Journal of Virological Methods, 38 (1992) 93-102 0 1992 Elsevier Science Publishers B.V. / All rights
VIRMET
93 reserved
/ 0166-0934/92/$05.00
01334
Psoralen preparation of antigenically intact noninfectious rotavirus particles William S. Groene
and Robert
D. Shaw
Departments of MedicinejGastroenterology. Northport Veterans Administration Medical Center, Northport, NY and the State University of New York at Stony Brook, Stony Brook, NY, U.S.A. (Accepted
IO January
1992)
Summary The use of the synthetic psoralen 4’-aminomethyl-4,5’&trimethylpsoralen hydrochloride (AMT) is described for the inactivation of infectious rotavirus, a member of the viral family Reoviradue with a double-stranded RNA genome. This method not only provides complete inactivation of the virus but leaves antigenically intact particles. The lack of viral replication following inactivation was determined with an immunohistochemical focus assay. The antigenic authenticity of the particles was determined by monoclonal antibody ELISA and a viral hemagglutination assay. Psoralen; Viral inactivation;
Rotavirus
Introduction Psoralens are plant-derived photochemicals that covalently bind to nucleic acids. In the absence of UV light they are inactive, but when stimulated by long-wave UV light, psoralen effectively cross-links nucleic acids, rendering uncoiling and therefore replication impossible. This inactivation process is complete and irreversible. Several derivatives of psoralen have been isolated and several synthetic psoralens with improved water solubility have been prepared. Psoralens have been used as probes of DNA and RNA structure, and have been used effectively to completely inactivate both RNA and DNA viruses. One important feature of this method of photochemical inactivation is Correspondence Affairs Medical
to: Robert D. Shaw, Research Service, Building 61 (151), Department Center (632), 79 Middleville Road, Northport, NY 11768-2290, U.S.A.
of Veterans
94
that the psoralen interacts only with the nucleic acids, leaving the viral proteins antigenically intact. (Hanson, 1983). The rotavirus genome consists of eleven segments of double-stranded RNA. This nucleic acid is contained within the core of the virion which also contains the viral proteins VPl, VP2, and VP3, and is surrounded by an inner shell consisting mostly of VP6; the outer capsid consists of VP4 and VP7 (Estes and Cohen, 1989). The diameter of the core is approximately 50 nm, and the whole virion about 70 nm. The genomic segments, which contain in the aggregate over 18,000 base pairs, are bent and packaged into the core in close association with the surrounding proteins (Estes and Cohen, 1989). Important neutralization epitopes on the outer shell proteins VP4 and VP7 have been extensively characterized, some of which are formed by complex interactions of discontinuous parts of each protein as well as some that include regions of both proteins (Shaw et al., 1986; Mackow et al., 1988a; Mackow et al., 1988b) (also F. Ramig, unpubl. data). The complex three-dimensional interactions of these outer shell proteins necessitates care in the interpretation of studies of viral structure or immune responses to individual proteins such as those expressed in baculovirus vectors (Mackow et al., 1990; Shaw et al., 1991). Also, co-expression of several viral proteins, resulting in partial reassembly of the viral particle, is currently being suggested as a method to create synthetic non-infectious vaccine candidates. These subunit vaccine candidates have also been proposed as carriers of synthetic peptides derived from other viral of such proteins (Ijaz et al., 1991). Any evaluation of the immunogenicity particles may be easily and effectively predicted by the immunogenicity of intact non-replicating virus, which will be complete with all structural features and protein interactions. We now describe the use of a synthetic psoralen derivative, AMT (4’aminomethyl-4,5’,8_trimethylpsoralen hydrochloride), to inactivate the doublestranded RNA genome of rhesus rotavirus. The inactivated rotavirus retains the ability to hemagglutinate, and is antigenically recognizable by rotavirusspecific monoclonal antibodies. This technique allows the convenient and reliable preparation of structurally intact but non-replicating rotavirus particles.
Materials and Methods Cell and virus growth Rhesus rotavirus was grown in MA104 cells as previously described (Shaw et al., 1985). The plaque titer of the virus was approximately 5 x 10’ PFU/ml. All of the virus was used as tissue culture supernatant and not purified or concentrated. The virus inoculum (2.5 x lo6 PFU) was incubated on the monolayers in T75 tissue culture flasks until CPE was complete (usually about 48 h), at which time the entire contents of the flask were briefly frozen at -80°C then thawed and pooled.
