Bernard Horowitz

Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-enveloped viruses by tri(n-butyl)phosphate detergent combinations.

Use of the organic solvent, tri(n‐butyl)phosphate (TNBP), and detergents for the inactivation of viruses in labile blood derivatives was evaluated by addition of marker viruses (VSV, Sindbis, Sendai, EMC) to anti‐hemophilic factor (AHF) concentrates. The rate of virus inactivation obtained with TNBP plus Tween 80 was superior to that observed with ethyl ether plus Tween 80, a condition previously shown to inactivate greater than or equal to 10(6.9) CID50 of hepatitis B and greater than or equal to 10(4) CID50 of Hutchinson strain non‐A, non‐B hepatitis. The AHF recovery after TNBP/Tween treatment was greater than or equal to 90 percent. Following the reaction, TNBP could be removed from the protein by gel exclusion chromatography on Sephadex G25; however, because of its large micelle size, Tween 80 could not be removed from protein by this method. Attempts to remove Tween 80 by differential precipitation of protein were only partially successful. An alternate detergent, sodium cholate, when combined with TNBP, resulted in almost as efficient virus inactivation and an 80 percent recovery of AHF. Because sodium cholate forms small micelles, it could be removed by Sephadex G25 chromatography. Electrophoretic examination of TNBP/cholate‐treated AHF concentrates revealed few, if any, changes in protein mobility, except for plasma lipoprotein(s).

Sterilisation of hepatitis and HTLV-III viruses by exposure to tri(n-butyl)phosphate and sodium cholate.

Blood product sterilisation with 0.3% tri(n-butyl)phosphate (TNBP)/0.2% sodium cholate (CA), a combination known to permit high recovery of factor VIII and factor IX, was evaluated for its effect on hepatitis B (HBV), non-A, non-B (NANB), and human T-lymphotropic type III (HTLV-III) viruses. 2 chimpanzees received factor VIII preparations contaminated with 10(4) chimpanzee infectious doses (CID50) of HBV and treated with TNBP/CA; neither had evidence of HBV infection during 9 months follow-up, but hepatitis B surface antigen (HBsAg) developed 5 and 6 weeks, respectively, after challenge with untreated inoculum. 2 chimpanzees were similarly exposed to 10(4) CID50 of Hutchinson NANB inoculum treated with TNBP/CA; neither became infected during 26 weeks of follow-up but both had characteristic NANB-associated ultrastructural changes 3-5 weeks after exposure to untreated inoculum. 2 chimpanzees inoculated with 80 ml of TNBP/CA-treated factor VIII derived from a pool of thirteen lots obtained from five US manufacturers remained free of any evidence of NANB infection during 32 weeks of follow-up. Subsequently, NANB infection developed in both animals 3-4 weeks after exposure to untreated inoculum. Exposure of HTLV-III diluted into a factor VIII preparation to TNBP/CA inactivated greater than or equal to 10(4.2) tissue culture infective doses within 20 min at 24 degrees C.

Virus safety of solvent/detergent-treated antihaemophilic factor concentrate

The safety of an antihaemophilic factor concentrate treated with the organic solvent tri-(n-butyl)phosphate and sodium cholate (factor VIII-SD) was assessed for transmission of non-A, non-B (NANB) hepatitis and human immunodeficiency virus (HIV). Patients enrolled in the study had no previous exposure to blood products made from plasma pools, although 5 had received small quantities of single-donor products. All but 1 had normal alanine aminotransferase (ALT) levels, none had markers of HIV infection, and all had been vaccinated against hepatitis B. After treatment with factor VIII-SD, serum ALT levels and HIV antibody were monitored for up to 1 year. 20 patients received 625 to greater than 40,000 U (total 163,000 U, median dose 3900 U), and 17 of these were followed up for at least 6 months: transmission of either NANB hepatitis or HIV was not observed.

