Analysis of human plasma products: polymerase chain reaction does not discriminate between live and inactivated viruses.

BACKGROUND: The viral safety of human plasma products is based on the careful selection of donors and donations and the removal and inactivation of human pathogenic viruses that could potentially contaminate human plasma. For the analysis of the final products for potential virus contamination, the use of polymerase chain reaction (PCR) has been proposed. To test whether this method can discriminate between infectious and inactivated viruses, the following studies were performed. STUDY DESIGN AND METHODS: Infectious and virus-inactivated preparations were titrated with specific PCR, using viruses such as hepatitis B virus (HBV), hepatitis C virus, bovine viral diarrhea virus, and poliovirus. The inactivation method employed was pasteurization (10 hours, 60 degrees C) or solvent/detergent (SD) treatment; in the case of HBV, there was consecutive treatment by both methods. RESULTS: Pasteurization of HBV and hepatitis C virus as well as SD treatment of HBV or pasteurization of HBV followed by SD treatment did not affect the detectability of these viruses by PCR, whereas an infectivity study in chimpanzees demonstrated that infectious hepatitis C virus was inactivated by pasteurization. Pasteurization also had no effect on the PCR titers of stabilized bovine viral diarrhea virus or poliovirus preparations, but it destroyed the infectivity of these viruses completely after only 4 hours' heat treatment. CONCLUSION: Pasteurization or SD treatment destroys the infectivity of the viruses tested, but neither significantly affects their detectability by specific PCR. Therefore PCR is not a suitable measure for testing the viral safety of finished plasma products that have been subjected to virus inactivation.

[Nanofiltration in production of Beriplex P/N: increasing the capacity of virus elimination while maintaining product quality]. / Nanofiltration bei der Herstellung von Beriplex P/N: Erhöhung der Kapazität zur Viruseliminierung unter Beibehaltung der Produktqualität.

For the manufacture of the PCC Beriplex P/N, nanofiltration was introduced into the production process of Beriplex HS providing an additional means to heat treatment for the clearance/inactivation of viruses. By nanofiltration, large enveloped viruses (HSV-1, HIV-1) were completely eliminated by a factor of more than 7 log10. While medium-sized enveloped viruses (HBV, BVDV) were cleared by a factor of approximately 4 log10, small non-enveloped viruses (poliovirus) were not removed. The product profile remained, no thrombogenic activities were detected.

[HIV safety of commercially produced human plasma proteins]. / HIV-Sicherheit industriell hergestellter humaner Plasmaproteine.

An important aim of the commercial manufacturing of human plasma proteins to be used as therapeutics is the HIV-safety of such products. This aim will be achieved by using (1) plasma donations of carefully selected, healthy donors, (2) by testing of each donation according to national and international requirements for antibodies or antigens specific for certain viruses, (3) by eliminating viruses by different purification procedures of the manufacturing process and (4) by inactivating viruses by a specific method included in the production process. Due to the current discussion in Germany this paper will particularly focus on HIV. As an example, the experimental studies of the manufacturing process of pasteurized factor VIII proving the elimination of HIV by various stages of the production process and its complete inactivation by pasteurization (= 10 h heat treatment of the stabilized, aqueous factor VIII-solution at 60 degrees C) is discussed. A cumulative reduction factor of > 10(16) is achieved by the different stages of the entire manufacturing process, including pasteurization. In fact, the HIV-inactivation is by some orders of magnitude higher than demonstrated by the special inactivation experiment, because the heat treatment of the production procedure consists of 10 hours instead of one hour as needed under experimental conditions for the complete inactivation of HIV. If the manufacturing procedure of a human plasma protein contains a method which completely inactivates HIV and which in total results in a cumulative reduction factor of > 10(12) for HIV, the final product is regarded as being free of infectious HIV.

Inactivation of hepatitis A virus by pasteurization and elimination of picornaviruses during manufacture of factor VIII concentrate.