95
Psoralen inactivation of virus
4’-Aminomethyl-4,5’,8-trimethylpsoralen (HCL) referred to as AMT was purchased from HRI Associates, Berkeley, CA. AMT was prepared as a 1 mg/ml stock solution in 50% ethanol, 50% water. 7 ml of viral tissue culture supernatant were added to each of 5 T25 flasks, producing a fluid layer about 3 mm deep. AMT was added to the flasks, which were then chilled on an ice bath for 15 min. The bottom and sides of the flasks were wrapped in foil and placed on top of an ice pack. The entire unit was then placed under the bulb of the UV light. The distance between the top of the flask and the bulb of the UV light was 7.5 cm. The UV light source (GBL-100C utilizing 100 W mercury reflector lamps, G.B. Gates and Co., Franklin Square, NY) emitted ligh! at a peak wavelength of 365 A and filtered out any light greater than 440 A. The AMT concentration and the duration of UV exposure was titrated. Immunochemical
focus assay of infectious virus The technique used was similar to a method previously described (Shaw et al., 1986). Ma104 cell monolayers were grown to confluency in 96-well plates. 100 ~1 virus samples (a checkerboard titration of amount of psoralen and time of UV exposure) were added to each well. Samples were run in triplicate. The plates were incubated at 37°C for 20 h. After the incubation, the virus was aspirated from each well. The plate was fixed by adding 200 ~1 of 100% ice-cold methanol to each well. 100 ,ul of guinea pig anti-RRV hyperimmune sera at 1: 1000 in PBS + 1% BSA was added to each well and incubated 30 min at 37°C. The plate was then washed two times with PBS. 100 ~1 of goat anti-guinea pig-horseradish peroxidase (KPL, Gaithersburg, MD) at 1:lOOO in PBS + 1% BSA was added to each well and incubated for 30 min at 37°C. The plate was then washed two times with PBS. 50 ~1 of the precipitating substrate 3-amino-9-ethyl-carbazole (AEC, Sigma Chemical Co., A5754) in NJ-dimethyl-formamide was added to each well. The plate was incubated for 1@15 min in the dark. The substrate was aspirated and 100 ~1 PBS + 0.1% sodium azide was added to stop the reaction. Hemagglutination assay Serial dilutions of virus samples were made in PBS containing 1% BSA in a 96-well round bottom microtiter plate. The titer is read as the reciprocal of the dilution of the greatest dilution that shows hemagglutination. ELBA
for Rotavirus detection The method used was modified from one published previously (Shaw et al., 1985). Immulon 2 microtiter plates were coated with Protein G-purified guinea pig IgG derived from rhesus rotavirus hyperimmune antisera. Serial dilutions of untreated RRV, psoralen-inactivated RRV, or mock virus (Ma104 cell lysate) were added. Optimal concentrations of capture antibody and RRV were predetermined in a checkerboard analysis. Excess binding sites were then blocked using PBS containing 2% FBS. 200 ~1 per well were added and the plates were incubated for 1 h at 37°C. The plates
96
were then washed 3 times with PBS. Each of the different virus samples was then detected using each of three different mouse monoclonal antibodies; these were designated 2G4 (anti-VP4), 7A12 (anti-VP4) and 4F8 (anti-VP7). The monoclonals were used in the form of mouse ascites diluted 1: 1000 in PBS (1% FBS). 50 ~1 per well were incubated for 3 h at 37°C. The plates were then washed 3 times with PBS. 50 ~1 per well of goat anti-mouse IgG labeled with biotin diluted 1: 1000 in PBS (1% normal goat serum) was then added and incubated for 1 h. The plates were then washed 3 times with PBS. 50 ~1 per well of Avidin D-horseradish peroxidase diluted 1: 1000 in PBS (1% FBS and 0.05% Tween 20) was then added and incubated for 1 h. The plates were then washed 3 times with PBS. 50 ~1 of the soluble substrate O-phenylene-diamine (Sigma #P8287) containing 2 ,ul of 30% hydrogen peroxide was then added and the reaction was allowed to continue in the dark for about 10 min. The reaction was terminated by the addition of 50 ~1 of a 3N sulfuric acid. The optical density at 490 nm was measured using a Biotek automated ELISA plate reader.
Results Immunochemical focus assay of viral infectivity Fig. 1 is a photomicrograph depicting the difference in viral infectivity between the AMT/UV-treated and untreated virus. In Fig. la, the darkly-staining MA104 cells contain replicating viral particles. Fig. lb shows the results of infection with an equal amount of the same virus pool after treatment with 30 pg/ml of AMT followed by 45 min of exposure to long-wave UV light. The lack of detectable viral antigen indicated that the virus was rendered completely noninfectious by the AMT/ UV treatment. Table 1 contains the results of an histochemical focus assay of several different viral samples, prepared by titrating the amount of AMT at various time intervals of UV light exposure. When no AMT was used and the virus was only exposed to light, the virus was not adversely affected and was still able to replicate. Conversely, when the virus was treated with AMT but not exposed to UV light, the virus remained infectious. As is evident in the table, the more AMT that is added, the less UV exposure time that is necessary to inactivate the virus.
Fig. 1. a. Ma104 cells monolayers in 24-well tissue culture dishes were exposed to 50 ~1 of the test virus (rhesus rotavirus), which was present at 10’ infectious particles/ml, for 14 h. Viral antigen was detected with an immunochemical detection assay as described in the text. The dark cytoplasmic staining in newly synthesized rotavirus antigen can be readily seen in this view of a portion of the monolayer. b. The result of the exposure of the monolayer to a portion of the same viral pool which had undergone treatment with psoralen (AMT, 30 pg/ml) and long-wave UV light (45 min). There was a total absence of detectable viral antigen in the entire monolayer.
98 TABLE I The effect of psoralen concentration and duration of UV exposure rotavirus as detected by an immunohistochemical focus assay
WMTI
Time” (min)
(pglml) 0
10
5 ::
4+ 4+ 4+
40
4+
3+ 3+ 2+ 3+ 3+
0
on the viability of rhesus
__~__ 20
30
40
50
3+ 3+ 2-k 2+
4+ 2+ 2+ 1+
1+
0
4+ 2+ 1+ 0 0
4+ 1+ 0 0 0
“The scale of 04 + is arbitrarily determined by the following parameters: the maximum obtainable antigen (no psoralen or UV treatment) designated 4+, which correlates to all cells stained; approximately 50% of cells stained, 3 + ; 25%, 2 + ; IO%, I + No stainable antigen whatsoever is designated 0.
Analysis of viral antigen authenticity Virus which was treated with 30 pg/ml of AMT for 45 min (well into the range determined to create inactive particles) was run in an ELISA assay to determine whether or not the inactivation procedure damaged the antigenic epitopes on the surface of viral particles. The monoclonal antibodies directed at the two major outer shell antigens of the RRV particle were used in an ELBA assay. The ELISA titers obtained using either untreated or treated virus did not differ among any of the antibodies used. Antibodies directed at different outer shell proteins and different epitopes on the same protein did not differ in titer between the treated of untreated virus. Hyperimmune sera also did not differ. Analysis of viral antigen hemagglutination Treated and untreated viral samples were compared in hemagglutination assays to determine whether the hemagglutination function of surface antigens was affected by AMTjUV exposure. The titers did not significantly vary between the treated (40 pg/ml of AMT for 50 min) and untreated samples as both demonstrated titers of 1:128256. The same titer was observed following treatment with either AMT or UV alone.
