Photodynamic inactivation of cell-free HIV strains by a red-absorbing chlorin-type photosensitizer

AND

~ O G Y

B~]~OLOGY

ELSEVIER

Journal of Photochemistry and Photobiology B: Biology 31 (1995) 171-177

Photodynamic inactivation of cell-free HIV strains by a red-absorbing chlorin-type photosensitizer Marc Grandadam ", Didier Ingrand b.., Jean-Marie Huraux a, B6atrice Aveline c, Olavio Delgado c, Christine Vever-Bizet c, Daniel Brault ~'* "Laboratoire de Virologie du CERVI, CNRS EP 57, H@ital de la Pitid-Salp~tri~re, 83 boulevard de l'H@ital, 75013 Paris, France b Laboratoire de Bacteriologic-Virologic-Hygiene, Hdpital Robert Debr~, rue Alexis Carrel, 51100 Reims, France c Laboratoire de Biophysique, INSERM U 201, CNRS UA 481, MusJum National d'Histoire Naturelle, 43 rue Cuvier, 75005 Paris, France Received 10 May 1995; accepted 30 June 1995

Abstract

We have investigated the photodynamic activity of a new chlorin-type photosensitizer on a reference human immunodeficiency vires type I (HIV-1) strain, two wild-type HIV-1 isolates and two drug-resistant HIV-1 isolates. This chlorin was highly effective for the inactivation of free viruses, as assessed by two different quantitative cell culture assays. In the absence of blood components, all the HIV strains, including wild-type and drug-resistant mutant isolates, were totally inactivated using 30 p,g ml - ~of chlorin and 0.75 J cm- 2 of 661 nm light. Successful killing of HIV-1 strains in either plasma or whole blood was also obtained by increasing the chlorin concentration moderately. Our results demonstrate the antiviral efficiency of this chlorin, suggesting the potential application of dye-sensitized photoirradiation to decontaminate blood products. Keywords: HIV-1; Drug-resistant mutants; Photoinactivation; Chlorin-type photosensitizer

1. Introduction

Although the interviewing of donors and serological screening have greatly reduced the contamination of blood by infectious agents, the risk of viral infections following blood transfusion still remains [1,2]. Cytomegalovirus (CMV), hepatitis B (HBV) and C (HCV) viruses and human immunodeficiency virus (HIV) are the major causes of blood transfusion-transmitted diseases. An important limitation of serological screening for HIV or HVC is the lack of response of the tests available at present in newly contaminated donors [ 1,2]. This limitation, as well as the emergence of new virus types [ 3], demonstrates the ongoing problem of potential contamination by transfused blood. Treatments involving heat or detergents have been successfully used to sterilize some blood-derived products, such as albumin and coagulation factors. However, they are not suitable for cellular blood components which are much more fragile. These problems have prompted research into new methods of virus inactivation which could be applied as a common prophylactic treatment of whole blood, platelets or red blood cells before transfusion. Photodynamic treatments, involving * Corresponding authors. 1011 - 1344/95 /$09.50 © 1995 Elsevier Science S.A. All rights reserved SSDI 1 0 1 1 - 1 3 4 4 ( 9 5 ) 0 7 2 0 1 - 2

the generation of short-lived reactive species on irradiation of a photosensitizer [4], are promising [5]. Several photosensitizers, including porphyrins and related compounds [68], merocyanine 540 [9], phthalocyanines [10], hypericin [11] and psoralens [12], have been investigated for their antiviral properties and the results have been reviewed [ 13]. However, severe requirements are needed for blood sterilization. In addition to the low toxicity and absence of mutagenic potential of the photosensitizer, the biological functions of the blood components must be preserved. In addition, possible interference of serum and light-absorbing material must be considered. In this respect, photosensitizers which absorb light above about 620 nm have received considerable attention because they can be activated in a region corresponding to minimal light absorption by haemoglobin. In this study, we have evaluated the efficiency of a chlorintype molecule [ 14] to photoinactivate, in a cell-free system, a series of HIV-1 strains suspended in buffer, plasma and whole blood. Although some reduction of efficiency is observed in the last two cases, complete inactivation of HIV1 can be achieved with a reasonable light dose. Wild-type and mutant strains, isolated from HIV-infected patients, also respond to the photoinactivation treatment.