Inactivation of viruses in blood with aluminum phthalocyanine derivatives

The inactivation of viruses added to whole blood and a red cell concentrate with aluminum phthalocyanine and its sulfonated derivatives was studied. A cell‐free form of vesicular stomatitis virus (VSV), used as a model, was completely inactivated (greater than 10(4) infectious units; TCID50) on treatment of whole blood with 10 microM (10 mumol/L) aluminum phthalocyanine chloride (AIPs) and visible light dosage of 88 to 176 J per cm2. At 44 J per cm2, complete VSV inactivation was achieved on raising the concentration of AIPc to 25 microM (25 mumol/L). Results at least as good were achieved on similar treatment of a red cell concentrate. Also inactivated were a cell‐associated form of VSV and both cell‐free and cell‐associated forms of human immunodeficiency virus; encephalomyocarditis virus, used as a model for non‐lipid‐enveloped viruses, was not inactivated by this procedure. This inactivation of cell‐free VSV suggests that a similar degree of inactivation could be achieved with a lower concentration of the sulfonated forms of aluminum phthalocyanine. Throughout the above studies, red cell integrity was well maintained, as judged by the absence of hemoglobin release (less than or equal to 2%) during the course of treatment or on subsequent storage. Red cell osmotic fragility was decreased on treatment of whole blood with AIPc. This study indicates that AIPc may be a promising method for the inactivation of viruses in cellular blood products.

New phthalocyanines for photodynamic virus inactivation in red blood cell concentrates.

Abstract Cationic phthalocyanines with either aluminum or silicon as the central metal were evaluated for their ability to inactivate viruses in red blood cell concentrates (RBCC) photodynamically. In addition, the virucidal potential of a substituted anionic phthalocyanine, aluminum dibenzodisulfophthalocyanine hydroxide (AlN2SB2POH) was evaluated and compared with that of the much studied anionic aluminum tetrasulfophthalocyanine hydroxide (AIPcS4OH). Based on the rate of inactivation of the lipid‐enveloped vesicular stomatitisvirus (VSV), the viruci dal potential of these phthalocyanines was: HOSiPCOSi(CH3)2(CH2)3N+(CH3)3I‐ (Pc 5) = SiPC[OSi(CH3)2‐(CH2)3N+(CH3)3I‐]2 (Pc 6) > AIPCOSi(CH,)2(CH2)˜N+(CH3)2(CH˜)11CH3I‐ (Pc 21) = A1N2SB2POH = AlPcS4 > HOSiPc[OSi(CH3)2(CH2)3N+(CH3)2(CH2)11CH31–]2(Pc 14) > AIPcOSi(CH3)2(CH2)3N+(CHS)3I‐ (Pc 2). Phthalocy anine ligand 14 and Pc 21 are new phthalocyanines, made by quaternizing known amino analogues. Compared to VSV, the rate of inactivation of Sindbis virus (another model lipid‐enveloped virus) was identical when treated in red blood cells (RBC) with Pc 5 and slightly higher when treated with Pc 6 and AlPcS4OH. Treatment of RBCC containing cell‐free human immunodeficiency virus (HIV‐1) with Pc 5 or AlPcS4OH required 15 min of irradiation to inactivate (>5 log10 reduction) the virus. The extent of HIV‐1 inactivation with AlN2SB2POH was 3.7 log10 after 60 min of red light exposure. The RBC integrity after photosensitization was measured by the ability of the cells to bind to plates coated with poly‐L‐lysine, (which reflects the retention of the RBC surface negative charges) and hemolysis of the cells over a 7 day storage period. The RBC damage using these criteria was most pronounced with Pc 5 and Pc 6 but could be reduced when treatment was in plasma instead of buffer. These data indicate that lipid‐enveloped viruses differ in their sensitivity to phthalocyanine photosensitization. Therefore, for virus sterilization of RBCC for transfusion the ability to inactivate human pathogenic viruses completely will have to be evaluated for each virus. The cationic Pc 5 appears to be a potentially useful virucidal agent.

IMPORTANCE OF TYPE I AND TYPE II MECHANISMS IN THE PHOTODYNAMIC INACTIVATION OF VIRUSES IN BLOOD WITH ALUMINUM PHTHALOCYANINE DERIVATIVES

The relative importance of type I and type II mechanisms in the photodynamic treatment of red blood cell concentrates (RBCC) to inactivate viruses was studied using aluminum phthalocyanine tetrasulfonate (AlPcS4), visible light and quenching or enhancing agents of reactive forms of oxygen. Treatment of a human RBCC with 10–13 μM AlPcS4 and 25–26 rnW/cm2 visible light resulted in the rapid and complete inactivation of added vesicular stomatitis virus (VSV). The addition of mannitol, glycerol, reduced glutathione (GSH), or superoxide dismutase (SOD), known quenching agents of type I mechanisms, had little to no effect on the rate of inactivation of VSV. Significant inhibition of VSV kill was observed on addition of tryptophan or sodium aide, known quenchers of type II mechanisms. Additionally, the rate of VSV kill was enhanced in the presence of D2O. Taken together, these results indicate a predominant role of singlet oxygen in the inactivation of VSV on photodynamic treatment of RBCC.