Discussion The use of a synthetic psoralen derivative, AMT, is described to inhibit completely the replication of rhesus rotavirus in tissue culture. This synthetic psoralen derivative is highly soluble in water and has a iWr of 293 Da. This method of viral inactivation was demonstrated to preserve the virion capsid structure by virtue of the ability of the virus to function as a hemagglutinin, and antigenic preservation of neutralization epitopes on the two outer capsid proteins was demonstrated by neutralizing monoclonal antibody recognition of psoralen treated virus. d
99
Psoralens block nucleic acid replication by forming covalent bonds with pyrimidines in single and double-stranded nucleic acids in the presence of longwave (320-380 nm) UV light - not short wave - or ‘germicidal’ UV. The psoralen molecule is bifunctional, resulting in the bridging of double-stranded structures and preventing the uncoiling required for replication or transcription (Hanson, 1983). Psoralens in general are non-toxic in the absence of UV light, permitting cell growth in culture and virus propagation (data not shown) (Hanson et al., 1978). If the photoinactivation procedure is excessive, however, photoreactions may potentially affect ribosomal proteins (Singh and Vadasz, 1978) enzymes (Veronese et al. 1982) membranes (Laskin et al., 1985, 1986) and unsaturated fatty acids (Specht et al., 1988). It is therefore useful to define the least conditions of photochemical inactivation consistent with complete inactivation. Previously, rotavirus inactivation with beta-propriolactone (BPL) has been reported. This method is effective for virus inactivation, but it also damages the outer protein antigens (Oflit et al., 1989). BPL reacts rapidly in water with nucleic acids but also hydroxyl, amino, carboxyl, sulphydryl, and phenolic groups associated with proteins (LoGrippo, 1958). Treatment of rhesus rotavirus with 0.15% or greater concentrations of BPL, which effectively eliminated the ability of the virus to grow in tissue culture, resulted in decreases in hemagglutination titer from 64 to 4 (an 8-fold reduction) (Offit et al., 1989). As rotavirus hemagglutination requires intact outer capsid structure and an erythrocyte recognition site on the VP4 protein, loss of hemagglutination implies damage to these structures (Mackow et al., 1988). In contrast, treatment of the same virus with AMT/UV also completely eliminated the ability of the virus to grow, but there was no decrease in the hemagglutination titer. Psoralens (including AMT) have been reported previously to cross-link RNA of reoviruses, also members of the viral family Reoviridae which share a similar genomic structure to rotaviruses (Nakashima and Shatkin, 1978). Reovirus type 3 inactivation was a function of both AMT concentration and duration of UV exposure. The AMT cross-linked the double-stranded RNA genome, but did not react with the reovirus proteins. However, even at the greatest concentrations and longest durations of irradiation, reovirus was not demonstrated to be completely inactivated. 2.8 x lo4 M AMT irradiated for 15 min resulted in a decrease in plaque-forming units from 2.0 x lo8 to < 104. For an assessment of the importance of viral replication as a determinant of immunity, it is vital to demonstrate complete inactivation of the virus as we here report for rotavirus. The concentration of AMT required to inactivate this rotavirus preparation is comparable to those required to inactivate other viruses. For instance, 5-10 pg/ml have been used to inactivate HIV (Sutjipto et al., 1990; Watson et al., 1990), 12 pg/ml added three times over 1 h was used for adenovirus (Wong and Hsu, 1988) and 25 pg/ml inactivated Rous sarcoma virus (Swanstrom et al., 1981). Reovirus was treated in the form of purified concentrated samples and
100
the authors failed to document complete inactivation with over 80 lug/ml (Nakashima and Shatkin, 1978). Times used for inactivation varied widely, but were generally not studied to minimize exposure. While many authors provide information describing the intensity of the light source, precise comparisons are still somewhat limited due to variations in the physical arrangements, temperature variations, clarity of media, and a multitude of other effects. Overall, the minimum conditions we report are typical: 10 pg/ml for 50 min or 40 pg/ml for 10 min. A prerequisite for effective viral inactivation by AMT is that the psoralen must be able to gain access to the rotavirus and then penetrate the doubleprotein capsid and cross-link the genome at the core. The penetration by AMT of cells and debris that harbor virus in crude preparations such as we have used in the present study is well-documented. In fact, AMT has been suggested for use in the decontamination of cells and tissues specimens infected with HIV prior to handling in a variety of environments (Watson et al., 1990). The psoralen must also penetrate the viral protein capsids to gain access to the nucleic acid core. The reconstructed electron-microscopic images published by Yeager et al. (1990) demonstrated aqueous pores that penetrated the outer capsids and appeared to be in communication with the viral core. These channels may explain the ease by which the psoralen molecules can gain access to the genome without perturbing the capsid structure. The antigenic condition of the outer capsid viral proteins was determined with an ELISA using several monoclonal antibodies, each directed at a different protein. The ability of monoclonal antibody 4F8 to recognize psoralen-treated virus is particularly compelling evidence of structural and antigenic integrity because the epitope on the outer capsid protein VP7 that is recognized by this antibody is conformationally determined. Binding is completely abrogated following disruption of capsid structure such as occurs with calcium chelation with EDTA, even if the disrupted VP7 is still present (Shaw et al., 1986; Mackow et al., 1988) (also R. Shaw and H. Greenberg, unpubl. data). Neither does this nor many other neutralizing monoclonal antibodies recognize VP7 in a Western blot format. We determined the relationship between the concentration of AMT and the duration of UV light exposure necessary to inactivate the rotavirus. As the amount of AMT is increased, the amount of UV exposure time is decreased and vice versa. This inverse relationship is in agreement with the findings of studies on reovirus inactivation (Nakashima and Shatkin, 1978). It is important to reiterate that these concentrations of AMT are effective for the inactivation of virus in unclarified tissue culture supernatants following cell lysis from viral cytopathic effect and a subsequent cycle of freezing and thawing. If higher densities of virus (either concentrated by centrifugation with cell debris or gradient-purified) are to be inactivated the concentration or duration will likely need to be increased accordingly due to increased amounts of RNA, or perhaps due to light-scattering effects of cell debris. Several other DNA and RNA viruses have been inactivated with the highly
101
water soluble AMT, and demonstrated to retain antigenic integrity. AMT has shown particularly good performance, however, against the more-diffcult-toinactivate RNA viruses. In many studies there were no efforts made to assess antigenic integrity, and some studies were published prior to the common availability of monoclonal antibodies, which obviously permit a superior assessment of antigenic characteristics. Viruses which have been shown to be vulnerable to AMT inactivation include reovirus (Nakashima and Shatkin, 1978), HIV (Watson et al., 1990), Adenovirus (Wong and Hsu, 1988), Vesicular stomatitis virus (Hearst and Thiry, 1977; Nakashima et al., 1979), herpes simplex type 1 (Hanson et al., 1978), Western equine encephalitis virus (Hanson et al., 1978), and Influenza A (Nakashima et al., 1979). Psoralen-inactivated antigenically intact rotavirus particles will be useful in experiments to determine the importance of viral replication in eliciting an intestinal immune response to the rotavirus. Such important determinants of the immune response such as binding, uptake, and processing by antigen presenting cells in the intestinal mucosa will be identical in all respects except for replicative capabilities.