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2. Materials and methods 2.1. Cells

Peripheral blood mononuclear cells (PBMCs) from healthy donors were used for the propagation of the primary HIV- 1 isolates. After 2-3 days stimulation with 3/~g mlphytohaemagglutinin (PHA), the PBMCs were cultured in RPMI 1640 medium supplemented with 2 mM glutarnine, 10% heat-inactivated foetal calf serum, intedeukin-2 (20 I.U. ml- i) and antibiotics. At a maximum, the density of PBMCs in the culture medium was 2 x 106 cells mlThe continuous T lymphoblastoid cell line, CEM, was maintained at 1 × 106 cells ml- ~in RPMI 1640 medium supplemented with 2 mM glutamine, 10% heat-inactivated foetal calf serum and antibiotics. HT4LacZ-1 cells, an HeLa clone cotransfected with the CD4 gene and an LTR fl-galactosidase construction [ 15] were kindly provided by D. Rocancourt (Institut Pasteur, Paris). Cells were cultivated in Dulbecco-modified Eagle medium containing 2 mM glutamine, 5% heat-inactivated foetal calf serum and antibiotics. All the cells were grown at 37 °C in humidified air conraining 5% CO2. 2.2. Viruses

The HIV- 1 Lai reference strain was expanded in CEM cells to obtain high titre virus stocks. Cell-free viruses from supernatants of infected cultures were precipitated overnight at + 4 °C using 50% ( w / v ) polyethylene glycol (PEG 6000) solution. After centrifugation at 3500g for 10 min at + 4 °C, the pellet was suspended in NTE buffer (NaCI, 100 mM; tris(hydroxymethyl)aminomethane (TRIS), 10 mM; ethylenediaminetetraacetic acid (EDTA), 10 mM; pH 7.4). This viral suspension was laid on a 30%-40% sucrose gradient and centrifuged at 35 000 rev min - ~for 2 h at + 4 °C (Kontron, TGA55, rotor SW41TI). The viral band was collected and washed in NTE buffer, and virions were sedimented by ultracentrifugation (35 000 rev rain- ~, 2 h). The pellet was resuspended in 0.5 ml NTE buffer and separated into 10/xl aliquots which were stored at - 80 °C. The 50% tissue culture infective dose (TCIDso) of this virus stock, determined by endpoint dilution assay, was l0 s TCIDso mlFour HIV- 1 isolates from infected patients hospitalized in Paris were studied: two wild-type HIV-1 isolates ( 100, 110) and two HIV-1 mutants resistant to zidovudine (218, 221), as assessed by a standardized antiviral drug susceptibility assay [ 16]. Their phenotypic changes were correlated with reverse transcriptase (RT) mutation at residue 215 (Thr2~5Tyr). Isolate 218, which presented the highest resistance, was characterized by an additional mutation at residue 67 ( Asp67Asn) [ 17 ]. All the HIV isolates were expanded in fresh PHAstimulated PBMCs and virus multiplication was evaluated by RT assay as already described [ 18 ]. Aliquots of clarified supernatants were stored at - 8 0 °C after RT activity had

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Fig. 1. Structureof the chlorin-typocompoundCHVD (two isomers) used in this study. reached at least 1 × 106 cpm ml- 1. The TCIDso m l - ~ of the virus stock was determined as described below. 2.3. Plasma and blood

Blood from healthy donors was obtained from the blood bank of the Piti6-Salp~,tfi~re hospital. Plasma was separated by centrifugation at 2000 rev min-~ for 20 min at room temperature, divided into 10 ml aliquots and stored at - 80 °C. Experiments on whole blood were carried out less than 4 h after venous puncture. 2.4. Chemicals

