Increasing West Nile virus antibody titres in central European plasma donors from 2006 to 2010.

We analysed by neutralisation assay 55 intravenous immunoglobulin preparations produced from human plasma collected in three central European countries, specifically Austria, Germany and the Czech Republic, from 2006 to 2010. The preparations from 2009 and 2010 contained increasing titres of neutralising antibodies against West Nile virus (WNV) in the absence of reported human WNV cases in these countries.

Safety and immunogenicity of the modified adult tick-borne encephalitis vaccine FSME-IMMUN: results of two large phase 3 clinical studies.

A prospective, randomised, multicentre, single-blind phase 3 study was performed to assess the safety of a vaccination schedule consisting of two vaccinations (21-35 days apart) with the tick-borne encephalitis (TBE) vaccine FSME-IMMUN "adults" (five consecutive lots) in comparison to another licensed TBE vaccine (Encepur), with polygeline) (two lots) in healthy volunteers (n=3966) aged 16-65 years. The safety of the third vaccination with FSME-IMMUN "adults" (6 months after the first vaccination) was investigated in a follow-up study on the same population (n=3705) and TBE antibody titres were analysed pre- and post-vaccination in a subgroup of volunteers (n=564). Following the first vaccination, the overall incidence of fever (> or =38.0 degrees C) was 0.8% in the FSME-IMMUN "adults" study group and 5.6% in the comparator study group; fever was mainly mild. The fever rate after the second vaccination was 0.6% and 0.5% in the two study groups, respectively. Local and systemic reactions after the first vaccination occurred with a lower frequency in the FSME-IMMUN "adults" study group than in the comparator group. Upon analysing the tolerability of the third vaccination with FSME-IMMUN "adults", similar results were determined in both study groups of volunteers previously vaccinated with FSME-IMMUN "adults" or with the comparator vaccine. The immunogenicity results demonstrated similar seroconversion rates (as determined by ELISA or neutralization test) before and after the third vaccination in the FSME-IMMUN "adults" group and in the comparator group respectively. The results of both studies demonstrate that: (1) FSME-IMMUN "adults" is safe and highly immunogenic, (2) all five production lots of FSME-IMMUN "adults" were consistent with respect to a low rate of adverse events, (3) FSME-IMMUN "adults" induces considerably lower adverse reaction rates than the comparator vaccine after the first vaccination, and (4) two vaccinations with the comparator vaccine can be successfully followed by a third vaccination with FSME-IMMUN "adults".

Development of a Vero cell-derived influenza whole virus vaccine.

Influenza vaccine production is dependent on the availability of embryonated hen eggs for virus growth. This is an extremely cumbersome system with many disadvantages with respect to selection of virus variants and the presence of adventitious viruses. We have developed an alternative cell culture system which allows rapid production of large volumes of vaccine. The WHO-approved Vero cell line was used in serum-free culture to grow many influenza strains to high titre. This system could be scaled-up to allow vaccine production with a 1200 litre fermenter. A purification scheme was developed which resulted in a high purity whole virus vaccine. This was demonstrated to be at least as immunogenic as a conventional egg-derived preparation.

New technologies for vaccines.

The impact of vaccination on the health of the world's people has been considerable. With the possible exception of clean water, no other development has had such a major effect on mortality reduction and population growth. During the last 200 years vaccination has controlled nine major diseases and has led to the eradication of one, i.e. smallpox. However, in many instances, the exact mechanisms of successful vaccines are not fully understood. Almost all of the vaccines in use today are of three types: live attenuated microorganisms, inactivated whole microorganisms, or split or subunit preparations. These have different strengths and weaknesses with respect to safety and efficacy, but traditional vaccine development methodologies have not yet led to the generation of a vaccine with all the characteristics required of the ideal vaccine. Thus the development of improved vaccines that overcome the difficulties associated with many of the currently available vaccines is a major goal of biomedical sciences. In addition, there is an urgent need for new vaccines against the many infectious agents that still cause considerable morbidity and, in some cases, mortality. As has been the case in many areas of biology, the application of recombinant DNA approaches to vaccinology has opened up whole new areas of possibilities. The details of these and other technologies and their application to vaccine development are described in this review.

A novel mammalian cell (Vero) derived influenza virus vaccine: development, characterization and industrial scale production.

Influenza virus for vaccine production are presently produced in embryonated chicken eggs. This conventional standard methodology is extremely cumbersome; it requires a huge amount of eggs and an extensive purification to reduce the amount of contaminating egg proteins and to minimize the risk of allergies against egg albumin. The shortage of eggs in a pandemic situation, the selection of egg-adapted variants and the presence of adventitious viruses has emphasized the necessity for production of influenza vaccines on a well characterized stable cell line. Our established Vero cell technology has been successfully adapted to large scale production of a variety of influenza virus strains. The production in 1200 litre fermenter cultures under serum-free conditions gave antigen yields comparable to the conventional embryonated egg technology. The development of a rapid and efficient purification scheme resulted in a safe high purity vaccine which was at least as immunogenic as conventional egg-derived vaccines in a mouse model. This vaccine has been shown to be safe and highly immunogenic in chimpanzees and to be capable of protecting ferrets against challenge with live virus. Clinical trials have now been initiated in the UK and Austria.