References Estes, M.K. and Cohen, J. (1989) Rotavirus gene structure and function. Microbial. Rev. 53, 41& 449. Hanson, C.V. (1983) Inactivation of viruses for use as vaccines and immunodiagnostic reagents. Medical Virology II. Elsevier, New York, pp. 45-78. Hanson, C.V., Riggs, J.L. and Lennette, E.H. (1978) Photochemical inactivation of DNA and RNA viruses by psoralen derivatives. J. Gen. Virol. 40, 3455358. Hearst, J.E. and Thiry, L. (1977) The photoinactivation of an RNA animal virus, vesicular stomatitis virus, with the aid of newly synthesized psoralen derivatives. Nucleic Acids Res. 4, 1339-1347. Ijaz, M.K.; Attah, P.S., Redmond, M.J., Parker, M.D., Sabara, M.I. and Babiuk, L.A. (1991) Heterotypic passive protection induced by synthetic peptides corresponding to VP7 and VP4 of bovine rotavirus. J. Virol. 65, 31063113. Laskin, J., Lee, E., Yurkow, E., Laskin, D. and Gallo, M. (1985) A possible mechanism of psoralen phototoxicity not involving direct interaction with DNA. Proc. Nat]. Acad. Sci. USA 82, 61856162. Laskin, J., Lee, E., Laskin, D. and Gallo, M. (1986) Psoralens potentiate ultraviolet light-induced inhibition of epidermal growth factor binding. Proc. Natl. Acad. Sci. USA 83, 821 l-8215. LoGrippo, G.A. (1958) Antigenicity of combined beta-propriolactone and UV-inactivated virus vaccines. J. Immunol. 80, 198-203. Mackow, E., Shaw, R., Matsui, S.M., Vo, P.T., Bass, D. and Greenberg, H.B. (1988a) Characterization of homotypic and heterotypic VP7 neutralization sites of rhesus rotavirus. Virology 165, 511-517. Mackow, E.R., Shaw, R.D., Matsui, SM., Vo, P.T., Benfield, D.A. and Greenberg, H.B. (1988a) Characterization of Homotypic and Heterotypic VP7 neutralization sites of rhesus rotavirus. Virology 165, 511-517. Mackow, E.R., Shaw, R.D., Matsui, S.M., Vo, P.T., Dang, M.N. and Greenberg, H.B. (1988a) The rhesus rotavirus gene encoding protein VP3: location of amino acids involved in homologous and heterologous rotavirus neutralization and identification of a putative fusion region. Proc. Natl. Acad. Sci. USA 85, 645-649.
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Mackow, E.R., Vo, P.T., Broome, R., Bass, D. and Greenberg, H.B. (1990) Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge. J. Virol. 64, 16981703. Nakashima, K. and Shatkin, A.J. (1978) Photochemical cross-linking of reovirus genome RNA in situ and inactivation of viral transcriptase. J. Biol. Chem. 253, 868@-8682. Nakashima, K., Chandra, P.K., Deutsch, V., Banerjee, A.K. and Shatkin, A.J. (1979) Inactivation of influenza and vesicular stomatitis virion RNA polymerase activities by photoreaction with 4’substituted psoralens. J. Virol. 32, 838-844. Offit, P.A., Greenberg, H.B. and Dudzik, K.I. (1989) Rotavirus-specific protein synthesis is not necessary for recognition of infected cells by virus-specific cytotoxic T lymphocytes. J. Virol. 63, 3279-3283. Shaw, R.D., Stoner-Ma, D., Estes, M. and Greenberg, H. (1985) Specific enzyme-linked immunoassay for rotavirus serotypes 1 and 3. J. Clin. Microbial. 22, 286-291. Shaw, R., Coulson, B., Kaljot, K., OfIit, P. and Greenberg, H. (1986) Antigenic mapping of the surface proteins of rhesus rotavirus. Virology 155, 434451. Shaw, R.D., Groene, W.S., Mackow, E.R., Merchant, A.A. and Cheng, E.H. (1991) VP4-specific intestinal antibody response to rotavirus in a murine model of heterotypic infection. J. Virol. 65, 3052-3059. Singh, H. and Vadasz, J.A. (1978) Singlet oxygen: a major reactive species in the furocoumarin photosensitized inactivation of E. coli ribosomes. Photochem. Photobiol. 28, 539-545. Specht, K., Kittler, L. and Midden, W. (1988) A new biological target of fumocoumarins: Photochemical formation of covalent adducts with unsaturated fatty acids. Photochem. Photobiol. 47, 537--541. Sutjipto, S., Pedersen, N.C., Miller, C.J., Gardner, M.B., Hanson, C.V., Gettie, A., Jennings, M., Higgins, J. and Marx, P.A. (1990) Inactivated simian immunodeficiency virus vaccine failed to protect rhesus macaques from intravenous of genital mucosal infection but delayed disease in intravenously exposed animals. J. Viral. 64, 229&2297. Swanstrom, R., Hallick, L.M., Jackson, J., Hearst, J.E. and Bishop, J.M. (1981) Interaction of psoralen derivatives with the RNA genome of rous sarcoma virus. Virology 113, 613-622. Veronese, F.O.S.R.B., Bordin, F. and Rodighiero, G. (1982) Photoinactivation of enzymes by linear and angular furocoumarins. Photochem. Photobiol. 36, 25-30. Watson, A.J., Klanieki, J. and Hanson, C.V. (1990) Psoralen/UV inactivation of HIV-l-infected cells for use in cytologic and immunologic procedures. AIDS Res. Hum. Retroviruses 6, 503513. Wong, M.L. and Hsu, M.T. (1988) Psoralen-cross-linking study of the organization of intracellular adenovirus nucleoprotein complexes. J. Viral. 62, 1227-1234. Yeager, M., Dryden, K.A., Olson, N.H., Greenberg, H.B. and Baker, T.S. (1990) Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction. J. Cell Biol. 110, 2133-2144.