The structure of the chlorin-type molecule (two isomers) used as photosensitizer is shown in Fig. 1. It was photochemically prepared from hydroxyethylvinyldeuteroporphyrin (HVD) which was derived from haematoporphyrin by partial dehydration [ 14]. HVD photosensitized its own transformation through attack of its vinyl chain by singlet oxygen [ 19]. A similar reaction was shown to yield photoprotoporphyrin from protoporphyrin. The reaction product was purified by chromatography on silica according to a procedure derived from Vever-Bizet et al. [20]. The preparation of this and related compounds has been described in more detail elsewhere [ 21 ]. In this study, a 1:1 mixture of the isomers (see Fig. 1 ) was used. In the following, this compound will be referred to as CHVD. Stock solutions of CHVD at a concentration of 2.5 mg m l - i in phosphate-buffered saline (PBS) were used. The absorption spectrum of CHVD in aqueous solution containing 2% Triton X-100 is reported in Fig. 2. 2.5. Photodynamic treatment

Photodynamic treatment was carried out using a 300 W xenon arc mounted in a lamp housing equipped with a rear reflector and a 3 in diameter fused silica condenser off/0.7 aperture (Oriel). After elimination of IR light by a water filter, the beam was reflected to the bottom by a 45 ° mirror. A 661 nm interference filter with a bandwidth of 10 nm at half height (Schott) was used to select a narrow irradiation

M. Grandadam et al. / Journal of Photochemistry and Photobiology B: Biology 31 (1995) 171-177

173

kept in the dark for the same time to serve as a control. All the experiments were performed at least in duplicate.

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2.6. Infectivity assays 4..-"

The infectious virus titres of the control and irradiated samples were determined in two independent ways: an RT assay which was used for all strains in the various fluids; a colorimetric assay based on the/3-galactosidase activity of infected HT4LacZ- 1 cells.

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Fig. 2. Absorption spectrum of CHVD dissolved in PBS containing 2% [ riton X-100.

band matching the absorption of the chlorin molecule. The condenser was set to produce a parallel collimated light beam with a diameter of about 7 cm, which made it possible to irradiate from above four wells of a 24-well plate. The uniformity of the light intensity was controlled by scanning the working area with a power meter (surface absorbing disc ~alorimeter from Scientech) equipped with a 2 cm diameter diaphragm. Differences of less than 5% were found between the positions occupied by the wells. The light intensity was 1.25+0.05 mW cm -2. As the light beam was parallel, no ~hadowing of the bottom wells by the walls occurred. Four virus samples were irradiated at the same time. In addition to the controls of beam uniformity outlined above, the tray was l,ently agitated to ensure equal irradiation of all the solution ~:ontained in each of the four wells. The volume of each :;ample was 200/~1, corresponding to a height of liquid of ,)nly 1 mm. Screen effects were thus minimized. The virus :;amples were irradiated for 10 min which corresponded to a otal fluence of 0.75 J cm -2. Each of the five HIV-1 strains was suspended in 200/zl PBS containing 0-30/~g ml-~ chlorin. This concentration :ange was expanded to 50/zg ml- t and 75/~g ml- t when '.he virus was suspended in plasma and whole blood respectively. The initial virus titres ranged from 1 × 104 to 1 x 106 I'CIDso m l - t . In all experiments, the photosensitizer was added after the virus had been suspended in the different media. Two samples were prepared for each concentration of the photosensitizer (including controls without photosensitizer) and incubated at + 4 °C for 45 min in the dark. One of the samples was placed in a well of a 24-well tissue culture tray and was irradiated as described above. The other was

2.6.1. RT assay Serial tenfold dilutions of the viral suspension in PBS, plasma or whole blood were realized in culture medium. An inoculum of 500/xl of each dilution was used to infect cells. After 2 h incubation at 37 °C, the cells were centrifuged, resuspended in fresh culture medium and seeded in quadruplicate in 96-well plates at a final concentration of 5 X 105 cells per well. A 50/zl aliquot of the supernatant was removed 4, 7 and 10 days after infection and replaced by the same volume of fresh medium. The samples were analysed by a radioactivity assay for RT activity as described by Schwartz et al. [ 18]. The virus titre was calculated according to Karber's method [22] and expressed as the TCID~o m l - ~. The cells used to propagate the virus were CEM cells and PBMCs for the HIV-1 Lai strain and primary isolates respectively. 2.6.2. ~-Galactosidase assay Serial tenfold dilutions of the viral suspension were inoculated on an HT4LacZ- 1 cell monolayer for 3 days. The virus titres were assessed from the number of fl-galactosidasepositive syncytia on microscopic observation. The method of endpoint dilution was used for quantification [ 15 ].

