Dr. Lena Scherba-Krugliak ^ H I V I N A C T I VAT I N G P R O C E D U R E S I N B L O O D P R O D U C T S Introduction The first case of transfusion-...
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Dr. Lena Scherba-Krugliak

^ H I V I N A C T I VAT I N G P R O C E D U R E S I N B L O O D P R O D U C T S Introduction The first case of transfusion-associated AIDS was described in 1982 in an infant after receiving a ransfu fon for erythroblastosis fetalis (1,2). The first 3 cases of AIDS in patients Sth hemonnilia were reported in 1982 after treatment with lyophilized factor VIII concentrate S RStoSSTSSe of transfusion-'associated AIDS resulted in great concern for he safe3Te Wood supply. Each lot of lyophilized Factor VIII concentrate is prepared from SLma f?om up to22 thXnd plasmapheresk donors (4). Primarily of AIDS concern,^ early ?98^prodTers of clotting facto? concentrates began a voluntary self-deferral program m order to screen out high risk gfoups. Obviously, such screerung was farfrom 100% effectiv^ The identification of the AIDS virus by Montegnier (5) and Gallo (6) in 1984 ledto ^develop ment of HIV antibody screening tests and test kits were licensed m early 1985 (7). I hus begin Sng in the spring of 1985, all blood and plasma collected in the US is being tested for HIV antibody. The identification of the AIDS virus also resulted in studies of methods of inactivating the vin? aSK?Resenttime, there are a number of commercially avai able clotting factor concentrates where a variety of viral inactivation procedures have been employed (8-18). It is the purpose of this review to discuss and to examine the clinical efficacy of HIV in activating procedures used in plasma products. Thermal inactivation of HIV *t)»K A significant portion of retroviruses withstand the procedures used to prepare factor V' VIII Retroviruses are resistant to freeze drying and can be kept in a lyophilized form tor at least one year without a decrease in titer. The lyophilization process was found to cause a reduction of infectious virus titer of only about 10-100 fold (20). The sensitivity of HIV to heat has been reported by several groups (19-24) At the re quest of the National Hemophilia Foundation Medical and Scientific Advisory Council, the Center for Disease Control (CDC) conducted thermal stability studies with the AIDS virus. Preliminary data of the CDC group in September 1984 formed the scientific basis for the sub sequent recommendation that heat-treated lyophilized antihemophilic Factor VIII and Factor IX c6ncentrates be used in the treatment of hemophilia (25). Recent extensive CDC data sup ported this recommendation (23). Methods and Materials Virus and cell lines Most of the investigators used either human T-cell leukemia line, H-9 or HIV was propagated in normal phytohemagglutin (PHA) stimulated lymphocytes. Aliquotes of virus suspension were stored in liquid nitrogen. Virus inocula used m the most studies had mfectivity titers of 105 (24). Viral infectivity assays In vitro systems for the determination of inactivation of the HIV virus have been developed. These systems depend on the ability of this virus to infect normal lymphocytes in culture system. Infectivity is determined by either the amount of reverse transcriptase activity of viral particles in the supernate of the cultured lymphocytes, or by indirect immunofloures_. cence assav or bv antisen-capture enzyme-linked immunosorbent assays (20,22)• 1 nese systems f^ a^made more sensitive bv using serial dilutions of virus inoculum such as the ID50 assay 23). The ID50 can be defined as the infectious viral titer that is the reciprocal of the dilution (in a loo scale) at which 50% of the cultures are positive for virus. The ID50 assay has been employed by researchers at the CDC Atlanta, to determine the effectiveness of thermal inac tivation of the AIDS virus.