Hepatitis A virus (HAV) infections have been reported among hemophiliacs who received factor VIII concentrates which had been purified by ion-exchange chromatography and treated by the solvent detergent (SD) method. Since the virus inactivation procedure of our manufacturing process is heat treatment of the stabilized, aqueous protein solution at 60 degrees C for 10 h (pasteurization), we investigated whether this method inactivated picornaviruses such as HAV and poliovirus type 1, which we routinely use as a test virus for non-enveloped viruses. HAV was substantially inactivated by pasteurization but the stabilizers used in the manufacturing process of the commercial products considerably delayed HAV inactivation. Residual infectious HAV was found even after 10 h heat treatment of the stabilized preparation. Thus HAV is more stable in the presence of stabilizers than poliovirus type 1. Furthermore, we studied stage by stage the elimination of poliovirus type 1 by the manufacturing procedure of these pasteurized factor VIII concentrates. Three other stages of the manufacturing process apart from pasteurization eliminated poliovirus by approximately three orders of magnitude each. Taking into account this efficient elimination of the picornavirus poliovirus and the substantial inactivation of HAV by pasteurization, we conclude that a high margin of safety exists for pasteurized factor VIII concentrates regarding HAV. This conclusion is supported by the fact that no HAV infection has been reported in hemophilia patients treated with pasteurized factor VIII concentrates. Furthermore, in a retrospective study, none of 95 patients subjected to a long-term treatment with pasteurized factor VIII concentrates had developed anti-HAV seroconversion as a result of this treatment.

Inactivation of hepatitis A virus by heat treatment in aqueous solution.

Hepatitis A virus infections have been reported recently among hemophilic patients in Italy and Germany, leading to speculation that infectious hepatitis A virus (HAV) might have been present in some factor VIII concentrates. In both cases, the implicated factor concentrates had been treated by a solvent/detergent method, which inactivates enveloped viruses but which would not be expected to inactivate HAV, a nonenveloped picornavirus. To determine whether HAV would be inactivated during pasteurization of factor VIII concentrate, an alternative method employed for virus inactivation, we determined the extent to which the infectivity of cell culture-adapted HAV, suspended either in cell culture medium or in a proprietary stabilizing buffer, was reduced by heat treatment at 60 degrees C for 10 hr. The titer of infectious HAV declined rapidly at 60 degrees C, but the stabilizer considerably delayed HAV inactivation. In cell culture medium, HAV was inactivated by > 3.6 log10 within 30 min, but 3.6 log10 inactivation of HAV was reached only after 6 hr in the presence of the stabilizer. Residual infectious HAV was present after even 10 hr of heat treatment in the stabilizer, indicating that < 5.2 log10 infectious HAV particles are inactivated under these conditions. In the presence of the stabilizer, HAV was significantly more stable than poliovirus type 1, which has been used to validate virus inactivation by pasteurization. We conclude that pasteurized factor VIII concentrate should pose little if any risk for transmission of HAV if pooled plasma used for its manufacture contained low levels of the virus.

Theoretical and technical concerns in inactivation/elimination of viruses in plasma derivatives.

To know the virus eliminating/inactivating capacity of the manufacturing process of a plasma protein, it is essential to analyse it by adding virus to the source material or to different materials obtained at various stages of the manufacturing procedure and then to determine the elimination/inactivation of this virus. To carry out such experiments properly, three prerequisites have to be fulfilled: (i) the manufacturing procedure must be scaled down as exactly as possible; (ii) relevant test viruses have to be selected for the spiking experiments and (iii) the resulting samples must be assayed properly for infectious virus. The successful reduction of a manufacturing procedure to a more than 1000-fold smaller scale has to be validated to prove that it corresponds to the production scale. The most important viruses of risk in human plasma are hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). HIV is the only one of these viruses which can be tested in vitro. A decision has therefore to be made concerning which other viruses should be used. The selection of test viruses depends on (i) the relationship of these candidates to the viruses of risk; (ii) the possibility of growing them to high titres in vitro and (iii) the availability of accurate infectivity assays. The use of highly sensitive assays is necessary to be able to determine small amounts of residual viruses. Since such assays are based on a 7 to 28 day incubation of the virus samples on cell cultures, these samples must be sterile and non-cytotoxic.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of HIV, HBV, HCV related viruses and other viruses in human plasma derivatives by pasteurisation.