3. Results For all the experiments, the titres of viral suspensions after photodynamic treatment were compared with their corresponding dark controls. As illustrated in Figs. 3-5 (see below), no significant antiviral effect of the photosensitizer in the dark was observed in the range of chlorin concentrations investigated. Light alone had no effect either (see results without photosensitizer in Figs. 3-5).

3.1. Photoinactivation of the HIV-I Lai reference strain in buffer The virucidal potential of CHVD was first evaluated on the HIV-1 Lai strain in a ceil-free system using PBS as medium. As illustrated in Figs. 3(a) and 3(b), both the RT and/3-galactosidase assays were indicative of a photosensitizer dose-dependent reduction of the virus titre. Using the HT4LacZ-1 target cells, a substantial reduction (at least 1 iog~o) of TCIDso was noted with a dose as low as 2.5 /s,g ml- m(Fig. 3(b) ). Both methods showed a 6 log~o reduction

M. Grandadam et al. /Journal of Photochemistry and PhotobiologyB."Biology 31 (1995) 171-177

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the virions suspended in buffer were approximately the same, regardless of the phenotypic characteristics of the various strains. In all the experiments described above, no RT activity was detected when cell cultures were followed up to 15 days.

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Fig. 3. Dose-dependent photoinactivation of the HIV-1 Lai reference strain by CHVD in PB S: ( a ) RT assay ( after 10 day culture ); ( b ) ~galaetosidase assay; O, non-irradiated control; I , irradiated samples (0.75 J era-2). Mean values from two or more experiments are presented. Standard errors are shown as bars unless they are contained in the symbols.

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of the virus titre using a chlorin concentration of 20/zg ml- ~. Indeed, within the sensitivity limits of our tests, no infective viruses were detected.

3.2. Photodynamic treatment of free virions in plasma and whole blood Photodynamic treatment of free viruses was also performed in human plasma and whole blood from healthy donors. The HIV-1 Lai strain stocks were directly diluted in these media. In these experiments, only the RT assay was used. As shown in Figs. 4(a) and 4(b), photoinactivation was dose dependent, but some residual viral activity was observed with 20/zg m l - t of photosensitizer. However, a 6 Ioglo virus inactivation was obtained in plasma and blood by increasing the chlorin concentration to 50/xg ml- I and 75 # g m l - mrespectively.

Four primary HIV-1 isolates characterized for their resislance to zidovudine (see Section 2) were tested with regard to their sensitivity to photoinactivation. As shown in Fig. 5, the chlorin concentration thresholds required to inactivate all

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Fig. 4. Dose-dependent photoinactivation of the HIV-1 Lal reference strain by CHVD in (a) plasma and (b) whole blood as determined from RT assay (after 10 day culture): O, non-irradiated control; n , irradiated samples (0.75 J cm-2). Mean values from two or more experiments are presented. Standard errors are shown as bars.

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3.3. Photoinactivation o f HIV- 1 primary isolates from patients

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Fig. 5. Dose-dependent photoinactivation (RT assay after 10 day culture) of primary isolates from HIV-I infected patients by CHVD in PBS: broken lines, non-irradiated control; full lines, irradiated samples (0.75 J cm-=). The wild strains i 00 ( O ) and l 10 ( [ ] ) are compared with the mutant strains 218 (<>) and 221 (A). No significant differences were found between two series of experiments. For clarity, only data from one series are presented.