^ Dry versus wet heat HIV inactivation Heat has been applied to clotting factor concentrates in various ways with different com binations of time and temperature exposure (19-24). Rates of thermal inactivation of HIV varied with temperature and differed in liquid state as compared to dry lyophilized virus suspensions (23). Multiple studies have confirmed the resistance of the lyophilized retroviruses to heat treatment as opposed to the heat-sensitiveiiquid phase (20,23). In CDC study HIV virus was added Jo liquid factor concentrate, which was then processed according to the standard manufacturing procedures and heated either wet or lyophilized, dry (23). The original inocula, preheated samples, and prepared final product were evaluated as to in vitro viral infectivity. These researchers determined that the thermal decay/kill curve showed a first-order rate constant, and that one log dilution, termed D10, of virus was inactivated in 30 seconds when liquid F VIII was heated at 60 C When virus was in activated in lyophilized F VIII, one log dilution was inactivated in 30 minutes. (Fig. 1) In general, the range of the temperature and time exposure used to inactivate HTV in lyophilized form are from 60°c for 20 hours to 68°C for 72 hours. HIV in the liquid phase of F VIII concentrate required only 56°C for 1 hour for complete viral inactivation (19,20). As the minimum heating time of "dry" product is 60°C for at least 10 hours, the extrapolated viral kill would be a minimum of 20 logs, which should provide a wide safety margin. (4). Significance of the initial virus titer in heat HIV inactivation Levy et al. have found that the initial virus titer differences may reflect small variations in virus survival. High titer retrovirus (10 ) lyophilized preparation showed very low levels of f*^ infectious after lyophilized heat treatment for 72athours et product al. experi• _ residual ments have shownparticles that heating F VIIIatfor60°C 72 hours 68°C (19). or theLevy liquid for 10 hours at 60°C will eliminate infectious HIV if it is not present in the plasma at more than 10 infectious particles per ml (20). Influence of stabilizers on heat HIV inactivation Thermal inactivation of HIV in factor concentrates requires inclusion of stabilizers, such as sugars, amino acids and salts to reduce unwanted protein denaturation and loss of biological activity. Levy et al have found that in the absence of sucrose, wet heat HIV inactivation was sig nificantly more potent (20). Horowitz et al. reported that the rate of virus inactivation at 60 C was decreased by at least 100 to 700 fold by inclusion of 2.75 M glycine and 50% sucrose or 3.0 M potassium citrate (21). The CDC study, however, has not confirmed significant differences in thermal decay of HIV in unstabilized liquid F VIII concentrate or that stabilized with 50% sucrose and 2M glycine - D10 = 30 sec. versus 32 sec. respectively (23). Hemostatic efficacy of heated factor concentrates Heat-treated factor concentrates have been available for clinical use in the United States for approximately 3 years. The hemostatic efficacy of heated factor concentrates has been ex tensively studied. Recovery and survival studies of F VIII have been carried out on all commer cial products available in the United States (Table 1) (8-12). These studies have had a similar design in which small numbers of hemophiliac volunteers have been infused usually in a double-blind format with both heated and untreated products. The crossover to the alternate infusate was done at a minimum of 7 days. The recovery and survival data all show approximate equivalence under the experimental conditions for both F VIII and F IX concentrates. Heldebrant et al. found no significant difference in the F VIII plasma recovery or plasma half-life of the factor (8). Horowitz et al. found no loss of F VIII activity in lyophilized preparation after {" 72 hours of heating at 60°C (21). The heat-treated concentrates appeared bioequivalent to the untreated concentrates with the additional benefit of inactivation of potentially present infec tious viruses. Nevertheless, there are anecdotal reports in the literature of hemorrhagic episodes not responding to heat-treated product, necessitating more careful investigation of clinical efficacy of heat-treated factor preparations (4).