Laboratory studies were made to verify the capacity and efficacy of the pasteurisation step (heat treatment at 60 degrees C for 10 hours in stabilized aqueous solution) in inactivating various pathogenic viruses (e.g. HIV, HBV, HCV related viruses, HAV, HSV, poliovirus). These experiments proved that pasteurisation effectively inactivates a broad spectrum of viruses with a range of different physicochemical properties. The results, summarized in inactivation kinetics, demonstrate that complete inactivation of high quantities of both enveloped (e.g. HIV, HCV related viruses) and non-enveloped (e.g. poliovirus) viruses takes place during pasteurisation. In clinical trials on the virus safety of plasma derivatives no case of AIDS, hepatitis B or C occurred. From these data we conclude that heat treatment at 60 degrees C in a stabilized aqueous solution confers a high margin of safety on human plasma protein preparations regarding the transmission of diseases such as AIDS or hepatitis.

Antiviral effects of different CD4-immunoglobulin constructs against HIV-1 and SIV: immunological characterization, pharmacokinetic data and in vivo experiments.

The CD4 cell surface antigen belongs to the immunoglobulin superfamily and is the primary receptor for the human immunodeficiency virus 1 (HIV-1). The high affinity interaction between HIV-1 and CD4 is mediated by the viral envelope glycoprotein gp120. Recombinant soluble CD4 (rsCD4) has been shown in vitro to be an effective inhibitor of HIV-1 and HIV-2 propagation in lymphoid cells. A variety of antibody-like molecules were constructed, consisting of different parts of the extracellular domain of CD4 fused to immunoglobulin constant regions. The fusion proteins were expressed in mammalian cell lines and purified via affinity chromatography. The specificity and anti-viral effects of the different CD4-immunoglobulin constructs against HIV were analysed by different immunological tests, i.e., immunofluorescence, neutralisation and in vitro assays. In pharmacokinetic studies, differences were found in serum half-life between the four- and two-domain CD4 constructs in cynomolgus monkeys and between glycosylated and deglycosylated CD4-Fc constructs in rabbits. In two in vivo experiments using the four-domain CD4-Fc in SIV-infected macaques, no beneficial effects were observed.

Immune response of chimpanzees after immunization with the inactivated whole immunodeficiency virus (HIV-1), three different adjuvants and challenge.

Purified whole virus preparations of HIV-1 were produced from supernatants of infected cells and concentrated 5000-fold. After inactivation with formaldehyde, the concentrates were combined with one of three different adjuvants, and used to immunize three groups of three chimpanzees each. The chimpanzees were monitored for HIV-specific humoral and cellular immune responses by ELISA, immunoblot, virus neutralization, delayed-type hypersensitivity, lymphocyte proliferation and antibody-dependent cell-mediated cytotoxicity. Weak and inconsistent responses were observed in animals that received HIV-1 formulated with alum as adjuvant, whereas HIV-1 formulated with incomplete Freund's adjuvant or an experimental adjuvant (BWZL) induced good humoral and cellular immune responses to the virus. The three animals that received HIV-1 with the BWZL adjuvant generated overall the best immune responses; therefore, 2 weeks after the sixth immunization these animals were challenged with infectious HIV-1. Despite the presence of good humoral and cell-mediated immunity, all three immunized animals and a control animal became infected within 4 weeks, as evidenced by repeated isolation of HIV-1 from peripheral blood mononuclear cells and anamnestic antibody responses. The new experimental adjuvant has to be further investigated in other vaccine trials and different animal models.

Follow-up of hepatitis C virus infection in chimpanzees: determination of viraemia and specific humoral immune response.

