Ann Hematol (1992) 65:224-228

Annals of

Hematology 9 Springer-Verlag 1992

Original article No evidence for neoantigens in human plasma after photochemical virus inactivation H. Mohr, J. Kniiver-Hopf, B. Lambrecht, H. Scheidecker, and H. Schmitt DRK-Blutspendedienst Niedersachsen, Institute Springe, 3257 Springe, Federal Republic of Germany Received March 16, 1992/Accepted August 5, 1992

Summary. Photodynamic virus inactivation of h u m a n fresh plasma mediated by visible light in the presence of the phenothiazine dyes methylene blue or toluidine blue was investigated to determine whether it influences functional, structural, and immunological properties of plasm a proteins. The activities of the coagulation factors I, VIII, IX, X, and XI were affected to a certain degree, while those of most other plasma proteins were not. The elution profiles obtained by ion exchange chromatography of untreated and photodynamically treated plasma were almost identical. Using a number of antisera against h u m a n plasma and single plasma proteins, different immunochemical techniques revealed identical patterns for untreated and treated plasma. Thus, there was no indication that the photodynamic virus inactivation procedure applied considerably influences the properties of plasma proteins. Key words: Photochemical virus inactivation - Phenothiazine dyes - H u m a n fresh plasma - Neoantigens

Introduction We recently described a photodynamic virus inactivation procedure to decontaminate h u m a n plasma intended for therapeutic use by illumination with visible light in the presence of low concentrations (in the range of 1 ~h//) of the phenothiazine dyes methylene blue (MB) and toluidine blue (TB) [17]. It has the advantage that the plasmas from single donors can be decontaminated in their original containers; furthermore, because the two dyes at the concentrations used are non-toxic, they do not have to be subsequently removed. Virus inactivation is probably restricted to lipid enveloped viruses, as is also the case with

Correspondence to: H. Mohr, DRK-Blutspendedienst Niedersach-

sen, Eldagsener Strasse 38, D-3257 Springe 1, Federal Republic of Germany

solvent/detergent treatment and other photoinactivation procedures [1, 6, 7, 14, 18, 19, 21, 22]. To a certain degree, the procedure affects the functional activities of clotting factors and other plasma proteins [17]. This is due to the fact that under the influence of light and oxygen, MB (and probably also TB) reacts with histidine residues and other amino acids [8, 15]. Therefore, the possibility exists that after photoinactivation plasma proteins are structurally modified to such an extent that neoantigens are formed. These could give rise to antibody production and allergic reactions in the recipients of such a plasma. The present investigations were concerned with this problem.

Materials and methods Photodynamic virus inactivation

Methylene blue was obtained from Neopharma, Aschau (FRG), toluidine blue from Aldrich, Steinheim (FRG). Human fresh plasma was isolated from blood donations. It was kept frozen at -30~ and, prior to use, thawed in a water bath at 27~ Photodynamic treatment was carried out as described previously [17]. Larger volumes of plasma (approximately 250 ml in polyvinylchloride plasma bags from Baxter, Munich) were illuminated with fluorescent tubes (L 36 W/77 from Osram, Munich) at a light intensity of approximately 15 000 Lux for 2 h. In small-scale experiments human or animal plasma at a volume of 2 ml in polystyrene tubes was illuminated using halogen bulbs (Halostar AR 111, 12 V, 75 W, also from Osram) at a light intensity of 150 000-160 000 Lux. Illumination time was 30 rain. Ion exchange chromatography

Chromatographic separations were carried out with the FPLC system from Pharmacia, Freiburg (FRG), using a DEAE Si 500 column (particle size: 10 ~tm, column size: 4.6 x 250 ram) which was purchased from Serva, Heidelberg (FRG). The working pressure was 286 psi. The column was equilibrated with 50 mM sodium acetate buffer, pH 5.0, which also served as washing and dilution buffer. The plasma sample (0.1 ml) was diluted 1:20 and applied to the column, which in turn was washed until the optical density at 280 nm was zero. Elution was carried out using a linear gradient from 0 to 1 M NaC1 in 50 mM Tris/HC1 buffer, pH 8.6. Total elution time was 50 min.