(^ Theoretical risks of heat HIV inactivation There are a number of theoretical risk problems connected with the use of heat-treated factor concentrate preparations. These include: 1. Failure of the primary objective of viral inactivation 2. Altered hemostatic efficacy 3. Denatured proteins resulting in: a. Anaphylactic or other immune reactions b. Immune complex mediated disease c. Neoantigen exposure resulting in inhibitor stimulation d. Reticuloendothelial overload of denatured protein Studies were carried out in an attempt to detect protein biochemical changes and denaturation \>otl at 4e in vitro level and b? animal studies. Double^^^unodiffusion n agarose eels using antisera raised in rabbits to heated and non heat-treated F VIII failed to fdenufV Sy neoanti^m on cross-reactivity, thus confirming their immunological identity^ ExoerSitT investigating protein denaturation have failed to reveal protein changes on SDSS c ^ S e l P e r m e a t i o n c h r o m a t o g r a p h y a n d e m i s s i o n fl u o r e s c e n c e spectra (26-29) Animal studies have shown equal tolerance to the infusion of heated and non-heated concentrates (26-29). Thus preliminary studies such as double immunodiffusion and animal studies failed to P reveal neoaAtlgen formation or protein changes, indicating a substantial safety margin. HIV seroconversion after use of heat-treated clotting factor concentrates The heat-treated clotting factor concentrates have been used clinically for over 3 years in the United States and for a longer period in Europe (4). Thirteen major non-U.S. Hemophilia ?eamientCenters (Europe, Cafada and Australia^were asked to provide information concernno HIVW occurring in patients receiving heat-treated concentrates only No 2seTof seroconversion following the use of donor-screened, heat-treated products were iden tified mrough this survey (30). Rouzioux et al followed a group of 18 ^™P¥^^ ™« treated exclusively with heat-treated products. None seroconverted over a period of at least 6 SSmrKer, there have teen two separate reports of single patients, previously ™cumemed as seronegative, becoming seropositive after infusion with heat- reated unscreened s^rXDroducts (32 33). Such reports indicate that despite both in vitro and in vivo data, longer iSJtoSiS»* required for the conclusion as to the efficacy of HIV inactivation by the heat-treatment process. Chemical HIV inactivation Chemical processes for HIV inactivation in plasma and its fractionated products have been also evaluated in the literature (16,17,24,33-36). Chemicals have been known to exert their antiviral action at different targets of the viral structure Xs! lipid solubilizing agents, such as ethyl ether and Triton X-100 detergent would be expected to inactivate envelope viruses, the surface active agents, such as Nonoxynol-9 and Amphotericin B act at the membrane level.^ (^^eraseTnhtors,'suchas retinoic acid and suramin will inhibit HIV replica tion. ~ Lipid solubilizing agents inactivating HIV in vitro An NIH otoud investigated HIV inactivation by lipid solubilizing agents such as chloroform et^NeS, formaldehyde, betapropiolactone, Triton X-100 and by ultraviolet light

in the presence of psoralen (Table II) (24). Their results indicate that a variety of chemical pro fit cedures are effective in reduction of HIV infectivity. Surfactants inactivating HIV in vitro.

