Critical factors influencing prion inactivation by sodium hydroxide.

BACKGROUND AND OBJECTIVES: Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases caused by aberrantly folded cellular proteins (PrP(Sc); prions) that are generally resistant to conventional pathogen-inactivation techniques. To ensure effective decontamination and inactivation of prions that could be present in source material, we investigated critical factors that influence prion inactivation by NaOH. MATERIALS AND METHODS: A decrease in prion infectivity correlates with the disappearance of the protease-resistant core of PrPSc (PrPRES) observed in biochemical assays. To model prion inactivation, hamster scrapie (strain 263K) brain homogenate (SBH) was incubated for specific periods of time in 0.1 m NaOH at 4 or 18 degrees C, with or without detergent. Neutralized samples were subjected to limited digestion with proteinase K (PK) and then analysed using an endpoint dilution western blot assay and antibody 3F4. Structural changes in prions exposed to NaOH were examined using differential immunoprecipitation. RESULTS: Treatment of SBH with 0.1 m NaOH for 15 min, in the absence of detergent, at 4 and 18 degrees C caused a reduction in the PrP(RES) signal of 3.5 and 4.0 log10 units, respectively, with some residual signal remaining. The presence of the detergent sarkosyl during a 60-min incubation in NaOH further enhanced PrPRES reduction to > or = 4.5 log10 units (i.e. below the limit of detection). NaOH treatment induced conformational changes in PrP that resulted in the exposure of a hidden epitope and enabled prion immunoprecipitation by antibody 3F4. CONCLUSIONS: The use of NaOH can effectively reduce prion levels in an in vitro inactivation assay. After pretreatment of SBH with detergent, NaOH completely eliminates the PrPRES signal. Detergent may liberate lipid membrane-protected PrPSc to improve access to NaOH, which can then inactivate PrPSc by altering its structure. In cases of unidentified exposure to PrPSc during manufacturing, sanitizing procedures combining the use of detergent and NaOH may help to ensure minimal levels of contamination carryover in products.

[The effect of immunoglobulin M-enriched intravenous immunoglobulins against bacterial infections and on the neutralization of bacterial toxins]. / Untersuchung zur Wirksamkeit von Immunoglobulin M-angereicherten, intravenösen Immunoglobulinen gegen bakterielle Infektionen und zur Neutralisation bakterieller Toxine.

Studies on the Efficacy of Immunoglobulin M Enriched Intravenous Immunoglobulins against Bacterial Infections and in Neutralization of Bacterial Toxins. An essential component of the new i.v. immunoglobulin product Pentaglobin is immunoglobulin M (IgM), which is concentrated in this preparation. The efficacy of this IgM containing preparation in comparison to conventional i.v. immunoglobulins was demonstrated in mouse protection tests and by in vitro neutralization of bacterial toxins. The IgM containing immunoglobulin preparation has significantly higher protection rates against gram-positive and gram-negative bacteria in mouse protection tests compared to immunoglobulin G (IgG) preparations. Bacterial toxins of Pseudomonas aeruginosa and Staphylococcus aureus are likewise more effectively neutralized by the IgM containing immunoglobulin preparation.

[Monitoring of sterilization procedures for plasma derivatives using bacteriophages]. / Kontrolle von Sterilisationsverfahren für Plasmaderivate mit Bakteriophagen.

Since 1968 the combination of beta-propiolactone (beta-PL) plus UV-inactivation for the sterilization of plasma derivatives is in use at Biotest. In 1981, a bacteriophage test system was established for routine monitoring of the efficacy of this sterilization procedure, using the bacteriophages phi x 174, phi e, Kappa and f2. In the period of 1981 to 1986, 88 control experiments were performed under production conditions demonstrating a mean inactivation of these test viruses of greater than or equal to 6.7 log10. This constant and high efficacy of the beta-PL/UV sterilization procedure guarantees the longstanding safety of beta-PL/UV sterilized blood derivatives. Bacteriophages are also useful experimental viruses for monitoring the efficacy of pasteurization processes.

Efficacy of intravenous immunoglobulin preparations against viral and bacterial infections in mouse protection tests.

