Molecular approaches to validate disinfectants against human hepatitis B virus.

Disinfection is an important measure to prevent hepatitis B virus (HBV) transmission by instruments. However, virucidal testing of disinfectants against HBV is difficult, because no simple quantitative infectivity assay exists. Since molecular changes of viral epitopes and the genome may indicate virus inactivation, we measured the alteration of these constituents with 0.065% peracetic acid (PAA) for exposure times up to 1 h. Plasma of a chronic HBV carrier with 10(9) HBV genomes/ml served as viral source in the form of a 10% dilution or of a purified HB-antigen preparation. Alterations of HBV epitopes were analyzed with four monoclonal antibodies in an enzyme-linked immunosorbent assay. Changes of the HBV genomes were determined by the inability to amplify the target sequence with a quantitative real-time polymerase chain reaction, either of a short fragment (189 bp) or of the full-length (3,200 bp). The determination of the epitope and genome alteration was quantified as log10 reduction factor (RF) with the parallel line bioassay. Under a high protein load of 10% human plasma, PAA induced a HBV genome alteration of RF = 1.5 after an exposure time of 60 min. Similar RFs were seen with the four HB epitopes. Without protein load, the alteration of these epitopes amounted to a RF of more than 3.5 within 30 min. Such inhibition of PAA activity by protein load was also seen in the virucidal tests with parvovirus. Although the RF were higher in the virucidal tests, the time-dependent dose-response curves for the epitope and genome alteration and for the infectivity inactivation followed the same inactivation kinetics. The molecular alteration and disintegration epitope and genome test may therefore be suitable to measure antiviral activity of disinfectants against HBV.

Long-term humoral and cellular immunity after vaccination with cell culture rabies vaccines in man.

To determine the duration of anti-rabies immunity, peripheral blood of 18 vaccinees was obtained between 2 and 14 years after immunization. Peripheral blood mononuclear cells (PBMC) and serum were tested for the presence of either rabies virus-specific antibodies or rabies antigen-specific proliferation. Neutralizing immunoglobulin class G anti-rabies virus antibodies could be detected in sera of all vaccinees, but not in 18 age- and sex-matched controls. Rabies antigen-induced proliferation of PBMCs from vaccinees was significantly higher than that of controls. The anti-rabies T and B cell response showed no time-dependent pattern. These results suggest the induction of a long-term immunity after rabies immunization according to pre- and post-exposure schedules with inactivated cell culture vaccines against rabies.

Methodological approaches to disinfection of human hepatitis B virus.

Three commercial disinfectants (two quaternary formulations and one phenolic) were tested against human hepatitis B virus (HHBV). The treated virus was assayed for infectivity by the chimpanzee assay and for morphological alteration by the Morphological Alteration and Disintegration Test. The same agents were tested against duck hepatitis B virus in a duck hepatocyte infectivity assay. It is apparent that human and duck hepatitis viruses were relatively susceptible to disinfection, becoming noninfectious after < or = 10 min of contact with the disinfectant. The Morphological Alteration and Disintegration Test accurately predicted activity in the two infectivity tests. The anti-human hepatitis B virus effect of the low-level quaternary ammonium germicides is a novel finding and suggest that members of the family Hepadnaviridae are relatively susceptible to chemical agents.

Enumeration of T, B and natural killer peripheral blood cells of patients with multiple sclerosis and controls.

The median percentages of peripheral blood immunoglobulin-positive (Ig+) lymphocytes (8%, n = 46), CD8+ (12%, n = 49) and CD57+ cell numbers (5%, n = 37) of patients suffering from multiple sclerosis (MS) were significantly (p less than 0.05) lower than the values of age- and sex-matched healthy individuals (Ig+ cells: 13%, n = 46; CD8+ cells: 17%, n = 49; CD57+ cells: 9%, n = 37). Comparison of calculations on decreased peripheral blood cell counts and increased brain cell counts in MS patients revealed that sequestration of blood cells into the MS brain is a possible explanation of these findings.

Alterations of the immune system following splenectomy in childhood.

After splenectomy due to blunt abdominal trauma, splenectomized children showed a restricted pattern of T-cell immunodeficiency compared to age and sex-matched normal children. Peripheral blood total (CD3) T-cell counts of 11 splenectomized children of 43%, double positive helper (CD4) inducer subpopulation (CD29) cell counts of nine splenectomized children of 7%, and phytohemagglutinin (PHA)-induced T-cell proliferation of 11 splenectomized children of 53,206 c.p.m. were significantly (p less than 0.05) lower than values of normal children (61% CD3 cells, n = 12; 13% CD4CD29 cells, n = 11; 107,832 c.p.m. PHA-induced proliferation, n = 12). The deficit of CD4CD29 cell numbers may be due to impaired maturation of these particular CD4 lymphocytes and may explain diminished PHA-induced proliferation in small children. The significantly higher B-lymphocyte counts of splenectomized children (21%, n = 11; 558 cells/mm3, n = 10) compared with 12 normal children (14%; 329 cells/mm3) may be due to loss of the reservoir function of the spleen.

[Experiences with transfusion of CMV tested blood preparations after bone marrow transplantation]. / Erfahrungen bei der Transfusion von CMV-getesteten Blutpräparationen nach Knochenmarktransplantationen.

Cytomegalovirus infections are frequent and often life-threatening in bone marrow transplant recipients. Among 112 of our patients after bone marrow transplantation (BMT), 28 developed clinical symptoms or laboratory test results of CMV infection, and 15 of them died. In our experience blood cell substitution with products from CMV negative blood donors does not completely prevent CMV infections. Other factors like disturbances of immunoreconstitution after BMT are relevant in promoting the development of CMV disease.