M. Grandadam et al. /Journal of Photochemistry and Photobiology B: Biology 31 (1995) 171-177

4. Discussion

Owing to the short lifetime of the reactive species generated by photosensitization, the primary biological damage is selectively limited to the structures labelled by the photosensitizer [4,23]. With regard to their action on viruses, photose.nsitizers can be classified into two groups [4,24]. The first group consists of molecules which bind to nucleic acids. Included in this group are positively charged drugs, such as n-~utral red, which interact with the phosphate backbone, or a:-omatic compounds, such as psoralen derivatives, which intercalate between nucleic acid bases. On light irradiation, these compounds induce oxidation of nucleic acid components or form covalent adducts respectively. The second group consists of lipophilic and negatively charged amphotcric substances. These compounds efficiently combat enveloped viruses, suggesting that they bind to the lipid viral eavelope and/or to surface glycoproteins impeding the early steps of the infectious cycle [ 13]. With regard to the problem of blood decontamination, the molecules of the first group may appear to be more attractive because they exhibit better selectivity for viruses compared with red blood cells or platelets which lack nuclei. However, the use of certain positively charged photosensitizers, which were shown about 20 years ago to photoinactivate the herpes simplex viruses, was discouraged. Indeed, the treatment was fl~und to induce the oncogenic potential of the viruses [25]. In the same way, although psoralens appear to be able to inactivate a large panel of viruses with minimal adverse effects to platelets, some concerns have been expressed regarding their mutagenic potential [ 26]. Although less specific, the photosensitizers of the second group are not likely to be mutagenic. Among them, merocyaaine 540, porphyrins and phthalocyanines have been investigated for their antiviral properties. All of these photosensitizers are believed to act via singlet oxygen generation and to induce damage to the viral envelope. Interestingly, meroeyanine may exhibit some selectivity towards certain virally infected cells [ 27 ]. However, the cellular components of blood, in particular platelets, are found to be altered in the conditions required to fully inactivate the viruses [28]. Also, this compound absorbs light in the same region as haemoglobin, which results in an important screenlug effect when blood is treated [ 9,27 ]. If whole blood or red cell concentrates are to be treated, red-absorbing photosens ~tizers must be definitively preferred. The porphyrin preparations, haematoporphyrin derivative and Photofrin®, already used in tumour photodynamic therapy, inactivate a number of viruses [6,7]. However, porphyrins must be activated around 630 rim, a wavelength still attenuated by haemoglobin. Moreover, these preparations are complex mixtures of various porphyrins, which makes the elucidation of the action mechanisms difficult. Among other red-absorbing photosensitizers, sapphyrins [29], phthalocyanines [10] and benzoporphyrins [ 8 ] have been tested on various viruses. Although

175

promising with model viruses, benzoporphyrins are unable to inactivate fully HIV strains [8]. A second point to be outlined is the effect of albumin. Hydrophobic photosensitizers are very efficiently bound by albumin, which reduces their availability to interact with the lipid envelope of viruses or with cellular membranes. Indeed, albumin protects blood components from the effect of merocyanine, but total virus inactivation is no longer possible [ 28 ]. It should be noted that the activity of other hydrophobic compounds, including psoralens, is also reduced in the presence of albumin [ 30]. The choice of the hydrophobic / hydrophilic balance of photosensitizers aimed at the viral decontamination of blood is not straightforward. Taking into account the above considerations, we have adopted a strategy based on the following points: (i) since, in many cases, studies on model viruses failed to predict the effects on HIV, investigations were carried out on these viruses; (ii) a photosensitizer of the chlorin type, absorbing light well outside the absorption bands of haemoglobin (see Fig. 2), was prepared as a pure and well-characterized compound [ 14,21] ; (iii) although this photosensitizer was intended for action on the viral envelope, it was chosen to be moderately hydrophobic in order to reduce the interactions with albumin. As shown in Fig. 1, some hydrophilic character was given to our chlorin by two ionizable carboxylic chains and other oxygen-bearing groups bound at the macrocycle periphery. Interestingly, a fairly good water solubility was also provided by these substituents. This compound can also be solubilized as a momomer in detergent micelles as exemplified in Fig. 2. As shown in Figs. 3-5, total HIV-1 inactivation is achieved whatever the suspension medium. Although some increase in chlorin concentration is required to obtain this result in plasma or blood, it is much less important than for certain other photosensitizers. For instance, the activity of merocyanine is reduced by a factor of about 200 in the presence of 5% albumin [28 ], which roughly corresponds to the albumin concentration in plasma. The merocyanine activity is also drastically reduced in red blood cell suspensions [ 27 ]. The same trends are likely to hold with other hydrophobic photosensitizers, including psoralens [ 30]. However, the inhibitory effect of albumin also depends to a large extent on the charge of the molecules. In a series of phthalocyanines, this inhibition was minimal with positively charged molecules [31]. A salient feature of our results is that all the HIV- 1 strains are inactivated. The genomic variations responsible for the resistance of HIV- 1 to the usual drugs targeted to RT do not induce resistance to photoinactivation, as shown by the behaviour of mutants 218 and 221. The mechanism of virus inactivation by our chlorin-type photosensitizer remains to be elucidated. Singlet oxygen, which is formed with a yield of 0.7-0.8 on CHVD irradiation [ 14], is the most probable reactive species. Owing to the presence of polar chains around the chlorin macrocycle, it is unlikely that this molecule will penetrate deep into the virus