225 Factor analysis The functional activities of plasma proteins were assayed using commercial test kits which were obtained from Merz & Dade, D0dingen (Switzerland) and from Behring, Marburg (FRG). Factor I was assayed according to von Clauss [4]. To estimate the concentrations of the other coagulation factors, one-stage assays were used. The coagulometer (Biomatic 4000) was from Sarstedt, Nt~mbrecht (FRG). The activities of plasminogen, alphaz-antiplasmin, Cl-inactivator, and antithrombin III were determined by chromogenic substrate assays. Antisera The antisera against human whole plasma and single plasma proteins, respectively, were obtained from Fresenius, Bad Homburg (FRG) and Behring, Marburg (FRG). In some experiments we used self-made antisera against untreated as well as photodynamically treated human plasma, which were raised in rabbits as follows: In the first week, each of two animals received three intravenous injections of the respective plasma (1 mg protein). One week later, they were boostered by subcutaneous administration of the fivefold amount of plasma (5 mg protein), which had been suspended in Freund's complete adjuvant. Two weeks later they were boostered again, this time by the subcuteanous administration of plasma (1 mg protein again) in incomplete Freund's adjuvant. After 2 weeks the rabbits were bled and the sera isolated. When rabbits were immunized with untreated or photodynamically treated autologous plasma, the following procedure was used: In the first week each of three animals initially received three times intravenously 0.5 ml of the respective plasma. In the following 6 weeks, at 2-week intervals, they were boostered by subcutaneous administration of the same amount of plasma. Again, 2 weeks later the rabbits were bled and the sera isolated. Immunochemical tests Immunodiffusion was carried out according to the procedure of Ouchterlony [24]. The antiserum was placed in the lower well of a 1070 agarose plate and was allowed to diffuse overnight against various antigens in the upper wells. After formation of the precipitation arcs, the agarose was soaked in PBS (phosphate buffered saline), pH 7.4, dried and stained with 0.25070 Coomassie blue R-250 in a solution that consisted of a mixture of methanol, water, and acetic acid at a ratio of 5 : 5 : 1. Crossed immunoelectrophoresis was performed as described by Clarke and Freeman [3]. A 1070 (w/v) agarose gel was used (Pharmacia-LKB, Freiburg, FRG). The working buffer was 7.3 m M Tris, 28 m M barbital buffer, pH 8.6, containing 2 m M azide. Gel bond film was used as support. For the first dimension 10-gl samples (protein content: 1 mg/ml) were applied and electrophoresed at 10 V/cm for 6.5 cm. As a marker, bromophenol blue was mixed with the samples. Second-dimension electrophoresis against rabbit anti-human plasma sera was performed at 2 V/cm for at least 16 h. The gel was then washed with PBS, dried and stained with Coomassic brilliant blue. Passive cutaneous anaphylaxis In vivo passive cutaneous anaphylaxis (PCA) is used to detect the presence of IgE antibodies. Its principle is as follows: If intradermally administered putative antiserum contains IgE antibodies, these bind to mast cells and sensitize them. In turn, the intravenous injection of the corresponding antigens mixed with a vital dye leads to the formation of antigen-antibody complexes, which triggers mast cell degranulation and mediator release at the site of the intradermal injection. The mediators locally increase vascular permeability and cause local extravasation of the dye. The amount of extravasated dye is proportional to the amount of antigen-specific IgE antibodies in the putative antiserum [2].

The PCA testing was carried out at the Laboratory of Pharmacology and Toxicology, Hamburg (FRG). Two groups of 20 female NMRI mice each received one intraperitoneal administration of the test and control substance, respectively. The administered volume was 10 ml/kg body wt. Aluminum hydroxide gel was used as adjuvant. The test substances were mouse plasmas illuminated for 30 rain in the presence of 1 and 10 g M MB, respectively. The animals in the positive control group were immunized with 2,4-dinitrophenyl-ovalbumin (DNP-OVA). Mice which were given untreated mouse plasma served as negative controls. Sera from five animals of each group were pooled on days 10, 15, 20, and 25 after administration. They were kept at < - 8 0 ~ until use for the PCA reaction in female Sprague-Dawley rats; 0.1 ml of the different mouse sera (diluted 1:1 with physiological NaC1) was injected intradermally into the shaved skin of three rats each (2, 3, or 4 wheals per animal). The MB-containing plasma used for antigen challenge was diluted 1:10 with physiological NaC1. Four hours after intradermal administration of the respective mouse serum the rats were challenged by intravenous injection of 1 ml antigen solution/kg body wt. containing 25 mg/ml Evans blue. As positive control DNP-bovine serum albumin was administered in the same way at a dose level of 5 mg/kg body wt. The negative control rats received the serum of the mice injected with untreated mouse plasma. The animals were killed after 30 min. The skin with the wheals was removed, placed into 4-ml formamide, and kept at 70~ for 24 h. Then the optical density of the formamide solution was read at 623 rim. The means of the absorption readings of the individual groups were compared with the control values using Student's t-test.

