[19] Inactivation of viruses in human plasma

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INACTIVATION OF VIRUSES IN H U M A N PLASMA

207

targets such as bacteria or viruses. Contrary to the well-known NDPO2, the new nonionic carrier DHPNO2 appears to be very effective in inactivating both nonenveloped viruses and intracellular enveloped viruses. It is also much more efficient than NDPO2 in killing bacteria. These differences can be related to the structure of the endoperoxides. The anionic carrier NDPO2 has no affinity for lipophilic membranes or negatively charged sites. Therefore, it releases 102 randomly in the aqueous medium. Thanks to its nonionic structure, DHPNO2 is able to penetrate into cell membranes where it generates 102 close to the intracellular targets. Hence, it might be a useful tool in investigating the in vitro activity of pure 102 on any intracellular target, such as DNA or mitochondria.

[19] I n a c t i v a t i o n o f V i r u s e s i n H u m a n

Plasma

By H. MOHR Introduction Human flesh frozen plasma (FFP) is widely used for therapeutic purposes and has a high safety margin based on two complementary approaches: 1. It is a single donor preparation isolated from donor blood or obtained by plasmapheresis. Each donation or plasma unit is tested for the absence of markers of human immunodeficiency virus (HIV) and the hepatitis viruses B and C (HBV, HCV), respectively.1'2 2. It is quarantined for up to 6 months in the frozen state. The plasma is not used before the respective donor or his new donation after this time period is retested and found to be negative for the viral markers mentioned. Nevertheless, there is a slight, but significant, residual risk of viral contamination as a result of donations in the preseroconversion window period of infectivity.3 The donor may also be infected with a virus that is not tested for, e.g.,

1 p. V. H o l l a n d , N. Engl. J. Med. 334, 1734 (1996). 2 W. K. R o t h , M. W e b e r , a n d E. Seifried, Lancet 353, 359 (1999). 3 G. N. Vyas, Transfusion 35, 367 (1995).

METHODS IN ENZYMOLOGY, VOL. 319

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parvovirus B19. 4 This justifies incorporating a step to ensure the viral safety of FFP. For plasma pool products, heat treatment in the liquid or freeze-dried state or treatment with detergents in combination with organic solvents is routinely used. 5-7 These procedures are, however, too labor and time consuming to process numerous single donor units of FFP. In this case, a realistic approach is photodynamic treatment.

Principle of P h o t o d y n a m i c V i r u s I n a c t i v a t i o n The principle of photodynamic virus inactivation of FFP is as follows: The plasma is mixed with a photoactive c o m p o u n d that has a preference for viral structures, e.g., the nucleic acid or m e m b r a n e components. The mixture is then illuminated with light of an appropriate wavelength, i.e., in the adsorption m a x i m u m of the photosensitizer. This activates the photosensitizer, and in the presence of oxygen, activated species of oxygen are generated, mainly the singlet form. Virus inactivation is accomplished by oxidative damage of the viral structures mentioned. In general, lipid-enveloped viruses are m o r e susceptible to photodynamic treatment than nonenveloped viruses 8-1° (Table I).

M e t h y l e n e Blue for P h o t o d y n a m i c T r e a t m e n t of P l a s m a H u m a n plasma has a high light absorption of between 300 and approximately 450 nm, but above 550-600 nm it is almost negligible (Fig. 1). Accordingly, the photosensitizer used for viral decontamination needs to have its own absorption above 550-600 nm. This property is fulfilled by methylene blue (MB, Fig. 2), whose adsorption m a x i m u m is about 660 nm (Fig. 1). An additional advantage is that MB is relatively nontoxic and has been used therapeutically for m a n y years, e.g., in the treatment of m e t h e m o g l o b i n e m i a and depression, u-13 Tolerated doses are in the range 4 C. Wakamatsu, F. Takahura, E. Kojima, Y. Kiriyama, N. Goto, K. Matsumoto, M. Oyama, H. Sato, K. Okochi, and Y. Maeda, Vox Sang. 76, 14 (1999). 5p. Murphy, T. Nowak, S. M. Lemon, and J. Hilfenhaus, J. Med. Virol. 41, 61 (1993). 6 p. Roberts and P. Feldman, Vox Sang. 73, 189 (1997). 7 B. Horowitz, M. E. Wiebe, A. Lippin, and M. H. Stryker, Transfusion 25, 516 (1988). s C. W. Hiatt, in "Concepts in Radiation Cell Biology," p. 57. Academic Press, New York, 1972. 9 E. Ben-Hur and B. Horowitz, Photochem. Photobiol. 62, 383 (1995). 10R. Santus, P. Grellier, J. Schr6vel, J.-C. Mazi6re, and J.-F. Stoltz, Clin. Haemorheol. 18, 299 (1998). 11R. M. Devine, J. A. van Heerden, C. S. Grant, and J. J. Muir, Surgery 94, 916 (1983).