1% nonoxynol-9 may have a protective effect against AIDS infection (37). £ rfSES^ Bto dolSl in the membrane of cells causes changes in cell jmSca^Siffloi and its binding to sterols of lipid-enveloped viruses causes loss of their infectivity (38). P^ntide-T is a svnthetic analog of an octapeptide, receptor like pattern of antigen T4 derived^Srope^deTn huma/brain, whicfi patently inhibits HIV at a concentration of lOOmM in vitro (35). Inhibitors of reverse transcriptase inactivating HIV in vitro. Wamin is a drue used in the therapy of Rhodesian trypanosomiasis, known to inhibit the J££Z2^^£^ of ^viruses Suramin was the first drug reported to have an in vitro inhibitory effect on HIV replication (34). ThP effect of retinoicacid on HIV replication in vitro was investigated in Japan Con comitant^ ofmfc^with 5ug/ml of retinoic acid and various concentrations of s^minrSdin the more effective inhibition of HIV replication than suramin alone (35). C* There are several commercially available factor concentrates that underwent HIV chemical inactivation. 1) TUtapmninlactone treated factor IX prothrombin complex concentrates are presently corrnnerci^uTaSkmEurope (marketed by Biotest) and are currently under trial in the United States (4). 2) F^rrnpio1artonf-"it"vmlPt lipht procedure has shown encouraging in vitrc> data with greater far^3.0log HIV inactivation. Betapropiolactone causes however, extensive Ztefn denaturation with some loss in potency of the prothrombin-complex proteins (4,13,14). It ?s notewoX that HIV is not inactivated by ultraviolet radiation alone in doses much higher than those usually employed in the laboratories of operating rooms. () 3) The organic solvent tri(n.h,.tynPhnsphate has been used together with the detergents TWnJfn nr «vfii,m cholate by researchers at the New York Blood Center to inactivate con^n^gviruser These agents are added either early or partway through the fractionation proces^ ?ogr 6 hou s. SodiunTcholate is the detergent used in the F VIII process and Tween-80 Tthe F IX process. In both systems, the added agents are removed from the processed proteins by a chromatographic step (16,17,32). Elimination of HIV during preparation of immunoglobulins Recently, the safety of immunoglobulin and hyperimmunoglobulin P^^tions have been the subject of discussions (42,43). Preparations conta.mng antibodies to HIV and possible causative agents of AIDS have been major concerns. The CDC and FDA after testing lots of immunoglobulin produced between 1982 and ^ 1985 found that, although the results were positive for HIV antibodies, th _ey were not infectious. V ■ Studies indicate that recipients of these immunoglobulin preparations did not later seroconvert to HIV positivity (44). Additionally, recent CDC evaluation of the Cohn-Oncley cold-ethanol fractionation

j fl f f K .

method determined that the process is effective in eliminating HIV infectivity. Reduction of in fectious retrovirus titers were JO5 to 10b-fold through Cohn-Oncley cold ethanol fractionation from plasma to fraction II, lf/to KT-fold through incubation at pH 4.0 and another 10 fold through incubation of the purified liquid immunoglobulin preparations at 27 C or 4S C (4j>). The results support the clinical and epidemiological evidence that therapeutic immunoglobulin preparations do not transmit AIDS virus. Conclusion 1 Multiple independent studies confirmed that thermal procedures of HIV inactivation provide large but not absolute margin of safety and expected efficacy of plasma preparations. Use of heat-treated products will reduce AIDS transmission by antihemophilic factor con centrates. 2. Chemical HIV inactivation procedures, described in the literature, are mostly in vitro experiments. Further information is required to define chemical HIV inactivation applicable to blood products. 3. Cohn-Oncley cole ethanold fractionation immunoglobulin preparation procedure is effective for elimination of infectious retroviruses. No transmission of AIDS has so far been observed after administration of specific gammaglobulin. 4. Most of the work with chemical and physical agents has been focussed on inactivation of HIV in plasma and not in cellular blood products. No information is available on HIV inac tivation in whole blood and cellular blood components.



1. Possible transfusion-associated aquired immune deficiency syndrome (AIDS). Calif Mnrh Mortal Wklv Rep 1982; 31:652.

2. Ammann AJ, Cowan MJ, Wara DW, et al. Acquired immunodeficiency in an infant: possible transmission by means of blood products. Lancet 1983; i:956. 3. Pneumocystis carinii pneumonia among persons with hemophilia A. Morb Mortal Wkly Rep 1982; 31:365. v 4. Gomperts ED. Procedures for the inactivation of viruses in clotting gactor concentrates. Am J Hematol 1986; 23:295. 5. Barre-Sinoussi F, Chemann JC, Rey R, et al. Isolation of a T lymphotropic retrovirus from a patient at risk for aquired immune deficiency syndrome (AIDS). Science 1983; 220:868. 6. Popovic M, Sarngadharan MG, Read E, et al. Detection, isolation and continuous production of cytopathic retroviruses (HTLV-HI) from patients with AIDS and preAIDS. Science 1984; 224:497. Sandler SG, Katz AJ. Impact of AIDS on blood services in the United States. Vox Sang 1984; 46:1. 7. McDougal JS, Jaffe HW, Cabridrilla CD, et al. Screening tests for blood donors presumed to have transmitted the aquired immunodeficiency syndrome. Blood 1985; 65:772.