Results

The fact that photodynamic treatment does not influence the functional activities of plasma proteins very much has a l r e a d y b e e n p u b l i s h e d [17]. T h e s e e x p e r i m e n t s were c a r r i e d o u t in test t u b e s at a v o l u m e o f 2 m l . A s Table 1 i n d i cates, s i m i l a r results w e r e o b t a i n e d u n d e r p r o d u c t i o n c o n d i t i o n s in w h i c h p l a s m a a l i q u o t s o f a p p r o x i m a t e l y 250 m l in p l a s t i c b a g s in t h e p r e s e n c e o f 1 ~ / M B were i l l u m i n a t e d w i t h f l u o r e s c e n t t u b e s f o r 2 h. T h e c o a g u l a -

Table 1. Activities of plasma proteins before and after photodynamic treatment of plasma (approximately 250-ml aliquots in plasma bags) in the presence of 1 IxM MB. Illumination time was 2 h. After treatment the plasma was stored deep frozen at - 3 0 ~ for 3 - 4 days and rethawed for testing. The values represent the means of three different plasmas Plasma protein

Factor I Factor II Factor V Factor VII Factor VIII Factor IX Factor X Factor XI Factor XII Plasminogen Alpha2antiplasmin Cl-Inactivator Antithrombin III

Before After Photodynamic treatment 2,13 m g / m l 1.42 U / m l 0.87 U / m l 1.11 U / m l 0.85 U / m l 0.58 U / m l 1.29 U / m l 1.26 U / m l 0.76 U / m l 84.6070

1.57 m g / m l 1.37 U / m l 0.79 U / m l 1.04 U / m l 0.58 U / m l 0.55 U / m l 1.03 U / m l 1.30 U / m l 0.66 U / m l 91.1070

119.4070 103.8~

109.0070 95.8070

112.4070

104.6070

Change (%)

- 26.3 3.5 -

9.2 6.3 31.8

5.2 - 20.8 + 3.1 13.2 + 7.7 -

-

8.7

-

7.7

-

6.9

226 Finally, each of three rabbits was repeatedly immunized with autologous untreated or photoinactivated plasma. After 9 weeks (and six immunizations) the putative antisera were isolated. Each was tested against untreated as well as photodynamically treated plasma by double immunodiffusion for the presence of antibodies against rabbit plasma proteins. As Fig. 4 demonstrates, no immunoprecipitation lines were detectable in the autologous (rabbit anti-rabbit) system, in contrast to the positive control, in which rabbit anti-human serum was diffused against human plasma. Table 2 shows the results of the PCA testing for the detection of IgE antibodies which could have been produced in mice by injection of light-treated mouse plasma containing 1 or 10 ~M MB. Except for the positive control, the PCA reaction was negative for all antisera used; i.e., no IgE antibodies were detected in the different mouse antisera, and hence no detectable allergens were produced in mouse plasma by the photodynamic virus inactivation with methylene blue and visible light.