VIRMET
93 reserved
/ 0166-0934/92/$05.00
01334
Psoralen preparation of antigenically intact noninfectious rotavirus particles William S. Groene
and Robert
D. Shaw
Departments of MedicinejGastroenterology. Northport Veterans Administration Medical Center, Northport, NY and the State University of New York at Stony Brook, Stony Brook, NY, U.S.A. (Accepted
IO January
1992)
Summary The use of the synthetic psoralen 4’-aminomethyl-4,5’&trimethylpsoralen hydrochloride (AMT) is described for the inactivation of infectious rotavirus, a member of the viral family Reoviradue with a double-stranded RNA genome. This method not only provides complete inactivation of the virus but leaves antigenically intact particles. The lack of viral replication following inactivation was determined with an immunohistochemical focus assay. The antigenic authenticity of the particles was determined by monoclonal antibody ELISA and a viral hemagglutination assay. Psoralen; Viral inactivation;
Rotavirus
Introduction Psoralens are plant-derived photochemicals that covalently bind to nucleic acids. In the absence of UV light they are inactive, but when stimulated by long-wave UV light, psoralen effectively cross-links nucleic acids, rendering uncoiling and therefore replication impossible. This inactivation process is complete and irreversible. Several derivatives of psoralen have been isolated and several synthetic psoralens with improved water solubility have been prepared. Psoralens have been used as probes of DNA and RNA structure, and have been used effectively to completely inactivate both RNA and DNA viruses. One important feature of this method of photochemical inactivation is Correspondence Affairs Medical
to: Robert D. Shaw, Research Service, Building 61 (151), Department Center (632), 79 Middleville Road, Northport, NY 11768-2290, U.S.A.
of Veterans
94
that the psoralen interacts only with the nucleic acids, leaving the viral proteins antigenically intact. (Hanson, 1983). The rotavirus genome consists of eleven segments of double-stranded RNA. This nucleic acid is contained within the core of the virion which also contains the viral proteins VPl, VP2, and VP3, and is surrounded by an inner shell consisting mostly of VP6; the outer capsid consists of VP4 and VP7 (Estes and Cohen, 1989). The diameter of the core is approximately 50 nm, and the whole virion about 70 nm. The genomic segments, which contain in the aggregate over 18,000 base pairs, are bent and packaged into the core in close association with the surrounding proteins (Estes and Cohen, 1989). Important neutralization epitopes on the outer shell proteins VP4 and VP7 have been extensively characterized, some of which are formed by complex interactions of discontinuous parts of each protein as well as some that include regions of both proteins (Shaw et al., 1986; Mackow et al., 1988a; Mackow et al., 1988b) (also F. Ramig, unpubl. data). The complex three-dimensional interactions of these outer shell proteins necessitates care in the interpretation of studies of viral structure or immune responses to individual proteins such as those expressed in baculovirus vectors (Mackow et al., 1990; Shaw et al., 1991). Also, co-expression of several viral proteins, resulting in partial reassembly of the viral particle, is currently being suggested as a method to create synthetic non-infectious vaccine candidates. These subunit vaccine candidates have also been proposed as carriers of synthetic peptides derived from other viral of such proteins (Ijaz et al., 1991). Any evaluation of the immunogenicity particles may be easily and effectively predicted by the immunogenicity of intact non-replicating virus, which will be complete with all structural features and protein interactions. We now describe the use of a synthetic psoralen derivative, AMT (4’aminomethyl-4,5’,8_trimethylpsoralen hydrochloride), to inactivate the doublestranded RNA genome of rhesus rotavirus. The inactivated rotavirus retains the ability to hemagglutinate, and is antigenically recognizable by rotavirusspecific monoclonal antibodies. This technique allows the convenient and reliable preparation of structurally intact but non-replicating rotavirus particles.
Materials and Methods Cell and virus growth Rhesus rotavirus was grown in MA104 cells as previously described (Shaw et al., 1985). The plaque titer of the virus was approximately 5 x 10’ PFU/ml. All of the virus was used as tissue culture supernatant and not purified or concentrated. The virus inoculum (2.5 x lo6 PFU) was incubated on the monolayers in T75 tissue culture flasks until CPE was complete (usually about 48 h), at which time the entire contents of the flask were briefly frozen at -80°C then thawed and pooled.