Our investigation indicates that pretreatment of human immunoglobulin for the elimination of the anticomplementary activity is associated with a loss of activity, the extent of which depends on the type of treatment applied. Laboratory preparations of human IgG were tested in a mouse protection assay using influenza A2-Taiwan virus, tetanus toxin and Salmonella typhimurium as the challenge. There was a 7-28% reduction in efficacy in an intravenous 7S preparation in comparison with an untreated 7S IgG. F(ab')2 fragments showed a 24-65% and Fab fragments an 80-100% reduction in efficacy. Two commercial human 7S products showed approximately 90% efficacy in the Salmonella assay; a commercial, pepsin-treated preparation showed 65-74% efficacy when compared with untreated 7S IgG.

Comparison of in vitro behaviour and in vivo efficacy of two 7S-immunoglobulin preparations for intravenous use.

Two intravenous 7S-immunoglobulin (IgG) preparations, beta-propiolactone(beta-PL)-treated (Intraglobin) and hydrochloric acid/pepsin-treated, were tested for their in vitro behaviour in haemagglutination, anticomplementary activity and binding to SPA-sepharose. These results were compared with the efficacy of these preparations in vivo, using mouse protection tests with Salmonella and Pseudomonas as a challenge. In spite of marked differences in the in vitro behaviour, both preparations showed almost the same efficacy in vivo. These results indicate that in vitro findings do not allow predictions of the in vivo efficacy of intravenous IgG preparations.

[Beta-propiolactone as a sterilizing agent in the manufacture of intravenous immunoglobulin preparations]. / beta-Propiolacton als sterilisierendes Agens bei der Herstellung eines intravenösen Immunoglobulin-Präparates.

It was demonstrated with phi X174, phi e and x phages that the beta-propiolactone step, which eliminated the anticomplementary activity of the intravenous immunoglobulin (IgG) preparation. Intraglobin, inactivates more than 10(6) viruses/ml. This means additional safety with respect to those viral diseases for which specific and sensitive diagnostic methods are not available at present.

[Properties and efficacy of a human immunoglobulin M preparation for intravenous administration]. / Eigenschaften und Wirksamkeit eines humanen Immunglobulin M-Präparates für die intravenöse Anwendung.

From Cohn fraction III a new immunoglobulin (Ig) preparation (Pentaglobin) was prepared. This preparation contains 72% IgG and is enriched in IgM (12%) and IgA (16%). Due to a treatment with beta-propiolactone it is suitable for intravenous application. This IgM-enriched immunoglobulin preparation is prominent in high antibody titers (passive hemagglutination) against gram-negative as well as gram-positive germs and shows significantly higher efficacy in mouse protection tests than intravenous standard IgG. The high IgM content of this preparation in particular is responsible for the binding of bacterial antigens. Thus a marked advancement in the treatment of bacterial infections is to be expected by this new intravenously tolerable immunoglobulin preparation.

Antibodies from colostrum in oral immunotherapy.

An immunoglobulin preparation for oral use was prepared from pooled bovine colostrum from more than 100 animals. The preparation has high antibacterial antibody titres, and a high capacity for the neutralization of bacterial toxins. It is well tolerated and highly effective in the treatment of severe diarrhoea, e.g. in AIDS patients. The preparation is spray-dried and stable at 2-8 degrees C.

Inactivation of lipid enveloped viruses by octanoic Acid treatment of immunoglobulin solution.

Inactivation of lipid enveloped viruses by treatment with octanoic acid has been investigated for three intravenous immunoglobulin preparations, using Human Immunodeficiency Virus, Bovine Viral Diarrhoea Virus, Sindbis Virus and Pseudorabies Virus as test viruses. At a concentration of 7.45 g octanoic acid per kg solution complete inactivation of lipid enveloped viruses to below detectable level (>5.36, >4.68, >6.25 and >5.55 log(10), respectively) was achieved within the first minutes of treatment. Octanoic acid treatment as described here, has been demonstrated as an effective and rapid virus inactivation procedure, which shows high robustness at the tested ranges of temperature, pH and protein content of the test material. However, pH must be considered as a critical parameter of treatment, as octanoic acid fails to inactivate lipid coated viruses at basic pH. At suitable conditions, e.g. pH<6.0 and a concentration of >3.7 g/kg, octanoic acid treatment gives reliable and highly effective inactivation of lipid enveloped viruses.