tion factors I, VIII, and X proved to be relatively sensitive to the photodynamic procedure. Within 2 h these proteins lost between 20% and 30% of their original activities. The other coagulation factors tested - plasminogen and the serum inhibitors alpha2-antiplasmin, Cl-inactivator and antithrombin III - were much more resistant (Table 1). Besides those shown in Table 1, the functional activities of other plasma proteins analyzed, e. g., that of coagulation factor XIII, were also only moderately influenced by photodynamic treatment. With plasma containing TB identical results were obtained. The factor activities were not influenced by dye treatment in the dark (at dye concentrations below 2 gtM) or by illumination in the absence of either dye (not shown). In all following experiments described, 2-ml aliquots of plasma were light treated in the presence of 1 pJV/MB or TB. The results obtained also suggest that the virusinactivation procedure might affect plasma proteins, but only to a slight degree. Figure 1 shows the elution profiles of untreated and photodynamically treated plasma, separated by anion exchange chromatography on a DEAE Si 500 column. They are almost identical, with the exception that in the profile of photoinactivated plasma the size of one small protein peak, which appears at 40 mM NaC1, is reduced. This reproducible effect of photodynamic treatment was observed in the presence of MB as well as of TB. Untreated and MB/light-treated plasma were also compared by immunoelectrophoresis according to Grabar and Williams and by crossed immunoelectrophoresis. We tested more than ten plasmas, using a number of different commercial antisera against human plasma and, additionally, self-produced antisera which had been raised in rabbits against untreated as well as photodynamically treated human plasma. One example of each test is shown in Figs. 2 and 3. In no case were any differences between the two protein patterns detected. In addition, antisera against single plasma proteins (albumin, coeruloplasmin, and orosomucoid) were used. Again, no differences between untreated and MB/light-treated plasma were found (not shown).

Discussion

Various methods are integrated into the production procedures for purified plasma proteins intended for therapeutic use, e.g., heat treatment in the liquid or in the lyophilized state (in the first case stabilizers have to be added), UV irradiation in the presence of beta-propiolactone, or treatment with organic solvents in combination with detergents [6, 9, 10, 13, 14, 25]. One or the other of the procedures mentioned can, in principle, also be used to decontaminate fresh plasma, assuming that at least the important plasma proteins survive the procedure. Of course, the stabilizers or the virus-disrupting agents have to be subsequently removed, because it is too costly to handle single plasma units by the procedures mentioned above. This makes it necessary to process pooled plasma preparations. However, because the production process includes no purification steps which might eliminate viruses not susceptible to the virus-in-

NaCI(M)

OD

28o

_ ..........................

.........

"1

0

i

5

- -

10

FP

untreated

i

15

i

!

20 25 30 E l u t i o n T i m e (rain) - -

FP

photoinactlvated

~

i

t

35

40

45

......

Gradient

]

(]

50

Fig. 1. Elution profiles obtained by anion exchange chromatographyof untreated (m) and human fresh plasma (FP) photoinactivatedin the presence of 1 ~ MB (--). Arrow indicates the position of the minor protein peak, whose size was reduced after photoinactivation

227

Fig. 2. Immunoelectrophoretic analysis, according to Grabar and Williams, of untreated human plasma (above) and plasma illuminated in the presence of 1 IxM MB. The human plasma proteins were immunoprecipitated with a rabbit anti-human plasma serum

Fig. 4. A double immunodiffusion of rabbit anti-human serum with nontreated (left upper well) human plasma and human plasma (right upper well) illuminated in the presence of 1 gM MB. B, C, D double immunodiffusion of rabbit anti-rabbit serum with nontreated rabbit plasma (B) and rabbit plasma treated with light in the presence of 1 gM MB (C) and TB (D). In each case the plasmas were placed in both upper wells and the corresponding antisera in the lower well of the test plate

Table 2. PCA reaction in rats. Examination of autologous methylene blue/light-treated plasma for the induction of IgE antibody production in mice Antigen used to immunize mice and to challenge rats

Fig. 3. Crossed immunoelectrophoresis of untreated (above) and photodynamically treated human plasma. The gel for the second dimension contained a rabbit anti-human plasma serum

Mouse plasma / 1 gM MB Mouse plasma / 10 gMMB Mouse plasma / untreated (negative control) DNP-OVA (positive control)

Optical density at 623 nm Days after immunization 10 15 20

25

0.089

0.124

0.105

0.117

0.118

0.117

0.120

0.118

n.d.

n.d.

n.d.

0.094

0.195"

0.256*

0.618"

0.833*

* Significantly different from negative control: p _< 0.01 n.d., Not done activation procedure used, despite the decontamination step, the virus safety of a pool preparation might be decreased rather than increased. Photoinactivation, i.e., the treatment of plasma or plasma proteins with light in the presence of a photosensitizing substance, is a potential alternative to the virusinactivation procedures commonly used. In the present context the phenothiazine dyes MB and TB, whose virusinactivating properties have been known for a long time [11, 12, 26, 27], are of special interest. Both are used for the treatment of methemoglobinemia and other diseases. The dosages at which they are repeatedly applied without intolerable side effects are in the range of 1 - 2 m g / k g body wt. [5, 16, 20, 23]. We found that for virus inactiva-

