathic effect on erythroid progenitor cells. Studies so far on the pathogenesis of B19-infection have been performed using bone marrow samples providing large amounts of erythroid progenitor cells. Extensive study, however, has been hampered by the limited access to bone marrow samples. We have designed a liquid culture method allowing the generation of large numbers of erythroid progenitor cells, initiating cultures with CD3- and CD14-poor peripheral blood mononuclear cells. Following a 12 d preincubation in liquid cultures containing recombinant human interleukin 3 (rhIl-3) and recombinant human erythropoietin (rhEpo), cells harvested from the liquid cultures were exposed to B19-containing plasma, followed by a further cultivation in liquid culture for up to 96 h. Cells expressing the CD13 and the glycophorin A (GlyA) antigens, respectively, were monitored sequentially by flow-cytometry, demonstrating a selective inhibition of GlyA-positive cells following B19-inoculation. Typical morphological changes were observed on cytocentrifuge-spots, and typical giant-cells were identified as staining for GlyA. Productive infection by B19 was demonstrable, as B19-DNA increased by about x 100 after 72 h of culture. The liquid culture method generating erythroid target cells for effective infection by B19 virus promises to be a useful and easily accessible tool for further research on B19 infection of haemopoietic cells.