Chimpanzees were inoculated intravenously with the H strain of hepatitis C virus (HCV), and analysed for viraemia using the polymerase chain reaction and for a humoral immune response using first and second generation anti-HCV ELISAs and an immunoblot assay (4-RIBA). In all seven chimpanzees studied, viraemia occurred several weeks before a significant increase in serum alanine transferase (ALT) activity, whereas the first circulating anti-HCV antibodies became detectable at the time of significant increase in ALT levels, provided the second generation ELISA or 4-RIBA was used. On the basis of the duration of viraemia the chimpanzees studied could be assigned to two different groups: those in which viraemia disappeared in conjunction with or shortly after seroconversion, and those remaining viraemic for many weeks after the appearance of antibodies. The clearance of HCV from the circulation did not correlate with the antibody pattern determined using 4-RIBA, i.e. the HCV-specific assays currently available do not enable us to predict whether an infected chimpanzee will develop persistent viraemia. Only two of the seven chimpanzees analysed developed anti-core protein (c-22) antibodies, which appeared at the same time as the first ALT peak, whereas all animals developed antibodies to the non-structural protein, c-33, and these antibodies persisted.

Virus safety of human immunoglobulins: efficient inactivation of hepatitis C and other human pathogenic viruses by the manufacturing procedure.

Human immunoglobulins are plasma derivatives with a low risk of transmitting viral infections. To the present, no proven case of human immunoglobulins transmitting human immunodeficiency viruses has been reported. However, there have been a few reports on the transmission of hepatitis C virus by these plasma proteins. To improve further the safety of both 5s iv human immunoglobulins and 7s im immunoglobulins, we introduced a 10-hour heat treatment of the aqueous solutions at 60 degrees C (i.e., pasteurization) into the manufacturing procedure. This treatment was not added to the manufacturing procedure of 7s iv immunoglobulin that already contained the S-sulfonation as a virus inactivating method. We now report on experimental data that show that the whole manufacturing procedures of the above immunoglobulins inactivate efficiently hepatitis C virus and that the specific virus inactivation methods alone, namely, pasteurization or S-sulfonation, also inactivate completely viruses of the flavivirus family, to which the hepatitis C virus belongs. The inactivation of the Flaviviridae bovine viral diarrhea virus, tick-borne encephalitis virus, and yellow fever virus by pasteurization or S-sulfonation was at least 10(5). The clearance of HCV achieved by the entire manufacturing process of each of these immunoglobulins was also at least 10(5). The experiments therefore show that pasteurization or S-sulfonation provides a high margin of safety to human immunoglobulins regarding the transmission of hepatitis C virus.

A rabies-specific human monoclonal antibody that protects mice against lethal rabies.

According to a recommendation from WHO (World Health Organisation) for prevention of a possible rabies infection, active vaccination has to be combined with application of immunoglobulin to get a fast protective effect. At present, preparations of purified human or equine rabies-specific immunoglobulin are used. We have generated a human rabies-specific monoclonal antibody (huMAb) by immortalization of human B-cells with Epstein Barr Virus (EBV), followed by fusion with a mouse myeloma cell. The resulting clone TW-1 secrets an IgG1 lambda huMAb which specifically reacts in ELISA with 5 laboratory rabies virus strains of serotype 1 and DUV3 (Duvenhage, serotype 4). Western Blot analysis revealed fine specificity for the G glycoprotein (gp67) of rabies virus. HuMAb TW-1 neutralizes rabies virus in vitro (RFFIT) as well as in vivo and protects rabies infected mice. Compared to polyclonal human rabies immunoglobulins, huMAb TW-1 is advantageous, because of its defined specificity and the very low amounts of total protein needed for therapeutic effects.

Protection against European isolates of tick-borne encephalitis virus after vaccination with a new tick-borne encephalitis vaccine.

A highly purified, inactivated tick-borne encephalitis (TBE) virus particle vaccine has been developed. In this study we report on the efficacy of this new vaccine to protect against TBE virus isolates from different geographical areas of Europe and the Asian part of the USSR.

Inactivation of retroviruses in biologicals manufactured for human use.