95
Psoralen inactivation of virus
4’-Aminomethyl-4,5’,8-trimethylpsoralen (HCL) referred to as AMT was purchased from HRI Associates, Berkeley, CA. AMT was prepared as a 1 mg/ml stock solution in 50% ethanol, 50% water. 7 ml of viral tissue culture supernatant were added to each of 5 T25 flasks, producing a fluid layer about 3 mm deep. AMT was added to the flasks, which were then chilled on an ice bath for 15 min. The bottom and sides of the flasks were wrapped in foil and placed on top of an ice pack. The entire unit was then placed under the bulb of the UV light. The distance between the top of the flask and the bulb of the UV light was 7.5 cm. The UV light source (GBL-100C utilizing 100 W mercury reflector lamps, G.B. Gates and Co., Franklin Square, NY) emitted ligh! at a peak wavelength of 365 A and filtered out any light greater than 440 A. The AMT concentration and the duration of UV exposure was titrated. Immunochemical
focus assay of infectious virus The technique used was similar to a method previously described (Shaw et al., 1986). Ma104 cell monolayers were grown to confluency in 96-well plates. 100 ~1 virus samples (a checkerboard titration of amount of psoralen and time of UV exposure) were added to each well. Samples were run in triplicate. The plates were incubated at 37°C for 20 h. After the incubation, the virus was aspirated from each well. The plate was fixed by adding 200 ~1 of 100% ice-cold methanol to each well. 100 ,ul of guinea pig anti-RRV hyperimmune sera at 1: 1000 in PBS + 1% BSA was added to each well and incubated 30 min at 37°C. The plate was then washed two times with PBS. 100 ~1 of goat anti-guinea pig-horseradish peroxidase (KPL, Gaithersburg, MD) at 1:lOOO in PBS + 1% BSA was added to each well and incubated for 30 min at 37°C. The plate was then washed two times with PBS. 50 ~1 of the precipitating substrate 3-amino-9-ethyl-carbazole (AEC, Sigma Chemical Co., A5754) in NJ-dimethyl-formamide was added to each well. The plate was incubated for 1@15 min in the dark. The substrate was aspirated and 100 ~1 PBS + 0.1% sodium azide was added to stop the reaction. Hemagglutination assay Serial dilutions of virus samples were made in PBS containing 1% BSA in a 96-well round bottom microtiter plate. The titer is read as the reciprocal of the dilution of the greatest dilution that shows hemagglutination. ELBA
for Rotavirus detection The method used was modified from one published previously (Shaw et al., 1985). Immulon 2 microtiter plates were coated with Protein G-purified guinea pig IgG derived from rhesus rotavirus hyperimmune antisera. Serial dilutions of untreated RRV, psoralen-inactivated RRV, or mock virus (Ma104 cell lysate) were added. Optimal concentrations of capture antibody and RRV were predetermined in a checkerboard analysis. Excess binding sites were then blocked using PBS containing 2% FBS. 200 ~1 per well were added and the plates were incubated for 1 h at 37°C. The plates
96
were then washed 3 times with PBS. Each of the different virus samples was then detected using each of three different mouse monoclonal antibodies; these were designated 2G4 (anti-VP4), 7A12 (anti-VP4) and 4F8 (anti-VP7). The monoclonals were used in the form of mouse ascites diluted 1: 1000 in PBS (1% FBS). 50 ~1 per well were incubated for 3 h at 37°C. The plates were then washed 3 times with PBS. 50 ~1 per well of goat anti-mouse IgG labeled with biotin diluted 1: 1000 in PBS (1% normal goat serum) was then added and incubated for 1 h. The plates were then washed 3 times with PBS. 50 ~1 per well of Avidin D-horseradish peroxidase diluted 1: 1000 in PBS (1% FBS and 0.05% Tween 20) was then added and incubated for 1 h. The plates were then washed 3 times with PBS. 50 ~1 of the soluble substrate O-phenylene-diamine (Sigma #P8287) containing 2 ,ul of 30% hydrogen peroxide was then added and the reaction was allowed to continue in the dark for about 10 min. The reaction was terminated by the addition of 50 ~1 of a 3N sulfuric acid. The optical density at 490 nm was measured using a Biotek automated ELISA plate reader.
Results Immunochemical focus assay of viral infectivity Fig. 1 is a photomicrograph depicting the difference in viral infectivity between the AMT/UV-treated and untreated virus. In Fig. la, the darkly-staining MA104 cells contain replicating viral particles. Fig. lb shows the results of infection with an equal amount of the same virus pool after treatment with 30 pg/ml of AMT followed by 45 min of exposure to long-wave UV light. The lack of detectable viral antigen indicated that the virus was rendered completely noninfectious by the AMT/ UV treatment. Table 1 contains the results of an histochemical focus assay of several different viral samples, prepared by titrating the amount of AMT at various time intervals of UV light exposure. When no AMT was used and the virus was only exposed to light, the virus was not adversely affected and was still able to replicate. Conversely, when the virus was treated with AMT but not exposed to UV light, the virus remained infectious. As is evident in the table, the more AMT that is added, the less UV exposure time that is necessary to inactivate the virus.
Fig. 1. a. Ma104 cells monolayers in 24-well tissue culture dishes were exposed to 50 ~1 of the test virus (rhesus rotavirus), which was present at 10’ infectious particles/ml, for 14 h. Viral antigen was detected with an immunochemical detection assay as described in the text. The dark cytoplasmic staining in newly synthesized rotavirus antigen can be readily seen in this view of a portion of the monolayer. b. The result of the exposure of the monolayer to a portion of the same viral pool which had undergone treatment with psoralen (AMT, 30 pg/ml) and long-wave UV light (45 min). There was a total absence of detectable viral antigen in the entire monolayer.
98 TABLE I The effect of psoralen concentration and duration of UV exposure rotavirus as detected by an immunohistochemical focus assay
WMTI
Time” (min)
(pglml) 0
10
5 ::
4+ 4+ 4+
40
4+
3+ 3+ 2+ 3+ 3+
0
on the viability of rhesus
__~__ 20
30
40
50
3+ 3+ 2-k 2+
4+ 2+ 2+ 1+
1+
0
4+ 2+ 1+ 0 0
4+ 1+ 0 0 0
“The scale of 04 + is arbitrarily determined by the following parameters: the maximum obtainable antigen (no psoralen or UV treatment) designated 4+, which correlates to all cells stained; approximately 50% of cells stained, 3 + ; 25%, 2 + ; IO%, I + No stainable antigen whatsoever is designated 0.