[19]

INACTIVATION OF VIRUSES IN HUMAN PLASMA

209

TABLE I LIPID-ENVELOPED AND NONEVELOPED VIRUSES TESTED IN PLASMA FOR THEIR SENSITIVITY TO MB/LI~HT TREATMENT

Name

Family

Characteristics

Inactivation Rate (Ioglo-steps)

Illumination time (min)

E n v e l o p e d Viruses HIV-1

**

10

HN'2

• "

~-15 15

sly

SO 6O

Non e n v e l o p e d viruses

o =

* tested at production conditions " 7 6 ml in cell culture flask

..... =4i~

so ; =

210

SINGLET OXYGEN

[ 19] 0 CO

m

0 ¢0

T~ 0

e~

S

m 0

>

J=

O

I •

0

0

0 o

uop, dJosqv ~,q6!-I

0

[ 19]

211

INACTIVATION OF VIRUSES IN HUMAN PLASMA

Methylene Blue (CH3)2N

N(CH3)2

(CH3)2N

NHCH3

(CH3)2N

NH2

CH3NH

NH2

H2N

NH2

Azure B

Azure A

Azure C

FIG. 2. Structures of MB and related phenothiazine dyes.

212

SINGLET OXYGEN

[ 19]

of 1-2 mg/kg body weight per day. For plasma treatment, a concentration of 1/zM is sufficient, i.e., approximately 300/xg/liter. 14'1s Another important aspect is that during illumination photoproducts are formed from the photosensitizer itself. In the case of MB they are well defined. The photoproducts are the demethylated analogs of MB, i.e., azure A, B, C, and thionine, respectively (Fig. 2), whose photodynamic and other properties are similar to those of MB itself. The virus inactivation properties of MB and other phenothiazine dyes in combination with visible light have been known for many years. Their use for the photodynamic treatment of sewage, tap water, vaccines, and blood plasma was proposed. 8'16-2° To date, however, only the treatment of single units of human plasma has been fully developed. Production of MB Plasma The procedure for manufacturing MB plasma is shown in Fig. 3. It is manufactured from fresh frozen plasma isolated from donor blood within 6-8 hr after donation. Alternatively, the plasma is obtained by plasmapheresis. The plasma units (approximately 300 ml) are shock-frozen and stored at ->-30 ° until further processing. They are thawed under gentle shaking in a water bath at 27-30 ° within 15-20 min. This is followed by visual inspection. Units that are hemalytic of lipemic are discarded for cosmetic reasons, as the plasma appears "dirty." The freezing/thawing cycle is important because this destroys contaminating leukocytes that might harbor cell-associated viruses such as HIV-1 or provirus integrated into the cellular genome. Freezing/thawing makes these structures accessible to MB/light. As an alternative, leukocytes can be depleted from the plasma by filtration through membrane filters. 21-24 It 12j. W. Harvey and A. S. Keitt, Br. J. Haematol. 54, 29 (1983). 13 G. J. Naylor, B. Martin, S. E. Hopwood, and Y. Watson, Biol. Psychiar 21, 915 (1986). 14 B. Lambrecht, H. Mohr, J. Kniiver-Hopf, and H. Schmitt, Vox Sang. 60, 207 (1991). 15 H. Mohr, B. Bachmann, A. Klein-Struckmeier, and B. Lambrecht, Photochem. Photobiol. 65, 441 (1997). 26j. R. Perdrau and F. R. S. Todd, Proc. Roy. Soc. London 112, 288 (1933). 17 C. W. Hiatt, E. Kaufman, J. J. Helprin, and S. Baron, J. Immunol. 84, 480 (1960). 18 C. Wallis and C. Melnick, Viology 23, 520 (1964). 19 G. S. L. Yen and E. H. Simon, J. Gen. Virol. 41, 273 (1978). 20 j. A. Badylak, G. Scherba, and D. P. Gustafson, J. Clin. Microbiol. 17, 374 (1983). 21 B. Rawal, B. T. S. Yen, G. N. Vyas, and M. Busch, Vox Sang. 60, 214 (1991). 22 D. Zucker-Franklin and B. A. Pancake, Transfusion 38, 317 (1998). 23 j. I. Willis, J. A. G. Lown, M. C. Simpson, and W. N. Erber, Transfusion 38, 645 (1998). 24 j. R. Rider, M. A. Winter, J.-M. Payrat, J.-M. Mathias, and D. H. Pamphilon, Vox Sang. 74, 209 (1998).

[19]

INACTIVATION OF VIRUSESIN HUMANPLASMA Store

FFP at -

3

213

~

Thaw plasma in water bath

(27 *C, approx, t5 min)

)ck for usabili (visual test)

no

Plasmais hemolytic, lipemic, bags are damaged

I

Add MB stock solution

i

(mix for 60 min at 8-12 °C)

~

\~

/J

Preincubate

Illuminate (60 min, > 45,000 Lux)

vacuum seal into outer

i Shock freeze within 30-40 min

FIG. 3. Production procedure for MB/light-treated fresh plasma. is the advantage of filtration that the freezing/thawing step can be omitted. After visual inspection the appropriate amount of a 5 0 / z M stock solution of MB is added to the plasma to achieve a final dye concentration of 1 /zM. When the plasma volume is about 250 ml, approximately 5.5 ml has to be added. The stock solution is in another plastic bag, which is positioned