Estimating the pathogen safety of manufactured human plasma products: application to fibrin sealants and to thrombin

BACKGROUND: Plasma fractionators have implemented many improvements over the past decade directed toward reducing the likelihood of pathogen transmission by purified blood products, yet little has been published attempting to assess the overall impact of these improvements on the probability of safety of the final product.

The use of tri(n‐butyl)phosphate detergent mixtures to inactivate hepatitis viruses and human immunodeficiency virus in plasma and plasma's subsequent fractionation

The treatment of plasma with organic solvent/detergent mixtures at the time of plasma collection or pooling could reduce the exposure of technical staff to infectious viruses and enhance the viral safety of the final product. Treatment of plasma for 4 hours with 2‐percent tri(n‐ butyl)phosphate (TNBP) at 37°C, with 1‐percent TNBP and 1‐ percent polyoxyethylensorbitan monooleate (Tween 80) at 30°C, or with 1‐percent TNBP and 1‐percent polyoxyethylene ethers, (Triton X‐ 45) at 30°C resulted in the rapid and complete inactivation of ≥ 104 tissue culture‐infectious doses (TCID50) of vesicular stomatitis and Sindbis viruses, which are used as surrogates. Treatment of plasma with TNBP and TNBP and Tween‐80 was shown to inactivate ≥ 104 TCID50 of human immunodeficiency virus. TNBP treatment of plasma contaminated with 106 chimpanzee‐infectious doses (CID50) of hepatitis B virus and 105 CID50 of non‐A, non‐B hepatitis virus prevented the transmission of hepatitis to chimpanzees. Immediately after treatment of plasma with 2‐percent TNBP, the recovery of factors VIII, IX, and V and antithrombin III was 80, 90, 40, and 100 percent, respectively. Recovery of all factors was ≥ 90 percent after treatment with TNBP and detergent mixtures. Treated plasma was fractionated by standard techniques into antihemophilic factor and prothrombin complex concentrates, immune globulin, and albumin. Prior treatment with TNBP or TNBP and detergent did not affect the separations of desired proteins. Therefore, it appears possible to inactivate viruses in plasma before the execution of standard fractionation procedures.

Virus inactivation in red cell concentrates by photosensitization with phthalocyanines : protection of red cells but not of vesicular stomatitis virus with a water-soluble analogue of vitamin E.

BACKGROUND: Photodynamic treatment of red cells (RBCs) with phthalocyanines and red light inactivates lipid‐enveloped viruses, such as vesicular stomatitis virus (VSV) and human immunodeficiency virus. To protect RBCs from photodynamic damage, type I free radical quenchers, such as mannitol, which did not affect virus inactivation, were added.

Tri(n-butyl) phosphate/detergent treatment of licensed therapeutic and experimental blood derivatives.

Abstract. Incubation of an AHF concentrate with 0.3% tri(n‐butyl)phosphate (TNBP) and 0.2% sodium cholate was shown to inactivate at least 10,000 infectious doses of lipid‐enveloped viruses, including hepatitis B and non‐A, non‐B viruses and HTLV‐III [Prince et al., Lancet i, pp. 706–710, 1986]. The use of TNBP/detergent combinations for virus sterilization was evaluated further to determine its effect on the structure and function of a wide variety of blood proteins. Vesicular stormatitis and Sindbis viruses were used as markers of virus inactivation. TNBP/detergent treatment did not significantly alter the function of AHF, factor VII, factor IX, factor X, fibrinogen, factor XIII, fibronectin, anti‐HBsAg and anti‐HA in normal immune serum globulin, haptoglobin, tumor necrosis factor, α‐interferon, and both native and chemically polymerized stroma‐free hemoglobin. As compared with partially purified derivatives, the extent of virus sterilization of plasma and component cryoprecipitate with 0.3% TNBP and 0.2% sodium cholate at ambient temperature could be improved by raising the TNBP concentration and temperature. Virus sterilization by TNBP/detergent mixtures appears to be generally applicable to blood protein derivatives.