Studies on the control of development. Accumulation of guanosine tetraphosphate and pentaphosphate in response to inhibition of protein synthesis in Bacillus subtilis.

Bacillus subtilis cells accumulate unusual phosphorylated substances at the end of logarithmic growth in a semi-synthetic medium. Two of these substances are guanosine 3'(2')-diphosphate 5'-diphosphate (ppGpp) and guanosine 3'(2')-diphosphate 5'-triohosphate (pppGpp) which, in contrast to amino-acid-starved Escherichia coli cells, are not degraded in sporulating cells of B. subtilis after the addition of chloramphenicol. Moreover, inhibition of protein synthesis in growing cells of B. subtilis causes accumulation of ppGpp and pppGpp, which is also in contrast to E. coli. This was shown by isolation and characterization of substances produced in these cells after the addition of chloramphenicol. Other inhibitors of protein synthesis acting at the ribosomal level also cause the accumulation of ppGpp and pppGpp. There is no difference between the action of antibiotics affecting 50-S and/or 30-S ribosomal subunits, since chloramphenicol, tetracycline erythromycin and neomycin cause the accumulation of almost equal amounts of these nucleotides. This apparently resolves the close connection between ppGpp accumulation and the rate of stable RNA synthesis, which was believed to exist also in B. subtilis because of the stringent response observed after amino acid starvation coupled with ppGpp accumulation. Antibiotics which inhibit protein synthesis differently than by affecting the ribosomes (puromycin) or which inhibit RNA (rifampicin) or DNA (nalidixic acid) synthesis do not cause ppGpp accumulation. The accumulation of ppGpp and pppGpp in the presence of charged tRNA provided by chloramphenicol treatment suggests that the signal for the synthesis of unusual nucleotides is an inhibition of the binding of tRNA (charged or uncharged) to the acceptor site of the ribosome. This activates the rel gene product which forms ppGpp and pppGpp from GTP and ATP. Sporulating cells of B. subtilis without chloramphenicol treatment produce besides ppGpp and pppGpp other unusual substances, which are likely to be highly phosphorylated nucleotides contained adenine as base moiety.

Validation of virus inactivation and removal for the manufacturing procedure of two immunoglobulins and a 5% serum protein solution treated with beta-propiolactone.

Intravenous immunoglobulins and serum protein solutions are manufactured from human plasma pools of healthy, screened donors. A step-by-step validation of virus removal and/or inactivation was performed for the manufacturing process, which includes cold ethanol fractionation, beta-propiolactone (beta-PL) treatment, UV irradiation, thermal inactivation and other chemical and physical purification steps. The total viral clearance factors achieved for the entire manufacturing process were by several magnitudes greater than the potential virus load of current plasma pools. Human immunodeficiency virus 1 (HIV-1) infectivity was reduced by > 13.4 log for 7S immunoglobulin, > 15.3 log for IGM enriched immunoglobulin and > 16 log for a 5% serum protein solution. In addition, high clearance rate for a broad spectrum of model viruses was demonstrated for all three blood derivatives being > 23.2 to > 27.8 log for pseudo rabies virus (PSR), > 12.3 to > 22.6 log for vesicular stomatitis virus (VSV) and 6.9-10.6 log for simian virus 40 (SV40). For the beta-propiolactone inactivation step Hepatitis C model viruses, e.g. equine arteritis virus (EAV) and bovine viral diarrhoea virus (BVDV) were also investigated.

Studies on the control of development. Correlation of initiucleotides in Bacillus subtilis.

