Safe biotechnology 9: values in risk assessment for the environmental application of microorganisms. The Safety in Biotechnology Working Party of the European Federation of Biotechnology.

Risk assessment for the deliberate release of microorganisms into the environment is traditionally carried out on a case-by-case basis. In a similar approach to that used when assessing human pathogenicity, we propose an alternative approach by introducing risk classes to facilitate or complement this type of risk assessment. These consider several sets of scenarios that address the different values that need to be protected. Examples of this approach include risk-class definitions for soil fertility and biodiversity.

Safe biotechnology. 8. Transport of infectious and biological materials. Working Party "Safety in Biotechnology" of the European Federation of Biotechnology.

The transport of infectious and biological material is regulated by a number of international organizations. This mini-review has been compiled to increase awareness within the scientific community of problems caused by differences in terminology (such as infectious materials/substances, biological products, diagnostic specimens, genetically modified microorganisms) and certain technical aspects of the main international guidelines, and to assist policy makers in the creation of harmonized guidelines. A list of relevant Internet resources has been compiled.

Safe biotechnology. Part 6. Safety assessment, in respect of human health, of microorganisms used in biotechnology.

The assessment of microorganisms in respect to human health is an important step for the introduction of new natural and genetically modified production strains to biotechnology. This report outlines the potential hazards posed by industrial microorganisms, important considerations related to pathogenicity, such as routes and portals of entry into the human body, mechanisms of spread of biological material and a definition of pathogenicity. Furthermore the most important steps in the assessment of pathogenicity of unknown strains are described. A short overview on characterization and in vitro and in vivo tests is presented. The hazard related to allergens and toxic metabolites is reviewed and the choice of methods and the handling of strains with unknown potential are discussed.

Biologically active protease 3C of human rhinovirus 1A is expressed from a cloned cDNA segment in Escherichia coli.

We produced the putative protease 3C of human rhinovirus 1A (HRV-1A), a minor group rhinovirus, by Escherichia coli expression of a segment of HRV-1A cDNA coding for 3A, 3B, 3C, and parts of 2C and 3D (delta 2C3ABC delta 3D). The protease 3C was expected to be processed by intramolecular Q-G cleavages from the virus-specific precursor polypeptide. While the N-terminal 3B-3C site was correctly cleaved, the C-terminal Q-G site of 3C was not processed. Western blotting with a site-specific polyclonal antipeptide antibody showed that not the mature 3C polypeptide, but the 3C-containing precursor 3C delta 3D was the only rhinovirus-specific protein. Mature 3C was obtained by introducing two stop codons at positions glycine-1 and glutamine-2 of 3D by site-specific mutagenesis. This mutant produced the mature 3C protease of HRV-1A. In contrast to poliovirus, the mature 3C protease of HRV-1A is a minor peptide in virus-infected HeLa cells. The 3C protein can be detected only by Western blotting with a polyclonal antipeptide 3C antibody but not by radiolabeling the viral polypeptide.

Transdominant repressors for human T-cell leukemia virus type I rex and human immunodeficiency virus type 1 rev function.

Human T-cell leukemia virus type I (HTLV-I) encodes a 27-kDa trans-acting gene product (Rex) which is involved in the regulated expression of transcripts coding for the viral structural proteins. We used oligonucleotide-directed mutagenesis to generate a series of mutant HTLV-I rex genes. Transient expression experiments demonstrated that 3 of 28 mutant proteins are functionally inactive on the homologous HTLV-I rex response element, whereas an additional 2 mutant proteins are functionally inactive on the heterologous human immunodeficiency virus type 1 rev response element. One of these mutants is able to suppress the function of the wild-type HTLV-I Rex protein in trans on the homologous rex response element sequence. Furthermore, all of these mutants are able to inhibit Rex function on the heterologous rev response element sequence. Intriguingly, only three of these mutants are able to inhibit the human immunodeficiency virus type 1 Rev protein in a dominant-negative manner.

Pharmacokinetics of an anti-cytomegalovirus hyperimmunoglobulin after single intravenous administration to healthy volunteers.