Human immunodeficiency virus may occur in human plasma and mammalian retroviruses in established cell lines used for the production of monoclonal antibodies or recombinant proteins. To avoid any risk of retrovirus infections being transmitted to human patients by human plasma proteins or other biologicals obtained from established cell lines, these products must be free of contaminating retroviruses. This can be achieved by excluding contaminated source material and by establishing manufacturing procedures which inactivate and eliminate retroviruses. Some methods were investigated, and it was proved that these methods not only completely inactivate retroviruses, but that they are at the same time mild enough not to destroy the biological activities of the substances to be used as therapeutics. In addition, the clearance of retroviruses by the entire manufacturing process of an immunoglobulin, human plasminogen, a murine monoclonal antibody and a recombinant human protein were studied. In each case cumulative clearance factors of greater than 10(12) were achieved for the retroviruses used in these spiking experiments. It can be concluded that these high clearance factors contribute a very high margin of safety to the biologicals manufactured.

Antibody- and complement-mediated lysis of HIV-infected cells and inhibition of viral replication.

HIV-1-positive antisera were tested for their ability to lyse HIV-1-infected cells in the presence of active complement. Cytolytic effects caused by sera derived from infected humans were slower than those observed with sera from immunised chimpanzees. Lytic but also negative sera were found among HIV-1-infected asymptomatic men as well as among clinical AIDS cases. Human antisera that lysed infected cells reacted similarly irrespective of whether the complement was heterologous or autologous. Analysis of complement-mediated lysis using defined antisera against recombinant HIV-1 env or core antigens suggested that gp160/gp120 and p24 can act as target antigens for an antibody- and complement-mediated cytolysis of infected cells. Complement alone reduced the spread of HIV-1 infection in CD4+ cells and the ability of HIV-1 and HIV-2 to form plaques in CD4-transfected HeLa cells. Co-operative effects of specific antibodies and complement were the most effective in inhibiting HIV infections.

Escherichia coli-derived envelope protein gD but not gC antigens of herpes simplex virus protect mice against a lethal challenge with HSV-1 and HSV-2.

Immunization studies with HSV-1 and HSV-2 envelope proteins expressed in Escherichia coli were performed. After active immunization of mice with a gD-1 antigen (Leu53-Ala312) expressed as a fusion protein, the animals were protected from a lethal challenge with HSV-1 and HSV-2. In addition, antisera from rabbits immunized with the same gD-1 antigen also conferred passive immunity to mice against a challenge infection with either HSV-1 or HSV-2. In contrast to these successful gD-1 protection experiments, various gC-1 and gC-2 fusion proteins from E. coli failed to induce protective immunity. Moreover, the mice sera from immunized animals were not able to react with the authentic, glycosylated gC-1 and gC-2 envelope proteins, whereas sera raised against authentic gC-1 and gC-2 glycoproteins do recognize the gC fusion proteins from E. coli. These results indicate, that E. coli might represent an ideal system for expressing gD antigens as a possible component of a HSV vaccine, whereas gC antigen cannot be produced in an immunocompetent form in E. coli.

Inactivation of HIV-1 and HIV-2 by various manufacturing procedures for human plasma proteins.

Human retroviruses causing AIDS (HIV-1, HIV-2) can occur in human plasma donations. Since HIV-contaminated plasma cannot be completely excluded by testing for anti-HIV-1 (routine plasma screening for anti-HIV-2 has not yet been established), a safeguard against AIDS in therapeutics derived from human plasma can only be achieved by introducing HIV inactivating/eliminating methods into the manufacturing process of plasma derivatives. To investigate the HIV inactivating efficiency of such methods, aliquots of infectious HIV-1 or HIV-2 concentrates were added to a protein preparation, the resulting HIV spiked preparation was then treated according to the method to be studied, and the amount of infectious HIV in this preparation was determined before and after treatment. Methods by which HIV-1 or HIV-2, respectively, were completely inactivated were ethanol fractionation according to the Cohn procedure, pepsin treatment, affinity chromatography, protein precipitation by various methods, and pasteurization (heat treatment at 60 degrees C in aqueous solution). The use of these methods for manufacturing human plasma derivatives resulted in products that were free of any infectious HIV-1 or HIV-2 and thus unable to transmit AIDS.