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).
Plasma levels of HDL apo A-I are reduced in individuals with low HDL cholesterol (HDL-C) concentrations as a result of increased fractional catabolic rates (FCRs). To determine the basis for the high apo A-I FCRs, seven subjects with low HDL-C levels (31.0±4.3 mg/dl) were compared with three subjects with high HDL-C levels (72.0±4.5 mg/dl). Each subject received autologous HDL that was labeled directly by the iodinemonochloride method (whole-labeled) and autologous HDL that was labeled by exchange with homologous radiolabeled apo A-I (exchange-labeled). Blood was obtained for 2 wk, specific activities determined, and FCRs (d-'+SD) estimated. In every subject, whether in the low or high HDL-C group, the exchange-labeled FCR was greater than the whole-labeled FCR. The exchange-labeled FCR was higher in the low HDL-C group (0.339±0.043) versus the high HDL-C group (0.234 ±0.047; P < 0.009). The whole-labeled FCR was also greater in the low HDL-C group (0.239±0.023) versus the high HDL-C group (0.161±0.064; P < 0.02). In addition, in both low and high HDL groups ultracentrifugation resulted in more radioactivity in d > 1.210 (as percentage of total plasma counts per minute) with the exchange-labeled tracer than with the wholelabeled tracer (12.55±4.95% vs. 1.02±0.38%; P < 0.003). With both HDL tracers, more radioactivity was found in d > 1.210 in the low versus the high HDL-C groups. When apo A-I catabolism was studied by perfusing isolated rabbit kidneys with whole-labeled HDL, there was twice as much accumulation (cpm/g cortex) of HDL apo A-I isolated from subjects with low HDL-C than from subjects with high HDL-C (P < 0.0025). Finally, HDL that had been isolated from subjects with high levels of HDL-C was triglyceride enriched and exposed to partially purified lipases before perfusion through kidneys. Threefold more apo A-I from modified HDL accumulated in the cortex compared with the unmodified preparation (P < 0.007). The results of these in vivo and ex vivo studies indicate that individuals with low HDL-C levels have more loosely bound, easily exchanged apo A-I and that this exchangeable apo A-I is more readily cleared by the kidney. (J. Clin. Invest.
Fresh frozen plasma (FFP) is prepared in blood banks world-wide as a by- product of red blood cell concentrate preparation. Appropriate clinical use is for coagulation factor disorders where appropriate concentrates are unavailable and when multiple coagulation factor deficits occur such as in surgery. Viral safety depends on donor selection and screening; thus, there continues to be a small but defined risk of viral transmission comparable with that exhibited by whole blood. We have prepared a virus sterilized FFP (S/D-FFP) by treatment of FFP with 1% tri(n-butyl)phosphate (TNBP) and 1% Triton X-100 at 30 degrees C for 4 hours. Added reagents are removed by extraction with soybean oil and chromatography on insolubilized C18 resin. Treatment results in the rapid and complete inactivation of greater than or equal to 10(7.5) infectious doses (ID50) of vesicular stomatitis virus (VSV) and greater than or equal to 10(6.9) ID50 of sindbis virus (used as marker viruses), greater than or equal to 10(6.2) ID50 of human immunodeficiency virus (HIV), greater than or equal to 10(6) chimp infectious doses (CID50) of hepatitis B virus (HBV), and greater than or equal to 10(5) CID50 of hepatitis C virus (HCV). Immunization of rabbits with S/D-FFP and subsequent adsorption of elicited antibodies with untreated FFP confirmed the absence of neoimmungen formation. Coagulation factor content was comparable with that found in FFP. Based on these laboratory and animal studies, together with the extensive history of the successful use of S/D-treated coagulation factor concentrates, we conclude that replacement of FFP with S/D-FFP, prepared in a manufacturing facility, will result in improved virus safety and product uniformity with no loss of efficacy.
The use of a human immunodeficiency virus (HIV) immune globulin (HIVIG) in prevention of HIV infection in chimpanzees was investigated in the hope of ultimate application to interruption of vertical transmission. In previous experiments, no protection was observed when relatively high challenge doses were used. This study shows that HIVIG protected against a challenge dose (10 CID50) tenfold lower than that used previously. The protected animal remained free of HIV infection as determined by cocultivation and by polymerase chain reaction (PCR), and did not mount a primary immune response detectable by enzyme-linked immunosorbant assay (ELISA) and neutralization assays. These results imply that HIV vaccines should induce neutralizing antibody and may not need to induce cell-mediated immunity in order to be protective against exposure to HIV. They also provide an experimental basis for the conduct of clinical trials to evaluate prevention of maternal-infant transmission by HIVIG.
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 (A1N2SB2POH) was evaluated and compared with that of the much studied anionic aluminum tetrasulfophthalocyanine hydroxide (A1PcS4OH). Based on the rate of inactivation of the lipid-enveloped vesicular stomatitis virus (VSV), the virucidal 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) > A1PcOSi(CH3)2(CH2)3N+(CH3)2(CH2)11CH3I- (Pc 21) = A1N2SB2POH = A1PcS4 > HOSiPc[OSi(CH3)2(CH2)3N+(CH3)2(CH2)11CH3I-]2 (Pc 14) > A1PcOSi(CH3)2(CH2)3N+(CH3)3I- (Pc 2). Phthalocyanine 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 A1PcS4OH. Treatment of RBCC containing cell-free human immunodeficiency virus (HIV-1) with Pc 5 or A1PcS4OH required 15 min of irradiation to inactivate (> 5 log10 reduction) the virus. The extent of HIV-1 inactivation with A1N2SB2POH 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.(ABSTRACT TRUNCATED AT 250 WORDS)
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