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M. Grandodom et al. /Journal of Photochemistry and Photobiology B: Biology 31 (1995) 171-177

envelope and eventually cross it to attain the capsid. Indeed, the HIV envelope with a phospholipid to cholesterol ratio of near unity is not fluid [32]. The crossing of such lipid bilayers, even by more hydrophobic molecules, is extremely slow [ 33 ]. However, the high curvature of the virus lipid envelope may induce distortions favouring incorporation in the external hemileaflet of the bilayer. Another interesting feature has been reported by Neurath and coworkers [ 34,35]. A series of carboxylic porphyrins and related compounds are found to inhibit, to some extent, the HIV-1 replication and the reaction between the envelope glycoprotein gpl20 and antibodies targeted to the V3 loop. It is postulated that porphyrins with anionic groups and some hydrophobic chains may interact with highly conserved, positively charged, hydrophobic sites of the V3 loop. It is worth noting that our chlorin is derived from a porphyrin (2(or 4)hydroxyethyl-4(or 2)-vinyl deuteroporphyrin IX) which presents good efficiency in these tests [35]. Within the chlorin concentration ranges used in our study, such interactions are not sufficient to compete with the adsorption of the viruses to the cellular receptors, as shown by the absence of a dark effect. However, they may be sufficient to increase the local concentration of the photosensitizer around glycoprotein gpl20, thus making it a target for attack by singlet oxygen. Although details of the mechanism are still unknown, the photoinactivation clearly involves the viral envelope. Indeed, poliovirus, a non-enveloped virus, is resistant to photoinactivation by CHVD (experiments not shown). The fact that zidovudine-resistant HIV-1 strains are not resistant to photoinactivation is also in agreement with mechanisms involving two different steps of the virus replication cycle.

5. Conclusions We have demonstrated the ability of our chlorin photosensitizer to inactivate HIV-1 (even drug-resistant mutants) in various media including biological fluids. Full inactivation is obtained by using a fairly low fluence of red light. These results suggest the possible use of this compound in the dyephotosensitized decontamination of blood, although additional strategies should be developed to take into account actively and latently infected cells. Photodynamic effects on cellular blood components are now under study to evaluate further the feasibility of such an approach. The possible reactivation of latently infected cells on light irradiation must also be taken into account if whole blood treatment is considered. This effect may be minimized since our compound is not expected to interact with nucleic acids and by using long wavelengths not absorbed by nucleic acids [36]. However, HIV-1 reactivation can also occur following oxidative stress due to singlet oxygen production [ 37,38]. Recent results on the dynamics of HIV-1 infection, which demonstrate continuous rounds of virus infection and replication and the rapid emergence of drug-resistant variants [39,40], are likely to renew interest in methods intended to reduce the virus titre

in blood. The potential of extracorporal photopheresis in the treatment of AIDS using psoralens has been investigated recently [41 ]. Obviously, red-absorbing photosensitizers would be more adequate for such approaches.

Acknowledgements The authors are grateful to Suman Dhami for helpful criticisms during manuscript preparation. This work was supported by grants from the Agence Nationale de Recherche sur le SIDA (ANRS) and Laboratoires Lederle-Cyanamid France.

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