214

SINCLETOXVCEN

[ 191

above the plasma-containing bag. Using a sterile docking system, the tubings of both bags are connected (Fig. 4). Flow of the dye solution into plasma is by gravity. The plasma unit is placed on a computer-assisted balance, and the addition of dye solution is stopped when the required amount has been added to the plasma. The manual addition of MB is cumbersome. In two alternative procedures, a fixed amount of dye is in the plasma bag system before isolation of the plasma, either in the form of a solution or as a pill, which is placed in the tubing between the blood bag and the plasma bag. When the plasma is transferred into its container, the dye-containing pill is dissolved simultaneously and mixed with the plasma. Because the plasma volume is variable in these two procedures, variations of the MB concentrations between 0.8 and 1.2/xM must be tolerated. In the next production step, the MB-containing plasma units are rotated overhead in the dark in a thrombocyte rotator at 10-12 ° for 60-65 min. This ensures complete mixing of dye and plasma, while at same time lowering the unit temperature to approximately 18°. This is an advantage because the plasma temperature increases by 5-7 ° during illumination. Thus, precooling prevents the plasma from overheating.

FIG.4. Additionof MB stock solutionto singleunits of plasma.

[19]

INACTIVATION OF VIRUSES IN HUMAN PLASMA

215

The following production step comprises the actual photodynamic treatment. The illumination device is a rectangular box with a glass plate on top. It is equipped with eight fluorescent tubes, e.g., Philips TL-M 115 W (33 RS) emitting white light and arranged horizontally below the glass plate. The device is air-cooled to remove excess heat. The plasma units to be treated are on the glass plate, with the labeled side up (Fig. 5). Up to 21 units can be illuminated at the same time. Minimal light intensity is 45,000 Lux. This is checked at different positions of the illumination area before and during each treatment cycle. Illumination time is 1 hr. After illumination the plasma units are labeled, sealed into outer plastic bags, and deep-frozen again at -<-30 °. Alternative light sources may also be used that are equipped with LEDs emitting red light with a wavelength in the range of the absorption maximum of MB (i.e., approximately 660 nm) or low-pressure sodium lamps emitting high-intensity yellow light at 595 nm. It has been demonstrated that illumination time in both cases may be shortened considerably to achieve complete virus inactivation while at the same time better preserving the activities of plasma proteins. 15 The efficacy of the procedure was proved using a variety of different lipid-enveloped and nonenveloped model viruses (Table 1). These included those virus types that are recommended by CPMP guidelines 268/95 ("Note

FIG. 5. Photodynamic treatment of MB containing plasma units.

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SINGLETOXYOEN

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for guidance on virus validation studies: the design, contribution, and interpretation of studies validating the inactivation and removal of viruses") and 269/95 ("Note for guidance on plasma-derived medicinal products"). These guidelines note which viruses should be used in validation studies on the inactivation and removal of viruses and how this should be done. It is necessary to test for HIV, a model for hepatitis C virus (HCV), for nonenveloped viruses and enveloped DNA viruses such as herpes viruses. Because there is no cell culture system to assay HCV, flavi, toga, and pestiviruses may be used as models. It is evident from Table I that these viruses especially are very sensitive to MB/light treatment and are inactivated within less than 1-5 min. Polymerase chain reaction studies using HIV-infected plasma also indicate that HCV is probably effectively inactivated. 2s 25 K. Mtiller-Breitkreutz and H. Mohr, J. Med. Virol. 56, 239 (1998).

[20] 3- ( 4 ' - M e t h y l - 1' - N a p h t h y l ) p r o p i o n i c A c i d , l',4'-Endoperoxide for Dioxygenation of Squalene and f o r B a c t e r i a l Killing By

MINORU NAKANO, YASUHIRO KAMBAYASHI,

and

HIDETAKA TATSUZAWA

Introduction In recent years, considerable interest has been focused on the reaction of singlet oxygen (IO2; lAg) with organic compounds and its toxicity toward living cells. Known 102 generating systems, such as NaOCI-H202,1 myeloperoxidase-hydrogen peroxide (H202)-halide ions, 2 and light-photosensitizer-O23 systems contain reactive oxygen species other than 102 and often generate free radicals, and therefore such systems cannot serve as pure 102 generating reactions. In the light of the aforementioned information, Saito et aL 4 synthesized a novel water-soluble naphthalene endoperoxide [3-(4'-methyl-l'-naphthyl)propionic acid, l',4'-endoperoxide; NEPO], producing 102 thermolyt1 T. Kajiwara and D. K. Kearms, J. A m . Chem. Soc. 15, 5886 (1976). 2 j. R. Kanofsky, Chem.-Biol. Interact. 70, 1 (1989). 3 R. C. Straight and J. D. Spikes, in "Singlet O2," p. 91. C R C Press, Boca Raton, FL, 1985. 4 I. Saito, T. Matsuura, and K. Inoue, J. A m . Chem. Soc. 103, 188 (1981).

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