By selecting blood donors with high cytomegalovirus (CMV) antibody titres, a plasma pool was obtained which was used to produce an IgG hyperimmunoglobulin by means of pepsin fractionation. After administration of approximately 100 mg/kg body weight to healthy subjects, the time course both of anti-CMV IgG antibody titres by ELISA and of virus neutralisation (VN) titres was followed for 15 days. Seronegative subjects showed an increase in CMV-IgG antibodies as well as a significant enhancement of VN. The time course of both titres was non-uniform. The decline of both titres was biphasic: CMV-IgG antibodies fell slowly during the first week and remained unchanged thereafter, whereas VN titres decreased markedly faster in the first than in the second week. In seropositive subjects, on the other hand, VN remained unchanged. CMV-IgG antibodies increased by approximately 3 times, followed by a similar biphasic decline as seen in seronegative subjects. Due to the differences between seronegative and seropositive subjects and to the non-uniform time course, no calculations of the elimination rate were feasible.

Polyvalent group B streptococcal immune globulin for intravenous administration: overview.

Immunoglobulin therapy is becoming an important modality for the prevention and treatment of bacterial and viral infection. Standard intravenous immunoglobulin (IVIG) is made from large pools of plasma obtained from normal blood donors. However, lot-to-lot variation in titers of antibody to specific microbial pathogens may diminish therapeutic efficacy. Since group B Streptococcus (GBS) is an important neonatal pathogen, a preparation with high titers of activity against GBS was prepared and studied. Plasma was obtained from volunteers immunized with a polyvalent GBS vaccine and was processed by the Swiss Red Cross for intravenous infusion. This high-titered GBS intravenous immunoglobulin (GBS-IVIG) was shown to be superior both in vitro and in vivo to standard IVIG. GBS-IVIG provided protection when therapy was delayed for up to 24 hours after infection. Standard IVIG did not protect animals against all GBS strains. However, GBS-IVIG enhanced survival from infection with all strains tested, even when the challenge dose of GBS was increased by 2 log units. Specific hyperimmune globulins to pathogens such as Haemophilus influenzae and Streptococcus pneumoniae have also been studied. Immunization of adult volunteers as a means of obtaining hyperimmune globulin appears to be an effective method of producing high-titered pathogen-specific preparations of IVIG.

A placebo-controlled dose response study of the reactogenicity and immunogenicity of a live cold-recombinant influenza B virus vaccine in healthy volunteers.

A live cold-recombinant influenza B virus vaccine (RB77) was given intranasally in a placebo-controlled, double blind study to volunteers in dosages of 10(7.9) EID50/ml, 10(7.25) EID50/ml, 10(5.7) EID50/ml. The tolerability, safety, and immunogenicity of the vaccine were investigated. No revertant virus was found in nasal swabs taken after immunisation. Local reactions were mild and showed a significant increase over the placebo only in the highest dose group. Systemic reactions were not different from the placebo. A significant increase in haemagglutinin inhibition titre was found in the highest dose group against the immunising strain (RB77) and the two wild strains B/TEC and B/Sing.

Quality control of protein drugs.

Testing of protein drugs must include assays for purity, identity, safety, and potency. The structure of the final product as well as potential impurities are strongly influenced by the type of producing organism (bacteria, yeast, mammalian or insect cells).

A convenient system for the serial quantitation of viable cells measuring luminescence of ATP.

Measurement of intracellular ATP using luminescence offers an alternative method for the routine determination of viable animal cells in a large number of cultures. Optimal conditions of the new technology are described as is its application in various test systems.

Intravenous immunoglobulin in the treatment of neonatal sepsis: therapeutic strategies and laboratory studies.

IGIV appears to have a promising future in treating and perhaps preventing neonatal bacterial infections. However, all IGIV lots do not contain equal amounts of pathogen-specific IgG with functional opsonic activity. To ensure effective therapy it will be important to inform physicians that the IGIV lots available for use contain functional antibacterial antibody to the responsible pathogens. To optimize therapy IGIV may need to be given early in the infection and doses may need to be repeated if pathogen-specific antibody is rapidly depleted. Further, clinical studies will be necessary to determine if IGIV will be a valuable adjunct to antibiotic therapy in neonatal GBS infections.

5-(Haloalkyl)-2'-deoxyuridines: a novel type of potent antiviral nucleoside analogue.

Syntheses of 5-(2-haloethyl)-2'-deoxyuridines, 5-(3-chloropropyl)-2'-deoxyuridines, and 5-(2-chloroethyl)-2'-deoxycytidine are described. The antiviral activities of these compounds were determined in cell culture against herpes simplex virus types 1 and 2. All compounds were shown to possess significant and selective antiviral activity. The most potent derivative, 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), inhibited HSV-1 at concentrations below 0.1 microgram/mL. It exerted measurable inhibitory effects on cell proliferation only at concentrations higher than 100 micrograms/mL. In vivo CEDU reduced the mortality rate of HSV-1-infected mice at concentrations lower than 5 mg/kg per day when given intraperitoneally and orally. Thus, it proved to be more effective in this in vivo model than the reference compounds (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 9-[(2-hydroxyethoxy)methyl]guanine (ACV).

