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.

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).

Selective solubilization of hemagglutinin and neuraminidase from influenza viruses.

A novel approach is described for the isolation of hemagglutinin and neuraminidase from influenza viruses. In contrast to published procedures, the two glycoproteins are selectively solubilized, leaving an intact viral subparticle which contains lipid, RNA and non-glycoproteins. The pronounced differences in size allow a simple separation of the solubilized proteins from the virus 'core'.

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).

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.

Viral antigens and impurities in influenza vaccines. Evaluation by immunoelectrophoretic methods.

Laurell electrophoretic methods were used to evaluate internal influenza virus antigens and immunogenic impurities (from the fertile egg) in influenza whole virus and subunit preparations. It could be found that subunit preparations obtained after selective solubilization of the surface antigens with cetyltrimethylammoniumbromide are essentially free from internal structure proteins, ribonucleoprotein, and matrix protein. In addition impurities (non viral antigen substances) are considerably reduced in comparison with whole virus preparations, when the core particle is separated by centrifugation. By testing hyperimmune sera against whole virus and subunit preparations, concerning antibody specificities to host components, it could be demonstrated that whole viruses stimulate more antibodies to the host components, than subunit preparations. This is especially true if antigens are tested which are associated with the chorioallantoic membrane.

Potency of influenza vaccines: mouse protection experiments in correlation to field studies in man.

The seroconversion rates have been studied following vaccination of human volunteers with two commercial influenza vaccines. Vaccine A did not give a significant increase of hemagglutination-inhibition titers. Vaccine B, on the other hand, raised the titers 2- to 8- fold, depending on the pretiters of the individuals. The potency of the same vaccines has been tested using mouse protection experiments: vaccine B gave significantly better protection rates, as measured by survival as well as by reduction of lung lesions. These results give additional evidence that the use of mouse protection experiments for the evaluation of different influenza vaccines is meaningful.

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.

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.

[Vaccination of infants and schoolchildren with an influenza subunit vaccine (author's transl)]. / Impfung von Klein- und Schulkindern mit einer Influenza Subunit Vakzine.

A new influenza subunit vaccine which contains only hemagglutinin and neuraminidase antigens was investigated for reactogenicity and immunogenicity in children aged between three and 15 years. Children under six years of age received either 500 IU or 1000 IU of the commercial vaccine, those aged from six to 15 years either 1000 IU or 2000 IU. The vaccines contained the virus strains recommended by the World Health Organisation for the vaccination season 1976/77. In a double blind study the vaccinees were allocated at random to the different dosage groups. The children were examined for reactions by the vaccinating physician 24 hours after vaccination. Serum hemagglutination inhibiting antibody titers were determined before vaccination and four weeks after vaccination. In the younger age-group additional antibody determination was made two weeks after a booster injection. A very low rate of side-reactions was observed in all dosage groups. The increase of the antigen content was not associated with a higher rate of side reactions. After the first vaccination a significant rise of antibody titers could be observed in all children. After the booster injection a further increase of these antibody titers was observed. The response of the younger age group to the dosages 500 and 100 IU did not different significantly. In contrast, in the older age group the increase of the dosage from 1000 to 2000 IU was connected with a better immune response. This was especially marked in the antibody titers against the influenza B-strain virus.

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.

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.

[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.

Clinical trials with a new influenza subunit vaccine in adults and children.

The reactogenicity and immunogenicity of a new influenza subunit vaccine containing essentially only hemagglutinin and neuraminidase has been studied in man. Studies in primed individuals demonstrated that the subunit vaccine induced antibody levels as high as those induced by a comparable whole virus vaccine, or a commercially available whole virus vaccine or by a split vaccine. The commercial whole virus vaccine caused systemic reactions, including fever and headache in 15% of volunteers. In contrast local and systemic reactions were significantly fewer after application of subunit vaccine. When unprimed individuals were vaccinated serological responses were, however, superior with whole virus vaccines. The subunit vaccine demonstrated good immunogenicity and a very low reactogenicity in children. Three months after vaccination, a number of the children were challenged intranasally with live attenuated influenza virus. All proved, as judged by virus isolation and antibody response to be resistant.

[A new influenza subunit vaccine: reactogenicity and antigenicity in comparison to split and whole virus vaccines (author's transl)]. / Eine neue Influenza-Subunit-Vakzine Verträglichkeit und Antigenität im Vergleich zu herkömmlichen Spalt- und Ganzvirusvakzinen

The reactogenicity and immunogenicity of a new influenza subunit vaccine essentially containing only haemagglutinin and neuraminidase was studied in man. The vaccine was compared to commercially available vaccines, an adjuvant containing tween-ether split vaccine (800 IU per dose), and a fluid whole-virus vaccine (2100 IU per dose). Two dosages (700 and 2100 IU) of the fluid subunit vaccine were compared. All vaccines contained the virus strains recommended by the WHO for the 1975/76 season. In a double-blind study 399 volunteers were randomly selected to receive one of the four vaccines. The volunteers were examined for side-effects 24 and 48 hr after vaccination. Antibodies inhibiting haemagglutination were determined prior to and four weeks after vaccination. The sudunit vaccine at 700 IU per dose caused significantly fewer local side effects than the comparable split vaccine, and resulted in significantly higher antibody titers against both influenza A strains. A comparison of the subunit and whole virus vaccines containing high dosages (2100 IU) showed striking differences in reactogenicity. Subunit vaccine was very well tolerated. whereas whole virus vaccine caused systemic reactions, including fever and headache, in 15% of the volunteers. No significant reactogenicity was seen with a high dosage of subunit vaccine (2100 IU) although this is a three-fold increase on the currently used European dosage. Antibody titers were significantly enhanced however.

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.