Unusual highly phosphorylated nucleotides are found in sporulating cells of Bacillus subtilis. Adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp (highly phosphorylated nucleotide I), and adenosine 3(2')-dephosphate 5'-triphosphate, pppApp (highllls are starved for carbon and nitrogen sources. These nucleotides are correlated with sporulation because only ribosomes from sporulating but not vegetative cells are able to synthesize ppApp and pppApp in vitro. Two other nucleotides, adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp (highly phosphorylated nucleotide IV), and a nucleotide with a tentative structure of ppZpUp (highly phosphorylated nucleotide III), where Z is an undetermined sugar, also seem to be involved in regulation of sporulation, especially initiation of sporulation. Sporulation can be initiated even in the presence of amino acids, salts, vitamins etc. in logarithmically growing or stationary-phase cells when carbon sources, i.g. glucose, are used up or artifically removed from the medium. A drastic increase in spore titer is observed 4--5 h later. Also, carbon starvation causes accumulation of the highly phosphorylated nucleotides pppAppp and ppZpUp. On the other hand, sporulation is prevented under the same conditions when excess glucose is maintained in the medium. Correlated with this inhibition of sporulation is the inhibition of formation of highly phosphorylated nucleotides, pppAppp and ppZpUp. Since synthesis of these nucleotides is closely related to sporulation, we anticipate that these substances can cause initiation of development in B. subtilis. Further evidence for our hypothesis on initiation of sporulation by highly phosphorylated nucleotides is that phosphate starvation also causes sporulation with prior accumulation of pppAppp and ppZpUp. Apparently, as long as phosphate is present to synthesize phosphorylated metabolites of glucose, formation of highly phosphorylated nucleotides is repressed. Derepression occurs when either lack of glucose or phosphate or both prevents synthesis of phosphorylated metabolites of glucose allowing synthesis of highly phosphorylated nucleotides. These nucleotides, representing the signal 'lack of glucose or phosphate', then somehow cause changes in gene activity, initiating the complex process of sporulation. Whether or not pppAppp alone or together wtih ppZpUp or even further substances (nucleotides, proteins etc.) is necessary for the above described processes will be answered with the help of suitable mutants lacking the ability to synthesize either one or both regulatory nucleotides. Guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp, are not involved in regulation of devlopment as is shown by using a normally sporulating mutant of B. subtilis, unable to synthesize these nucleotides.

Inactivation of the Hutchinson strain of hepatitis non-A, non-B virus in intravenous immunoglobulin by beta-propiolactone.

beta-propiolactone (beta-PL) treatment has been evaluated for its ability to inactivate 10(3.5) chimpanzee infectious doses (CID50) of the Hutchinson strain of hepatitis non-A, non-B virus (HNANBV). Two chimpanzees were inoculated with a beta-PL-treated immunoglobulin solution to which this dose of the titrated virus had been added prior to beta-PL treatment. beta-PL treatment was performed in accordance with the production procedure used for a licensed intravenous immunoglobulin preparation. Neither animal developed hepatitis. When subsequently challenged with the same spiked immunoglobulin solution that had not been beta-PL treated, both animals developed clear-cut hepatitis non-A, non-B. The results of this experiment demonstrate that beta-PL treatment is effective for the inactivation of hepatitis non-A, non-B virus in intravenous immunoglobulin.

Immunoglobulin substitution therapy in a patient with primary hypogammaglobulinaemia and anti-IgA antibodies.

Permanent immunoglobulin substitution therapy was performed in a 44-year-old patient with common variable immunodeficiency, recurrent respiratory tract infections, total absence of serum IgA and a high titre of class-specific anti-IgA antibodies. An IgA-depleted i.v. immunoglobulin (IG) preparation was used. Infusions were well tolerated by the patient although minor anaphylactoid symptoms regularly occurred. Anti-IgA antibody titres rose during the first 4 months of treatment and gradually fell during the following 8 months. Regular IG substitution therapy led to a substantial improvement in the patient's health and quality of life.

Inactivation of the Hutchinson strain of non-A, non-B hepatitis virus by combined use of beta-propiolactone and ultraviolet irradiation.