Studies in man with a cold-recombinant live influenza B virus vaccine.

A cold recombinant live influenza B virus vaccine was tested in man. In comparison to a placebo, reactogenicity attributable to virus infection was slight or moderate. No revertant viruses were shed, and there was no evidence of transmission to the placebo group who were housed in close contact with the vaccinees. Serological responses to initial inoculation were moderate; 60% of vaccinees showing twofold increases in serum hemagglutination inhibition (HAI) titers gave a geometric mean titer (GMT) of 1:13. Three weeks after the first vaccination, both the vaccine and the placebo group were revaccinated with homologous live virus vaccine. The group previously given vaccine was resistant to reinfection as judged from clinical reactions and virus shedding and the GMT increased only slightly to 1:16.3. In contrast, the former placebo group responded; mild symptoms were seen, the majority shed viruses and 50% showed twofold increases in serum HAI titers to a geometric mean titer of 1:17.4.

Parameters of humoral and cellular immunity of influenza A/USSR/90/77 (H1N1) virus in various age groups.

After a local epidemic of A/USSR influenza, immunologic parameters related to influenza A/USSR/90/77 (H1N1) virus were studied in four age groups: elderly persons (greater than or equal to 64 years), healthy adults (20-44 years), children (six to 13 years), and neonates (who served as controls). Sera from the first three groups had nearly equivalent titers of hemagglutination-inhibiting antibody (geometric mean titers, 1:8-1:11), which were greater than those in cord blood of neonates. Lymphocyte proliferation responses to influenza A/USSR viral antigens among study groups were similar, with mean stimulation ratios (to whole virus) of 4.2-5.4; neonatal cord blood samples were unresponsive (stimulation ratio, 1.1). In contrast, the magnitude of antibody-dependent cellular cytotoxicity against baby hamster kidney target cells infected with influenza A/USSR virus was significantly greater with lymphocytes from adults and elderly persons (P less than 0.05) than with those from children.

Lymphocyte responses to hemagglutinin and neuraminidase subunits of influenza virus.

The responses of peripheral lymphocytes to purified hemagglutinin and neuraminidase subunits and other components of an influenza. A virus were measured in 21 normal adults and compared with antibody titers. All had influenza antibodies and demonstrated influenza antigen recognition by lympho-proliferative responses. There was a significantly greater response to the two purified influenza virus surface antigens, hemagglutinin and neuraminidase, than to either whole intact virus or its separated subviral core. No correlation between magnitudes of antibody titers and lymphocyte responsiveness was observed.

[A new influenza subunit vaccine: hemagglutinating antibodies one year after vaccination (author's transl)]. / Eine neue Influenza Subunit Vakzine: Hämagglutinations-hemmende Antikörper ein Jahr nach der Impfung.

The antibody response to a new influenza subunit vaccine was compare d one year after vaccination with the responses induced by two other influenza vaccines. The subunit vaccine was given either in a high dose form containing 2100 IU, or in a low dose form containing 700 IU. As comparison a split vaccine was used containing 800 IU and AI(OH)3 as adjuvant and a whole virus vaccine containing 2100 IU. Of the 399 vaccinated subjects which had taken part in this study 151 were available for hemagglutination inhibiting (HAI) antibody determinations one year after vaccination. Protection rates assessed for the respective groups on the assumption that serum HAI titers of 1 : 32 or greater confer protection. With the high dose of subunit vaccine 85% of volunteers were considered still to have protective titers one year after vaccination, compared with 77% of those who received the whole virus vaccine. Although the high dose subunit vaccine and whole virus vaccine induced similarly high protective levels lasting at least one year, the reactions observed on vaccination were significantly less with the subunit preparation. The lower dose of subunit vaccine induced lower levels of protection (60%) after one year, and lower mean HAI titers than the high dose subunit vaccine. Nevertheless protection was superior to that of the split virus adjuvant vaccine. The addition of adjuvant thus does not seem materially to improve the immune response to influenza virus antigens. An increase of antigen content can however be seen as a practical alternative for achieving higher antibody levels. The subunit vaccine would appear to be particularly suitable in this respect as even with a higher dose there is no increase in reactogenicity.