Analysis of viral antigen authenticity Virus which was treated with 30 pg/ml of AMT for 45 min (well into the range determined to create inactive particles) was run in an ELISA assay to determine whether or not the inactivation procedure damaged the antigenic epitopes on the surface of viral particles. The monoclonal antibodies directed at the two major outer shell antigens of the RRV particle were used in an ELBA assay. The ELISA titers obtained using either untreated or treated virus did not differ among any of the antibodies used. Antibodies directed at different outer shell proteins and different epitopes on the same protein did not differ in titer between the treated of untreated virus. Hyperimmune sera also did not differ. Analysis of viral antigen hemagglutination Treated and untreated viral samples were compared in hemagglutination assays to determine whether the hemagglutination function of surface antigens was affected by AMTjUV exposure. The titers did not significantly vary between the treated (40 pg/ml of AMT for 50 min) and untreated samples as both demonstrated titers of 1:128256. The same titer was observed following treatment with either AMT or UV alone.
Discussion The use of a synthetic psoralen derivative, AMT, is described to inhibit completely the replication of rhesus rotavirus in tissue culture. This synthetic psoralen derivative is highly soluble in water and has a iWr of 293 Da. This method of viral inactivation was demonstrated to preserve the virion capsid structure by virtue of the ability of the virus to function as a hemagglutinin, and antigenic preservation of neutralization epitopes on the two outer capsid proteins was demonstrated by neutralizing monoclonal antibody recognition of psoralen treated virus. d
99
Psoralens block nucleic acid replication by forming covalent bonds with pyrimidines in single and double-stranded nucleic acids in the presence of longwave (320-380 nm) UV light - not short wave - or ‘germicidal’ UV. The psoralen molecule is bifunctional, resulting in the bridging of double-stranded structures and preventing the uncoiling required for replication or transcription (Hanson, 1983). Psoralens in general are non-toxic in the absence of UV light, permitting cell growth in culture and virus propagation (data not shown) (Hanson et al., 1978). If the photoinactivation procedure is excessive, however, photoreactions may potentially affect ribosomal proteins (Singh and Vadasz, 1978) enzymes (Veronese et al. 1982) membranes (Laskin et al., 1985, 1986) and unsaturated fatty acids (Specht et al., 1988). It is therefore useful to define the least conditions of photochemical inactivation consistent with complete inactivation. Previously, rotavirus inactivation with beta-propriolactone (BPL) has been reported. This method is effective for virus inactivation, but it also damages the outer protein antigens (Oflit et al., 1989). BPL reacts rapidly in water with nucleic acids but also hydroxyl, amino, carboxyl, sulphydryl, and phenolic groups associated with proteins (LoGrippo, 1958). Treatment of rhesus rotavirus with 0.15% or greater concentrations of BPL, which effectively eliminated the ability of the virus to grow in tissue culture, resulted in decreases in hemagglutination titer from 64 to 4 (an 8-fold reduction) (Offit et al., 1989). As rotavirus hemagglutination requires intact outer capsid structure and an erythrocyte recognition site on the VP4 protein, loss of hemagglutination implies damage to these structures (Mackow et al., 1988). In contrast, treatment of the same virus with AMT/UV also completely eliminated the ability of the virus to grow, but there was no decrease in the hemagglutination titer. Psoralens (including AMT) have been reported previously to cross-link RNA of reoviruses, also members of the viral family Reoviridae which share a similar genomic structure to rotaviruses (Nakashima and Shatkin, 1978). Reovirus type 3 inactivation was a function of both AMT concentration and duration of UV exposure. The AMT cross-linked the double-stranded RNA genome, but did not react with the reovirus proteins. However, even at the greatest concentrations and longest durations of irradiation, reovirus was not demonstrated to be completely inactivated. 2.8 x lo4 M AMT irradiated for 15 min resulted in a decrease in plaque-forming units from 2.0 x lo8 to < 104. For an assessment of the importance of viral replication as a determinant of immunity, it is vital to demonstrate complete inactivation of the virus as we here report for rotavirus. The concentration of AMT required to inactivate this rotavirus preparation is comparable to those required to inactivate other viruses. For instance, 5-10 pg/ml have been used to inactivate HIV (Sutjipto et al., 1990; Watson et al., 1990), 12 pg/ml added three times over 1 h was used for adenovirus (Wong and Hsu, 1988) and 25 pg/ml inactivated Rous sarcoma virus (Swanstrom et al., 1981). Reovirus was treated in the form of purified concentrated samples and
100
the authors failed to document complete inactivation with over 80 lug/ml (Nakashima and Shatkin, 1978). Times used for inactivation varied widely, but were generally not studied to minimize exposure. While many authors provide information describing the intensity of the light source, precise comparisons are still somewhat limited due to variations in the physical arrangements, temperature variations, clarity of media, and a multitude of other effects. Overall, the minimum conditions we report are typical: 10 pg/ml for 50 min or 40 pg/ml for 10 min. A prerequisite for effective viral inactivation by AMT is that the psoralen must be able to gain access to the rotavirus and then penetrate the doubleprotein capsid and cross-link the genome at the core. The penetration by AMT of cells and debris that harbor virus in crude preparations such as we have used in the present study is well-documented. In fact, AMT has been suggested for use in the decontamination of cells and tissues specimens infected with HIV prior to handling in a variety of environments (Watson et al., 1990). The psoralen must also penetrate the viral protein capsids to gain access to the nucleic acid core. The reconstructed