Standardization of an enzyme immunoassay for the in vitro potency assay of inactivated tissue culture rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera.

A non-competitive enzyme-linked immunoassay (ELISA) has been standardized to supplement the in vivo potency test used for the quality control of inactivated tissue culture vaccines against rabies. The essentials of the ELISA were: fixation of the virus in different dilutions of vaccine on the surface of microtitre plates; testing of the reference and up to six test vaccines on one plate; incubation with polyclonal antisera to rabies virus glycoprotein containing an excess of antibody; further incubation with a species-specific anti-IgG coupled to peroxidase; a final incubation with a substrate. The incubation periods were 1 h, 1 h and 30 min both at +37 degrees C. The relative potency determinations were made graphically or by a computer using a parallel line bioassay in which the potencies of the vaccines of unknown potency were tested against the reference preparation on a single microtitre plate. Under these conditions inactivated rabies vaccines of different types (virus strains, cell substrates, inactivation and concentration procedures) were tested for potency. Furthermore, it was possible with this in vitro method to assay adjuvanted vaccines, in process samples such as tissue culture supernatants with live or inactivated rabies virus, concentrates, and vaccines undergoing thermal stability tests. The rabies glycoprotein antigen-antibody reaction was highly specific according to the results and the glycoprotein content was measured quantitatively. The potency determined by the in vitro ELISA correlated with the in vivo NIH protection potency test. The lower limit of detection of the ELISA was 0.015 IU/ml. Quantitative antigen determination was possible with both homologous and heterologous antisera to rabies virus glycoprotein when vaccines of the same virus strain were tested. When the potencies of vaccines of different virus strain specificity were calculated, it was necessary to take into account the strain-specific antigenicity. Even so vaccines of high potency were found to give a stronger reaction with a heterologous serum than did weak vaccines with a homologous antiserum. Stability tests made on inactivated tissue culture vaccines such as vaccine from the human diploid cell strain (HDCS), from purified chicken embryo cell (PCEC) or from purified Vero cell rabies vaccine (PVRV), showed high stability of the glycoprotein antigen even after four months of storage at +37 degrees C or 24 h at +56 degrees C, provided that the vaccines were stored in a lyophilized state. The antigenicity of liquid vaccines was inactivated after a few hours at +56 degrees C. For tropical areas, therefore, only lyophilized vaccines should be considered.

Effect of strain differences on the potency testing of rabies vaccines in mice.

Antigenic differences between several strains of rabies virus, namely CVS, SAD and Flury (LEP) strains, were studied in cross-challenge experiments or cross-neutralization tests performed on sera of mice immunized with vaccines containing each strain. A typical wild fox virus strain was also included as challenge virus. The strain differences affected the relative potencies of the three vaccines in the European Pharmacopoeia mouse protection test for veterinary rabies vaccines, in that higher antigenic values were obtained when the vaccine strain was homologous to the challenge virus. This observation was confirmed by neutralizing antibody induction in mice.

[Cytomegalovirus hyperimmune globulin and cytomegalovirus-tested blood products--a pilot study in bone marrow graft recipients]. / CMV-Hyperimmunglobulin und CMV-getestete Blutprodukte--eine Pilotstudie bei Knochenmarktransplantatempfängern.

Nineteen bone marrow transplant recipients had cytomegalovirus prophylaxis using a commercial hyperimmune globulin. Seronegative donors were selected for blood component substitution. The incidence of seroconversion was 25%. One patient acquired fatal cytomegalovirus associated interstitial pneumonitis. The value of this combined cytomegalovirus prophylaxis in bone marrow transplant recipients in comparison to sole passive immunization is recommended to be the subject of a controlled trial.

Comparison of the morphological alteration and disintegration test (MADT) and the chimpanzee infectivity test for determination of hepatitis B virucidal activity of chemical disinfectants.

The morphological alteration and disintegration test (MADT) as a key indicator for Hepatitis B Virus (HBV) inactivation was compared with the chimpanzee infectivity test using three suspensions of HBV differing in the degree of inactivation as judged by electronmicroscopic studies. The results of the MADT and the chimpanzee studies correlated well. Thus, the MADT, evidently, can replace the laborious, costly and time-consuming animal studies for the evaluation of chemical disinfectants for hepatovirucidal activity.

[Alterations of humoral and cellular immunity after splenectomy (author's transl)]. / Veränderungen des humoralen und zellulären immunsystems nach Splenektomie.

In twenty-seven splenectomized patients, who had undergone splenectomy for trauma (n=13) and thirty-one immunologically healthy persons, matched for age and sex, the following immunological parameters were examined: Serum immunoglobulins M, A, G, D and E, complement factors C3, C4 and the serum haemolytic complement activity (Ch 50), as well as the ratio and in vitro reagibility of B-and T-lymphocytes measured by transformation responses to the non-specific mitogens Phytohämagglutinin, Concanavalin A and Pokeweed-Mitogen. The phagocytic capacity of polymorphonuclear neutrophils was tested by the Nitroblue tetrazolium test. Total peripheral lymphocytes, B- und T-lymphocytes from splenectomized patients were significantly higher than in healthy controls (p less than 0.01). The PHA response in splenectomized patients was significantly depressed (p less than 0.01) as compared to nonsplenectomized controls. Also IgM serum concentration was found to be significantly lower in these patients (p less than 0.01). No differences were observed between patients, who had been splenectomized 7 months to 5 years or 6 to 14 years ago. The comparison of persons splenectomized for traumatological or haematological disorders showed statistically no significant differences. The importance of the spleen for the primary immune response is discussed.