tion in plasma only 1 gM, i.e., approximately 300 gg/1 is sufficient [17]. This is probably low enough to leave the dyes in the plasma after virus inactivation. In addition, because they are excited at wavelengths between about 600 and 660 nm [11], the plasmas of single donors can be decontaminated in those plastic bags in which they are isolated when the blood donations are processed. Due to the cationic nature of MB and TB, they bind not only to virus surfaces and to nucleic acids, but also to plasma proteins [8, 15]. Therefore, it has to be ruled out that the photoinactivation process modifies and inactivates plasma proteins and exposes neoantigenic struc-

228 tures w h i c h m i g h t be h a r m f u l to the recipients o f the p l a s m a . T h a t this p o s s i b i l i t y exists was d e m o n s t r a t e d by I n a d a et al., w h o f o u n d t h a t i l l u m i n a t i o n in the presence o f 2 0 - 1 0 0 g M M B c o m p l e t e l y i n h i b i t e d the activity o f the c o a g u l a t i o n factor I, f i b r i n o g e n [15]. O u r own d a t a show t h a t at 1 ~LA4M B the clotting activity o f factor I was r e d u c e d by a b o u t 25~ within 2 h, a n d t h o s e o f t h e factors V I I I a n d X to a similar degree. T h e f u n c t i o n a l activities o f m o s t o t h e r p l a s m a proteins tested were o n l y slightly d i m i n i s h e d , if at all. O t h e r a n a l y t i c a l a p p r o a c h e s also gave n o i n d i c a t i o n s t h a t w h e n w h o l e p l a s m a was t r e a t e d the virus-inactivation p r o c e d u r e led to m a j o r structural alterations o f plasm a proteins. T h e elution profiles o b t a i n e d by c a t i o n exchange chromatography of untreated and photoinactivated p l a s m a were a l m o s t identical. T h e n a t u r e o f the protein(s) present in t h a t m i n o r p e a k , w h o s e size was red u c e d after the p l a s m a h a d b e e n treated, has yet to be det e r m i n e d . Using a n u m b e r o f different antisera a g a i n s t h u m a n p l a s m a a n d single p l a s m a proteins we were u n a b l e to detect any differences in the i m m u n o e l e c t r o p h o r e t i c properties of untreated and photoinactivated plasma. T h e s a m e negative result was o b t a i n e d using antisera t h a t h a d been raised a g a i n s t either t y p e o f p l a s m a . Moreover, n o a n t i b o d y f o r m a t i o n was detected in rabbits which for p r o l o n g e d times were r e p e a t e d l y i m m u n i z e d with a u t o logous photoinactivated plasma. I n a d d i t i o n , P C A testing revealed t h a t there was n o i n d i c a t i o n o f IgE f o r m a t i o n after the a d m i n i s t r a t i o n o f M B / l i g h t - t r e a t e d a u t o l o g o u s p l a s m a into mice, even if the M B c o n c e n t r a t i o n was 10 g M , i.e., ten times higher t h a n usual. This latter result, t o g e t h e r with the d a t a obt a i n e d f r o m o u r in vitro investigations, suggests t h a t virus i n a c t i v a t i o n o f h u m a n p l a s m a by i l l u m i n a t i o n in the presence o f M B or TB does n o t i n d u c e n e o a n t i g e n i c structures o n p l a s m a proteins.

Acknowledgements. We thank U. Grundmann, P. Sch~fer, C. Schmidt, and B. Schr6der for their technical assistance, and J. Linneweber for her assistance in preparing the manuscript.

References 1. Alter H J, Morel PA, Dorman BP, Smith GC, Creagan RP, Wiesehahn GP, Corash L, Popper H, Eichberg JW (1988) Photochemical decontamination of blood components containing hepatitis B and non-A, non-B virus. Lancet 8627: 14461450 2. Aronson DL, Menach6 MD (1987) Prevention of infectious disease transmission by blood and blood products. Prog Haematol 15:221-241 3. Clarke MHG, Freeman T (1968) Quantitative immunoelectrophoresis of human serum proteins. Clin Sci 35:403-413 4. Clauss A v o n (1957) A rapid physiological coagulation method for determination of fibrinogen. Acta Haematol 17:237-246 5. Daunderer M (1980) Antidot-Therapie: Toluidinblau bei Meth~imoglobin~imie. Fortschr Med 98:462-464 6. Edwards CA, Piet MPJ, Chin S, Horowitz B (1987) Tri(n-butyl) phosphate/detergent treatment of licensed therapeutic and experimental blood derivatives. Vox Sang 5 2 : 5 3 - 5 9

