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Reliable Inactivation of Hepatitis B Virus

Organization name

TransMIT Gesellschaft für Technologietransfer mbH


New test system for the detection of titer reductions by more than 4 log10 steps

How effective are cleaning agents and disinfectants or inactivation procedures against human hepatitis B virus? The new in vitro test with hepatocyte cultures isolated from Tupaia sp. (Southeast Asian tree shrews) and highly concentrated hepatitis B virus fractions demonstrates the efficacy of inactivation procedures achieving titer reductions by more than 4 log 10 dilution steps, thus meeting the guidelines of the Robert Koch Institute and the German Association against Virus Diseases for the first time. Not all cleaning agents or inactivation procedures are as effective in reducing HBV infectivity as peracetic acid which was used in the figure below.

Unique selling points

The test procedure demonstrates the efficacy of substances and/or methods which eliminate infectivity using several quantitative measurement parameters (HBsAg, HBeAg, mRNA, cccDNA) for the detection of HBV infection after a titer reductionby more than 4 log 10 stepsand is, thus, the first HBV infectivity test to meet the new guidelines of the Robert Koch Institute (RKI) and the German Association for the Control of Virus Diseases (DVV) for the efficacy of disinfectants and virus-inactivating procedures. Previous procedures were too insensitive and investigated only the destruction of HBV structural elements, which however does not necessarily correlate with infectivity. Alternatively, RKI and DVV approve of the bovine virus diarrhea virus (BVDV) as a general model for enveloped viruses as a test for the effectiveness of disinfectants. This virus, however, has an entirely different structure so that substancese valuated by these means cannot be explicitly considered as effective against HBV. Duck HBV, which is more closely related, is occasionally used as a model virus for HBV, but the transferability of results to human HBV is not assured here as well.

The infection is performed with purified human hepatitis B virus which was isolated from serum or plasma of HBV infected humans.

In contrast to recombinant hepatitis B virus isolated from transfected cell cultures, these viruses show higher infectivity and the characteristic resistance to substances and methods eliminating infectivity which is missing in recombinant hepatitis B viruses from cell cultures as a result of the culturing conditions. The new test system, therefore, much better reflects the actual infectivity of HBV. The comparability of virus preparations from the plasma of different donations is confirmed by DNA sequencing, protein and antigen analysis, as well as via biophysical characterization of HBV particles.

Utilization of hepatocytes from Tupaia sp. with reproducible viability and high susceptibility for HBV.

In addition to humans, only primates can be infected with HBV. Up to now, only highly differentiated hepatocytes isolated from fresh human liver could be infected with HBVin vitro. Besides the extremely limited availability, the variable susceptibility of these cultures for HBV caused further problems. Surprisingly, hepatocytes from freshlyisolated liver tissue of Asian tree shrews are also susceptible for HBV. Breeding the animals on our own ensures the availability in sufficient amounts. For the isolation of hepatocytes from Tupaia liver, a feasible standardized procedure was developed which also allows a large scope of experiments. The relevance as in vitro model for HBV infection was demonstrated by international publications in leading scientific journals (J Virol. 77:9511-21; Gastroenterology 129:234-45).

Areas of application

Surface or instrument disinfection for the elimination of pathogens.
Blood of HBV-infected donors may contain up to 1010 virus particles/ml. Since even 1-10 virusparticles can evidently lead to hepatitis B after percutaneous application in vivo, eventhe smallest blood spots are dangerous if the infectivity was not eliminated or at least significantly reduced. The detection of anti-HBV efficacy is thus of great importance for the certification of disinfectants and cleaning agents in hygiene. The majority of users explicitly request a scientifically proven anti-HBV efficacy. Based on questionable surrogate tests, several manufacturers of disinfecting agents declare an anti-HBVefficacy for their products which, however, is denied by the RKI and the DVV. Now atest system is available which allows the experimental determination of anti-HBV efficacy with reasonable expenditure.

Virus safety of blood and plasma derivatives. Another important field of applicationis the validation of virus safety for therapeutic agents which are produced from human blood or tissue. Even though excellent diagnostic procedures are available, a residualin fectivity of blood products and plasma derivatives cannot entirely be excluded. Methods for the elimination and inactivation of potentially present HBV are thus highly desirable. A quantitative proof of efficacy could only be performed in experimentally inoculated chimpanzees up to now, which, however, is not feasible. Here, the procedure discloses new perspectives.

Measurement of HBV neutralizing antibodies. Incubation with neutralizing antibodies against the antigen epitopes of HBV envelope proteins, a preventive and therapeutic method of HBV inactivation, is applicable in vivo. The new method is excellently suitable for the quantitative test of validity of Hepatitis B immunoglobulin and also for testing of new or improved active hepatitis B vaccines. The concentration of anti-HBs antibodies quantified by commercially available immunoassays is not always proportional to the neutralization titer, as experiments demonstrated.

Patent status

A German priority application was filed in November 2007.

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