Vaccine technology: looking to the future.

The impact of vaccination on the health of the world's peoples has been considerable. With the possible exception of clean water, no other development has had such a major effect on the reduction of mortality and on population growth. During the last 200 years, vaccination has controlled nine major diseases and has led to the eradication of one, ie smallpox. However, in many instances the exact mechanisms of successful vaccines are not fully understood. Almost all of the vaccines in use today are of three types: live attenuated micro-organisms, inactivated whole micro-organisms, or split or subunit preparations. These have different strengths and weaknesses with respect to safety and efficacy, but traditional vaccine development methodologies have not yet led to the generation of a vaccine with all the characteristics required of the ideal vaccine. Thus, the development of improved vaccines that overcome the difficulties associated with many of the currently available vaccines is a major goal of biomedical sciences. In addition, there is an urgent need for new vaccines against the many infectious agents that still cause considerable morbidity and, in some cases, mortality. As has been the case in many areas of biology, the application of recombinant DNA approaches to vaccinology has opened up whole new possibilities. The details of these and other technologies and their application to vaccine development are described in this review.

Development of a mammalian cell (Vero) derived candidate influenza virus vaccine.

Influenza vaccine production is dependent on the availability of embryonated hen eggs for virus growth. This is an extremely cumbersome system with many disadvantages with respect to selection of virus variants and presence of adventitious viruses. We have developed an alternative cell culture system which allows rapid production of large volumes of vaccine. The World Health Organisation (WHO) approved Vero cell line was used in serum-free culture to grow a multitude of influenza strains to high titre. This system could be scaled-up to allow vaccine production with a 1200 litre fermenter volume. A purification scheme was developed which resulted in a high purity whole virus vaccine. This was demonstrated to be at least as immunogenic as a conventional egg-derived preparation in a mouse model.

Natural IgM antibodies in baby rabbit serum bind high-mannose glycans on HIV type 1 glycoprotein 120/160 and activate classic complement pathway.

Serum from rodents and felines has been found very effective in complement-dependent lysis of HIV-1, even in nonimmunized animals, but the effector molecules in animal serum and target structures on HIV-1 envelope gp120/160 responsible for complement activation were not determined. We have found that the natural anti-carbohydrate-specific IgM antibodies present in baby rabbit serum were able to lyse effectively the CD4+ T cells coated with the whole virus or with a recombinant gp120/160, irrespectively of the virus strain or glycoprotein expression system. When the high mannose-type glycans on gp160 were enzymatically removed by endoglycosidase F or blocked with the specific lectins, the complement activation and subsequent cell lysis were abolished. IgM-depleted baby rabbit serum was not able to lyse the gp120/160- and/or whole virus-coated target cells. These results suggest that the target structures for complement-activating and naturally occurring IgM antibodies in baby rabbit serum are high-mannose residues on HIV-1 envelope glycoprotein.

Inactivation of hepatitis A virus in plasma products by vapor heating.

BACKGROUND: The transmission of hepatitis A virus (HAV) has been associated with the use of a number of solvent/detergent-treated factor VIII concentrates and possibly a factor IX concentrate. These reports have emphasized the necessity of using virus-inactivation methods for plasma products that are capable of inactivating nonenveloped viruses such as HAV. STUDY DESIGN AND METHODS: A simple, highly accurate titration procedure for HAV, which allows extensive kinetic investigations of virus-inactivation procedures, has been developed. This system has now been used to evaluate the efficacy of vapor heating in inactivating HAV after the addition of the virus to a range of human plasma products. RESULTS: It was demonstrated that HAV was significantly more thermostable than other picornaviruses, which reinforced the fact that such viruses cannot be used as model viruses for HAV-inactivation studies. A one-step vapor-heating procedure was demonstrated to inactivate between 5.9 and > 6.3 log10 of HAV in different products. A two-step vapor-heating procedure had the capacity to inactivate between > 8.7 and > 10.4 log10 of HAV. Both procedures were more effective in inactivating HAV than was the pasteurization procedure used for virus inactivation in human albumin solutions. CONCLUSION: These data demonstrate the efficacy of vapor heating in inactivating high-titer HAV after the spiking of plasma products with virus. This study confirms and explains the results of controlled clinical trials and long-term clinical usage with respect to the lack of HAV transmission by such vapor-heated products.

Determination of the inactivation kinetics of hepatitis A virus in human plasma products using a simple TCID50 assay.