8. Heldebrant CM, Gomperts ed, Kasper CK, et al. Evaluation of two viral inactivation methods for the preparation of safer factor VIII and IX concentrates. Transfusion 1985;25:510-515. 9. Hyland Therapeutics Division, TravenoL Laboratories, INc, Glendale, California. "Hemofil-T Antihemophilic Factor (Human)," 10. Armour Pharmaceutical Co., Kankakee, Illinois. "H.T. Factorate and H.T. Factorate Generation II. Summary of Pre-Clinical and Clinical Data." 11. StagnaroT. Personal communication. 12. Cutter Biological, Berkeley, California. "Heat-Treated Konyne-H.T. Factor IX Complex (Human)." 13. Prince AM, Stephan W, Brotman B. Inactivation of Non-A, Non-B virus infectivity by a Beta-propriolactone/ultraviolet irradiation treatment and aerosil adsorption procedure ^ used for preparation of a stabilized human serum. Vox Sang 1984; 46:80-85. 14. Heinrich D, Kotitschke R, Berthold H. Clinical evaluation of the hepatitis safety of a Beta-propriolactone/ultraviolet treated factor IX concentration (PPSB). Thrombos Res 1982;28:75-83. 15. Heimburger N, Karges HE. Factor VIII concentrate, hepatitis safe: preparation and properties. Paper presented at the XVI International Congress of the World Federation of Hemophilia, Rio de Janeiro, 1984. 16. Horowitz B, Wiebe ME, Lippin A et al. Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-enveloped viruses by tri(n-butyl) phosphate detergent combinations. Transfusion 1985;25:516-22. 17. Horowitz B, Prince AM, Lippin A, et al. Inactivation of hepatitis viruses by treatment of AHF concentrates with tri(n-butyl)phosphate (TNBP) and sodium cholate (CA). International Congress of Thrombosis and Hemostasis, San Diego, 1985 (Abstract).


18. Eibl H. "Virus-Inactivated Factor Concentrates. Immuno International Study Update, 1985." Personal communication. 19. Levy JA, Mitra GA, Mozen MM. Recovery and inactivation of infectious retroviruses from factor VIII concentrates. Lancet 1934; ii:722. 20. Levy J A, Mitra GA, Wong MF, et al. Inactivation by wet and dry heat of AIDSassociated retroviruses during factor VIII purification from plasma. Lancet 1985; ii:1456. 21. Horowitz B, Wiebe ME, Lippin A, et al. Inactivation of viruses in labile blood derivatives. II. Physical methods. Transfusion 1985; 25:523. 22. Pettriciani JC, McDougal JS, Evatt BL. Case for concluding that heat-treated licensed, antihemophilic factor is free from HTLV-III. Lancet 1985; ii:890. 23. McDougal JS, Martin LS, Cert SP et al. Thermal inactivation of the acquired immunodeficiency syndrome virus, human T lymphotropic virus-III/lymphadenopathyassociated virus, with special reference to antihemophilic factor. J Clin Invest 1985;76:875-77. 24. Quinnan GV, Wells MA, Wittek AE, et al. Inactivation of human T-cell lymphotropic virus, type III by heat, chemicals, and irradiation. Transfusion 1986; 26:481. 25. National Hemophilia Foundation Medical Scientific Advisory Council. Recommendations concerning AIDS and therapy of hemophilia (revised October 13, 1984). National Hemophilia Foundation, New York. 26. Aronson DL. Bureau of Biologies Summary Basis of Approval: Antihemophilic Factor (Human) and Factor IX complex (Human). Ref. Nos. 82-196, 83-470, Hyland Therapeutics Division of Travenol Laboratories, Inc. 27. Aronson DL. Bureau of Biologies Summary Basis of Approval: Antihemophilic Factor /^^

(Human) and Factor IX complex (Human). Ref. Nos. 83-341 and 84-289, Miles Laboratory (Cutter Biological).