7. Fratanoni JC, Prodouz KN (1990) Viral inactivation of blood products. Transfusion 30:480-481 8. Fujii Y, Kobashi K, Nakai N (1984) Photo-oxidation of histidine residues in rat M1- and L-type pyruvate kinases. J Biochem 95:1289-1296 9. Gellis SS, Neefe JR, Stokes J jr, Strong LE, Janewy CA, Scatchard G (1947) Chemical, clinical and immunological studies on the products of human plasma fractionation: 36. Inactivation of the virus of homologous serum hepatitis in solutions of normal human serum albumin by means of heat. J Clin Invest 27:239-244 10. Heimburger H, Schwinn H, Mauler R (1980) Factor VIII concentrate, hepatitis safe: progress in the treatment of hemophilia. Die gelben Hefte 20:165-174 11. Hiatt CW (1972) Methods for photoinactivation of viruses. In: Concepts in radiation cell biology. Academic, New York, pp 5 7 - 82 12. Hiatt CW, Kaufman E, Helprin JE, Baron S (1960) Inactivation of viruses by the photodynamic action of toluidine blue. J Immunol 84:480-484 13. Hollinger FB, Dolana G, Thomas W, Gyorkey F (1984) Reduction in risk of hepatitis transmission by heat treatment of human factor VIII concentrate. J Infect Dis 150:250-262 14. Horowitz B, Wiebe ME, Lippin A, Stryker MH (1985) Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-enveloped viruses by tri(n-butyl)phosphate detergent combinations. Transfusion 25:516-522 15. Inada Y, Hessel B, Blombaeck B (1977) Photooxidation of fibrinogen in the presence of methylene blue and its effect on polymerization. Biochim Biophys Acta 532:161-170 16. Kiese M, Loercher W, Weger N, Zierer A (1972) Comparative studies on the effects of toluidine blue and methylene blue on the reduction of ferrihaemoglobin in man and dog. Eur J Clin Pharmacol 4:115-118 17. Lambrecht B, Mohr H, Knt~ver-Hopf J, Schmitt H (1991) Photoinactivation of viruses in human fresh plasma by phenothiazine dyes in combination with visible light. Vox Sang 60: 207-213 18. Lewin AA, Schnipper LE, Crumpacker CS (1980) Photodynamic inactivation of Herpes simplex virus by hematoporphyrin derivative and light. Proc Soc Exp Biol Med 163: 81-90 19. Mathews JL, Newman JT, Sogandares-Bernal F, Judy MM, Skiles H, Leveson JE, Marengo-Rowe A J, Chanh TC (1988) Photodynamic therapy of viral contaminants with potential for blood banking applications. Transfusion 28: 81-83 20. Naylor GJ, Martin B, Hopwood SE, Watson Y (1986) A twoyear double-blind cross-over trial of the prophylactic effect of methylene blue in manic-depressive psychosis. Biol Psychiatry 21." 915-920 21. Neyendorff HC, Barrel DL, Levy JG (1990) Development of a model to demonstrate photosensitizer-mediated viral inactivation in blood. Transfusion 30:485-490 22. O'Brien JM, Montgomery RR, Burns WH, Gaffney DK, Sieber F (1990) Evaluation of merocyanine-540-sensitized photoirradiation as a means to inactivate enveloped viruses in blood products. J Lab Clin Med 116:439-447 23. Osterloh J, Olson K (1986) Toxicities of alkyl nitrites. Ann Intern Med 104:727 24. Ouchterlony O (1962) Diffusion-in-gel methods for immunological analysis. II. Prog Allergy 6:30-154 25. Stephan W (1982) Fractionation of cold sterilized plasma. A new concept of production of non-infectious plasma proteins. Arzneimittelforschung 32" 799-801 26. Wallis G, Melnick JL (1964) Irreversible photosensitization of viruses. Virology 23" 520-527 27. Wallis C, Melnick JL (1965) Photodynamic inactivation of animal viruses: a review. Photochem Photobiol 4:159-170