A beta-propiolactone/ultraviolet irradiation procedure (beta PL/UV) has been evaluated for its ability to inactivate 30,000 chimpanzee infectious doses of the Hutchinson strain of non-A, non-B (NANB) virus. The chimpanzees were inoculated with plasma to which this dose of the titrated virus had been added prior to application of the beta PL/UV process in accordance with a procedure used for licensed blood derivatives in Germany. Neither animal developed hepatitis. When subsequently challenged with the same contaminated plasma, which had not been sterilized, both animals promptly developed typical NANB hepatitis. This study extends the high (approximately 10(7)-fold) process efficiency of the beta PL/UV procedure previously reported for hepatitis B virus to a blood-borne NANB virus.

Inactivation of HIV in plasma derivatives by beta-propiolactone and UV irradiation.

The inactivation of HIV in human plasma and plasma derivatives by combined treatment with beta-propiolactone and UV-irradiation was investigated. beta-propiolactone inactivated greater than or equal to 3.5 log10 and UV greater than or equal to 2.8 log10 HIV in plasma and beta-propiolactone greater than or equal to 3.5 log10 in cryoprecipitate and UV irradiation greater than or equal to 4.5 log10 in factor VIII concentrate.

Treatment of diarrhoea in human immunodeficiency virus-infected patients with immunoglobulins from bovine colostrum.

Diarrhoea and weight loss are found in more than 50% of patients with the acquired immunodeficiency syndrome (AIDS). In some patients the symptoms can be very severe, leading to death even in the absence of opportunistic infections. In 30% of these patients, enteric pathogens cannot be identified, and approximately only half of the identifiable aetiologic agents of diarrhoea in patients infected with the human immunodeficiency virus (HIV) were treatable with antibiotics. Immunoglobulins from bovine colostrum (Lactobin, Biotest, Dreieich, FRG) contain high titers of antibodies against a wide range of bacterial, viral and protozoal pathogens as well as against various bacterial toxins. Lactobin (LIG) is quite resistant to 24-h incubation with gastric juice. In a multi-center pilot study 37 immunodeficiency patients with chronic diarrhoea [29 HIV-infected patients, 2 patients with common variable immunodeficiency (CVID), one unidentified immunodeficiency, five patients with graft versus host disease (GvHD) following bone marrow transplantation] were treated with oral LIG (10 g/day for 10 days). Good therapeutic effects were observed. Out of 31 treatment periods in 29 HIV-infected patients 21 gave good results leading to transient (10 days) or long-lasting (more than 4 weeks) normalisation of the stool frequency. The mean daily stool frequency decreased from 7.4 to 2.2 at the end of the treatment. Eight HIV-infected patients showed no response. The diarrhoea recurred in 12 patients within 4 weeks (32.4%), while 19 patients were free of diarrhoea for at least 4 weeks (51.3%). In 5 patients intestinal cryptosporidiosis disappeared following oral LIG treatment. LIG treatment was also beneficial in 4 out of 5 GvHD patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Improvement of virus safety of a S/D-treated factor VIII concentrate by additional dry heat treatment at 100 degrees C.

In order to increase the virus safety of a solvent/detergent-treated Factor VIII concentrate in regard to non-lipid coated viruses and to respond to the continuous discussion about reports on hepatitis A transmission by Factor VIII preparations, we have investigated the effect of a terminal dry heat treatment (30 min 100 degrees C) on HAV and various other viruses. By this treatment Hepatitis A virus was inactivated below detectable level after a few minutes (> 5.3 log10). Other RNA viruses such as the Human Immunodeficiency Virus (> 6.6 log10), bovine viral diarrhoea virus (> 6.6 log10) and vesicular stomatitis virus (> 5.8 log10) were also inactivated below detectable level. Pseudo rabies virus and reovirus Type 3 are inactivated by 5.7 and > 6.0 log10, respectively. SV40 and bovine parvo virus showed significant resistance to dry heat treatment. We conclude that the involvement of two strong virus inactivation steps, acting by different mechanisms, improves the virus safety of Factor VIII concentrates without destroying the Factor VIII activity. Moreover, the terminal 100 degrees C heat treatment for 30 min represents an effective measure to inactivate non-lipid enveloped viruses, in particular hepatitis A, which is resistant to solvent/detergent treatment.