The transmission of hepatitis A virus (HAV) associated with use of FVIII concentrates has been reported in a number of European countries. All of these cases were associated with products inactivated by use of solvent detergent treatment. These reports have emphasized the necessity of evaluating virus inactivation methodologies for their ability to inactivate HAV. Such studies had previously been hampered by the difficulties associated with titration of HAV, because of the minimal cytopathic effect of most strains of virus on tissue culture cells. We have developed a simple, rapid, TCID50 virus titration system using a cytopathic strain of HAV which allows extensive kinetic studies of HAV inactivation. This has been compared with the standard radioimmunofocus forming (RFF) assay which is presently used for HAV titration. The reproducibility of the TCID50 assay was demonstrated to be equal to that of the RFF assay and the 95% confidence intervals for titres determined by both assays were also equal. The thermal stability of the cytopathic strain was studied and shown to be equivalent to that of a noncytopathic strain. The kinetics of HAV inactivation by heating in aqueous solution were compared to those of HIV-1 and a number of model viruses. It was demonstrated that HAV was highly stable, with 5 hours heat treatment at 60 degrees C in aqueous solution being required to inactivate 5.8 log10 virus. In contrast to heating in aqueous solution, lyophilization followed by 1 hour vapor heating at 60 degrees C was sufficient to inactivate 5.9 log10 HAV.

At what stage should virus inactivation be carried out?

On an industrial scale blood products are produced from plasma pools consisting of donations from thousands of donors. As a number of pathogens may be present in the donor before symptoms of an infectious disease become evident, such pathogens, particularly viruses, may be transmitted in human blood products. The level of virus contamination can be substantially reduced by a range of serological tests and by polymerase chain reaction screening. Viruses can also be removed and/or inactivated during the course of the plasma fractionation process. However, specific virus inactivation steps must be included in the manufacturing process for most blood products. The stage at which these steps should be introduced and the efficacy and drawbacks of various procedures are described.

Minimum safety requirements for preclinical testing.

A wide range of biological products for medicinal use can now be produced by novel biotechnology processes. These products include naturally occurring human proteins and peptides such as hormones, cytokines, blood products as well as monoclonal antibodies of murine and human origin and bacterial and viral antigens for use as vaccines. However, there are potential safety concerns that arise from the novel processes used in their manufacture and from the complex structural and biological characteristics of the products. The acute and repeated dose toxicity testing, pharmacodynamic and immunological testing and a range of product-specific biochemical and safety tests required for these products are described.

Vaccines in AIDS. Present state of knowledge.

The identification of a virus, HIV-1, as the etiological agent of AIDS has opened the door to the development of a vaccine against this disease, for both prophylactic and therapeutic purposes. A number of different vaccine strategies have been developed in pursuit of this aim. The first major advances in this direction were the reports that some candidate vaccines could prevent infection of chimpanzees with HIV-1. Human trials have now been initiated in HIV-1 seronegative and seropositive individuals and the details of the vaccine strategies involved and the results obtained to date are described.

[Comparison of virus isolation and antigen enzyme immunoassay in the detection of HIV]. / Vergleich von Virusisolierung und Antigenenzymimmunoassay zum Nachweis von HIV.

Virus isolation from peripheral blood lymphocytes and antigen detection in serum by ELISA were compared in 75 patients with a serologically confirmed HIV infection. 25 per cent of the lymphocyte cultures were HIV positive, but only 2 of the 14 patients were also antigen positive. In addition, infectious virus was not detected in 15 antigen positive patients, thus revealing only a low degree of correlation between the two assay systems.

[Molecular biology of human T-lymphotropic retroviruses (HTLV)]. / Die Molekularbiologie menschlicher T-lymphotroper Retroviren (HTLV).

The first human retroviruses have been discovered during the past seven years. They cause two diseases which involve disturbances of the growth of the T4-lymphocyte. This target cell type, which is central to the regulation of the immune system is induced by human T-lymphotropic virus type I (HTLV-I) to excessive proliferation (leukaemia) and by HTLV-III/LAV (lymphadenopathy associated virus) to premature death (acquired immune deficiency syndrome [AIDS]). Both also seem to be indirectly involved in several other disorders. The genetic structures of these retroviruses and the mechanisms by which they usurp host-cell functions are novel among retroviruses. The continuous increase in the number of AIDS cases for whom no effective therapy is currently possible mandates attempts at developing primary prevention by a vaccine. Based on past attempts at developing vaccines against retroviruses, the most feasible configuration will be the glycoprotein linked to its transmembrane protein. Any virus preparation containing nucleic acids could be considered less safe. Potential problems exist in that there is extensive heterogeneity among various HTLV-III isolates, particularly in the env-gene. This fact and the known relationship of HTLV-III to some Lentiviruses suggest that functional antigenic variation could be encountered. The methodology of developing a vaccine against the retroviruses causing AIDS should also be helpful in designing vaccine strategies against human leukaemia and lymphomas caused by other members of this virus family.