28. Aronson DL. Bureau of Biologies Summary Basis of Approval: Antihemophilic Factor (Human). Ref. No. 83-367, Armour Pharmaceutical Co. 29. Aronson DL. Bureau of Biologies Summary Basis of Approval: Antihemophilic Factor (Human) and Factor IX complex (Human). 'Ref. No. 84-069, Alpha Therapeutics Corp. 30. Survey of non-U.S. Hemophilia Treatment Centers for HIV seroconversions following therapy with heat-treated factor concentrates. Morb Mortal Wkly Rep 1987; 36:121. 31. Rouzioux CS, Chamaret L, Montagnier V, et al. Absence of antibodies to AIDS virus in haemophiliacs treated with heat-treated Factor VIII concentrate. Lancet 1985; i:271. 32. Van Der Berg W, Tencate, Breidervald C, et al. Seroconversion to HTLV-III in hemophiliacs given heat-treated factor VIII concentrates. Lancet 1986;i:803. 33. White GC II, Matthews TJ, Weinhold KJ, et al. HTLV-III seroconversion associated f^ with heat-treated factor VIII concentrates. Lancet 1986; i:611. 34. Mitsuya H, Popovic M, Yarchoan R et al. Suramin protection of T-cell in vitro against infectivity and cytopathic effect of HTLV-III. Science 1984;226:172-74. 35. Nakashima H, Harada S, Yamamoto N. Effect of retinoic acid on the replication of human immunodeficiency virus in HTLV-I positive MT-4 cells. Med Microbiol Immunol 1987:176:189-98. 36. Pert CB, Hill JM, Ruff MR. Octapeptides deduced from the neuropeptide receptor like pattern of antigen T4 in brain potently inhibit the human immunodeficiency virus receptor binding and T-cell infectivity. Proc Natl Acad Sci 1986;83:9254-58. 37. Hicks DR, Martin LS, Getchell JP et al. Inactivation of HTLV-III/LAV-infected cultures of normal human lymphocytes by nonoxynol-9 in vitro. Lancet 1985;ii: 1422-23. 38. Schafner TP, Pelscia OJ, Pontani D et al. Antiviral activity of amphotericin B methyl ester: inhibition of HTLV-III replication in cell culture. Biochem Pharmacol f * 1 9 8 6 ; 3 5 : 4 11 0 - 1 3 .

r 39. Tedder RS, Utley A, Cheinsong-Popov R. Safety of immunoglobulin preparation containing anti-HTLV-III (letter). Lancet 1985; i:815. 40. Gocke DJ, Raska K, Pollack W, et al. HTLV-III antibody in commercial immunoglobulin (letter). Lancet 1986; i:37. 41. Safety of therapeutic immune globulin preparations with respect to transmission of human T-lymphotropic virus type III/lyphademopathy-associated virus infection. Mortal Morbid Wkly Rep 1986; 35:231. 42. Mitra GM, Wong MF, Mozen MM, et al. Elimination of infectious retroviruses during preparation of immunoglobulins. Jj^nsfusion 1986; 26:394.


TA B L E I ,,, ' rT Viral inactivation procedures and recovery of Factor Vlll Percent recovery Method of viral Product FVIII inactivation Heat, dry, 60°C, 72 hr.


Cutter Biological (12) Heat, dry, 68°C, 72 hr.


Armour (10)

Heat, dry, 60°C, 30 hr.


Alpha Therapeutics (8)

Heat, dry, suspended in n-Hepatane, 60°C, 20 hr.


Immuno- Vienna (18)

Steam, under nitrogen 60°C, 10 hr. or 80°C, 1 hr.


Hyland Travenol (9)


TABLE II Chemical HIV inactivation (24) Inactivation method


* Infectivity IVIU/ml

None Chloroform Ethyl ether, 4°C

0 15 min. Ihr.


None Betapropriolactone 0.1%, 4°C Formaldehyde 1:1000, 4°C

0 18 hr. 18 hr.

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