electron-microscopic images published by Yeager et al. (1990) demonstrated aqueous pores that penetrated the outer capsids and appeared to be in communication with the viral core. These channels may explain the ease by which the psoralen molecules can gain access to the genome without perturbing the capsid structure. The antigenic condition of the outer capsid viral proteins was determined with an ELISA using several monoclonal antibodies, each directed at a different protein. The ability of monoclonal antibody 4F8 to recognize psoralen-treated virus is particularly compelling evidence of structural and antigenic integrity because the epitope on the outer capsid protein VP7 that is recognized by this antibody is conformationally determined. Binding is completely abrogated following disruption of capsid structure such as occurs with calcium chelation with EDTA, even if the disrupted VP7 is still present (Shaw et al., 1986; Mackow et al., 1988) (also R. Shaw and H. Greenberg, unpubl. data). Neither does this nor many other neutralizing monoclonal antibodies recognize VP7 in a Western blot format. We determined the relationship between the concentration of AMT and the duration of UV light exposure necessary to inactivate the rotavirus. As the amount of AMT is increased, the amount of UV exposure time is decreased and vice versa. This inverse relationship is in agreement with the findings of studies on reovirus inactivation (Nakashima and Shatkin, 1978). It is important to reiterate that these concentrations of AMT are effective for the inactivation of virus in unclarified tissue culture supernatants following cell lysis from viral cytopathic effect and a subsequent cycle of freezing and thawing. If higher densities of virus (either concentrated by centrifugation with cell debris or gradient-purified) are to be inactivated the concentration or duration will likely need to be increased accordingly due to increased amounts of RNA, or perhaps due to light-scattering effects of cell debris. Several other DNA and RNA viruses have been inactivated with the highly
101
water soluble AMT, and demonstrated to retain antigenic integrity. AMT has shown particularly good performance, however, against the more-diffcult-toinactivate RNA viruses. In many studies there were no efforts made to assess antigenic integrity, and some studies were published prior to the common availability of monoclonal antibodies, which obviously permit a superior assessment of antigenic characteristics. Viruses which have been shown to be vulnerable to AMT inactivation include reovirus (Nakashima and Shatkin, 1978), HIV (Watson et al., 1990), Adenovirus (Wong and Hsu, 1988), Vesicular stomatitis virus (Hearst and Thiry, 1977; Nakashima et al., 1979), herpes simplex type 1 (Hanson et al., 1978), Western equine encephalitis virus (Hanson et al., 1978), and Influenza A (Nakashima et al., 1979). Psoralen-inactivated antigenically intact rotavirus particles will be useful in experiments to determine the importance of viral replication in eliciting an intestinal immune response to the rotavirus. Such important determinants of the immune response such as binding, uptake, and processing by antigen presenting cells in the intestinal mucosa will be identical in all respects except for replicative capabilities.
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Mackow, E.R., Vo, P.T., Broome, R., Bass, D. and Greenberg, H.B. (1990) Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge. J. Virol. 64, 16981703. Nakashima, K. and Shatkin, A.J. (1978) Photochemical cross-linking of reovirus genome RNA in situ and inactivation of viral transcriptase. J. Biol. Chem. 253, 868@-8682. Nakashima, K., Chandra, P.K., Deutsch, V., Banerjee, A.K. and Shatkin, A.J. (1979) Inactivation of influenza and vesicular stomatitis virion RNA polymerase activities by photoreaction with 4’substituted psoralens. J. Virol. 32, 838-844. Offit, P.A., Greenberg, H.B. and Dudzik, K.I. (1989) Rotavirus-specific protein synthesis is not necessary for recognition of infected cells by virus-specific cytotoxic T lymphocytes. J. Virol. 63, 3279-3283. Shaw, R.D., Stoner-Ma, D., Estes, M. and Greenberg, H. (1985) Specific enzyme-linked immunoassay for rotavirus serotypes 1 and 3. J. Clin. Microbial. 22, 286-291. Shaw, R., Coulson, B., Kaljot, K., OfIit, P. and Greenberg, H. (1986) Antigenic mapping of the surface proteins of rhesus rotavirus. Virology 155, 434451. Shaw, R.D., Groene, W.S., Mackow, E.R., Merchant, A.A. and Cheng, E.H. (1991) VP4-specific intestinal antibody response to rotavirus in a murine model of heterotypic infection. J. Virol. 65, 3052-3059. Singh, H. and Vadasz, J.A. (1978) Singlet oxygen: a major reactive species in the furocoumarin photosensitized inactivation of E. coli ribosomes. Photochem. Photobiol. 28, 539-545. Specht, K., Kittler, L. and Midden, W. (1988) A new biological target of fumocoumarins: Photochemical formation of covalent adducts with unsaturated fatty acids. Photochem. Photobiol. 47, 537--541. Sutjipto, S., Pedersen, N.C., Miller, C.J., Gardner, M.B., Hanson, C.V., Gettie, A., Jennings, M., Higgins, J. and Marx, P.A. (1990) Inactivated simian immunodeficiency virus vaccine failed to protect rhesus macaques from intravenous of genital mucosal infection but delayed disease in intravenously exposed animals. J. Viral. 64, 229&2297. Swanstrom, R., Hallick, L.M., Jackson, J., Hearst, J.E. and Bishop, J.M. (1981) Interaction of psoralen derivatives with the RNA genome of rous sarcoma virus. Virology 113, 613-622. Veronese, F.O.S.R.B., Bordin, F. and Rodighiero, G. (1982) Photoinactivation of enzymes by linear and angular furocoumarins. Photochem. Photobiol. 36, 25-30. Watson, A.J., Klanieki, J. and Hanson, C.V. (1990) Psoralen/UV inactivation of HIV-l-infected cells for use in cytologic and immunologic procedures. AIDS Res. Hum. Retroviruses 6, 503513. Wong, M.L. and Hsu, M.T. (1988) Psoralen-cross-linking study of the organization of intracellular adenovirus nucleoprotein complexes. J. Viral. 62, 1227-1234. Yeager, M., Dryden, K.A., Olson, N.H., Greenberg, H.B. and Baker, T.S. (1990) Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction. J. Cell Biol. 110, 2133-2144.