Effect of measles virus antibodies on a measles SSPE virus persistently infected C6 rat glioma cell line.

Maintenance of measles (SSPE-Lec) virus persistently infected C6 rat glioma cells in medium containing polyclonal measles antiserum resulted in the loss of detectable expression of all measles virus proteins. Removal of these cells from antiserum, however, led to a re-expression of virus proteins and the production of infectious virus. Cloning of antibody-modulated non-expressing cells in the presence of antiserum showed that re-expression of virus proteins was not due to an incomplete curing process following the addition of antiserum, as a large number of non-expressing cell clones developed the capacity to express measles virus antigen at different periods after removal of antiserum. Irradiation of persistently infected cells to give a non-growing culture showed that modulation was not mediated by a selection and outgrowth of a small percentage of non-expressing cells originally present in the culture. Antibody directed against C6 membrane proteins did not lead to modulation and it was also shown that only monoclonal antibodies with neutralizing activity could affect intracellular antigen expression. Immunoglobulin Fab fragments with neutralizing activity also had modulating activity. Although all modulated cell clones were more susceptible to homologous virus infection than control C6 cells, it was not possible to rescue any defective measles virus which may have been maintained in the culture.

Effect of antibody-induced modulation of measles (SSPE) virus membrane proteins on beta-adrenergic receptor-mediated adenylate cyclase activity.

The effect of measles virus antiserum on the expression of viral glycoproteins on the membranes of measles (SSPE) persistently infected C6 rat glioma cells was studied. There was a gradual loss of virus membrane antigen from these cells until no antigen was detectable 18 days after initiation of antiserum treatment. At this stage approximately 25% of cells still displayed intracellular virus antigen which was also lost after further cell passages. There was an accompanying recovery of the previously reported disrupted catecholamine-dependent beta-adrenergic receptor-stimulated cAMP synthesis in antiserum-treated cells which coincided with the loss of viral antigen from the membrane. This was determined to be due to a recovery of fluoride-stimulated activity of the cAMP synthesising adenylate cyclase enzyme to normal values. Thus we demonstrate that the impairment of this important cell function was due to insertion of viral antigen in the cell membrane rather than its accumulation in the cytoplasm.

Establishment of persistent infection in mouse cells by Sindbis virus and its temperature-sensitive mutants.

The ability of wild-type (wt) Sindbis virus and six temperature-sensitive (ts) mutants to establish persistent infection in mouse L cells and a line of mouse embryo (ME) cells was determined. The wt established persistent infection in both ME cells and L cells at 39 degrees C. At 30 degrees C the wt established persistent infection in L cells but not ME cells, which did not recover from the initial infection. For the ts mutants, both cell lines survived the initial infection at 39 degrees C (the restrictive temperature) but the virus was eventually eliminated. At 30 degrees C (the permissive temperature) in L cells all mutants established persistent infection. In ME cells at 30 degrees C, RNA- mutants (unable to synthesize virus-specified RNA at 39 degrees C) established persistent infection whereas the cells did not recover from infection with RNA+ mutants (able to synthesize virus-specified RNA at 39 degrees C). The wt virus was less cytopathic in L cells than in BHK or ME cells. Interferon was produced by both L and ME cells at 30 degrees C and 39 degrees C, but its activity could not be detected in either cell line at 30 degrees C. It is proposed that establishment of persistent infection is dependent on reduced cytopathogenicity in the early stage of infection, and that further evolution of the virus then occurs to a less cytopathic form. Elimination of the virus at 39 degrees C is probably due to the action of interferon.

Demyelination in mice resulting from infection with a mutant of Semliki Forest virus.

Twelve of 34 weanling mice (35%) developed lesions in the brain and spinal cord following i.p. infection with 10(2) p.f.u. of a mutant of Semliki Forest virus (SFV). Six of 12 mice examined 13 days post infection (p.i.) showed meningo-encephalomyelitis with focal spongiform lesions in the grey and white matter. The spongiform lesions were characterised by necrosis of putative oligodendrocytes, myelinic vacuolation and mononuclear cell infiltration. Only one of six mice examined 21 days p.i. and one of six mice examined 28 days p.i. showed lesions which comprised reactive and dystrophic changes in the white matter. Spongiform lesions and pycnotic nuclei were not seen at these times. Viral nucleocapsids were seen in the early stages of the disease in putative necrotic oligodendrocytes. Mature virus particles were not seen. This was in contrast to mice infected with virulent wild-type SFV when lesions were more severe and were accompanied by large numbers of immature and mature virus particles. It is suggested that the demyelination in mice infected with mutant SFV results primarily from selective destruction of oligodendrocytes by the mutant virus.