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Efficient Delivery of Circulating Poliovirus to the Central Nervous System Independently of Poliovirus Receptor
VIROLOGY
229, 421–428 (1997) VY978450
ARTICLE NO.
Efficient Delivery of Circulating Poliovirus to the Central Nervous System Independently of Poliovirus Receptor WEI-XING YANG,* TETSUYA TERASAKI,*,1 KAZUKO SHIROKI,† SEII OHKA,† JUNKEN AOKI,* SHUICHI TANABE,* TATSUJI NOMURA,‡ EIJI TERADA,§ YUICHI SUGIYAMA,* and AKIO NOMOTO†,2 *Faculty of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan; †The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan; ‡Central Institute for Experimental Animals, 1430 Nogawa, Miyamae-ku, Kawasaki, Kanagawa 216, Japan; and §Institute of Laboratory Animal Sciences, School of Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228, Japan Received October 28, 1996; returned to author for revision December 16, 1996; accepted January 10, 1997 The transgenic (Tg) mice carrying the human gene for poliovirus receptor (PVR) are susceptible to poliovirus intravenously (IV) inoculated as well as intracerebrally or intraspinally inoculated. Thus, IV-inoculated poliovirus may invade the central nervous system (CNS) through the blood–brain barrier (BBB). To know the contribution of PVR to tissue distribution and BBB permeability of IV-inoculated polioviruses, these dissemination processes were investigated and compared between the Tg mice and non-Tg mice. Distribution profile of IV-inoculated poliovirus in various tissues of the Tg mice is similar to that in non-Tg mice. The data suggest that tissue distribution of the virus occurs independently of the transgene for PVR. The amount of poliovirus delivered to the CNS suggested the existence of a specific delivery system of the virus to the CNS. Virus accumulation in the CNS of the Tg mice was measured up to 7.5 hr after the IV inoculation. The viruses, regardless of whether the virulent or attenuated strain, seem to accumulate at a constant rate of approximately 0.2 ml/ min/g tissue. Similar phenomena were observed when the viruses were inoculated into non-Tg mice. The rates of the virus accumulation in the CNS are more than 100 times higher than that of albumin, which is considered not to permeate through the BBB via a specific transport system, and only three times lower than that of monoclonal antibody against transferrin receptor (OX-26), which is a potential candidate as a drug delivery vehicle specific to the CNS. These data suggest that polioviruses permeate through the BBB at a fairly high rate, independently of PVR and virus strains. q 1997 Academic Press
INTRODUCTION
by primary multiplication in the alimentary mucosa. After viral multiplication in tissues such as the tonsils and Peyer’s patches, the virus moves into the deep cervical and mesenteric lymph nodes and then into the blood. If viremia is established, the virus invades the central nervous system (CNS) and paralytic poliomyelitis occurs as a result of the destruction of neurons in the CNS, especially the motor neurons in the anterior horn of the spinal cord and neurons of the motor cortex and brain stem. Although many tissues are exposed to the virus during the viremic phase, most tissues do not support poliovirus replication. Thus poliovirus seems to have a distinct tropism with regard to tissues and cell types. Little is known, however, about mechanisms for the virus dissemination characteristic of poliovirus. Humans are the only natural host of poliovirus, although poliovirus can be experimentally transferred to monkeys, in which it also causes a paralytic disease. Other animal species are not susceptible to most poliovirus strains. Because of the characteristic species specificity of poliovirus, monkeys have been used as the only animal model for poliomyelitis, and inoculation of the virus into the monkey CNS has been the only method to evaluate neurovirulence levels of poliovirus. The virus-specific tropism must be controlled by host
Poliovirus, known to be the causative agent of poliomyelitis, is a human enterovirus that belongs to the family Picornaviridae and is classified into three stable serotypes, 1, 2, and 3. The poliovirion is an icosahedral, nonenveloped particle that is composed of 60 copies of each of four capsid proteins, VP1, VP2, VP3, and VP4, and a single-stranded RNA genome of positive polarity. The three-dimensional structure of the virion particle (Hogle et al., 1985) as well as the primary structures of the RNA genome and the viral proteins (Kitamura et al., 1981; Racaniello and Baltimore, 1981) have been elucidated. The three-dimensional structure has revealed depressions called ‘‘canyon’’ on the surface of the virion, which have been suggested to be attachment sites for poliovirus receptor (PVR) on the surface of permissive cells. The life cycle of poliovirus was first described in the 1950s (Bodian, 1955; Sabin, 1956). Poliovirus infection is initiated by oral ingestion of the virus, which is followed 1 Present address: Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki-aza, Aoba-ku, Sendai 980-77, Japan. 2 To whom correspondence and reprint requests should be addressed. Fax: 81-3-5449-5408. E-mail: [email protected].
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Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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YANG ET AL.
factors that support the virus replications. Among these host factors, a specific cell surface receptor is considered to be one of the most important factors that determine host range of the virus. Indeed, the transgenic (Tg) mice carrying the human PVR gene were shown to be susceptible to all three poliovirus serotypes, and the infected mice showed clinical signs resemble those of humans and monkeys (Ren et al., 1990; Koike et al., 1991). In the CNS of the infected Tg mice, poliovirus antigens were detected only in neurons, and neuronal damages were observed (Koike et al., 1991, 1994b; Ren and Racaniello, 1992a; Horie et al., 1994). Clinical and pathological similarities of poliovirus infection in the Tg mice to those in monkeys suggest that the Tg mice become an additional animal model for poliomyelitis (Horie et al., 1994). To control poliomyelitis, attenuated poliovirus strains of all three serotypes have been developed and effectively used as oral live poliovirus vaccines, that is, Sabin 1 (serotype 1), Sabin 2 (serotype 2), and Sabin 3 (serotype 3) (Sabin and Boulger, 1973). They have a very poor capacity to replicate in the CNS of monkeys and show a very weak monkey neurovirulence, whereas the virulent strains replicate well in the CNS and show a strong monkey neurovirulence. The virulent and attenuated phenotypes of poliovirus observed in monkeys appeared to be preserved in the Tg mice, since much larger doses of virus were required for the attenuated strains to cause paralysis compared with those of the virulent strains. In fact, relative neurovirulence levels of polioviruses estimated in the Tg mice showed a good correlation with those in monkeys (Horie et al., 1994). The mouse neurovirulence tests involving intracerebral (IC) and intraspinal (IS) inoculation routes have been established to date (Horie et al., 1994; Abe et al., 1995a; 1995b). Different neurovirulence levels are also observed between the virulent and attenuated poliovirus strains when the viruses are intravenously (IV)-inoculated into the Tg mice (Koike et al., 1994b). Using this inoculation route, we investigate dissemination of poliovirus from the blood stream to the CNS of the Tg and non-Tg mice. Our data indicate that tissue distribution of the virus occurs independently of human PVR. Furthermore, we show that IVinoculated poliovirus accumulates in various tissues of the CNS, independently of human PVR, at a fairly high rate. The results suggest that poliovirus is able to permeate through the blood–brain barrier (BBB) of mice without the aid of human PVR. MATERIALS AND METHODS Transgenic mice The Tg mouse line ICR-PVRTg21 (Koike et al., 1991), in the hemizygous stage of 8 to 10 weeks of age, was used as an animal model for poliomyelitis. The copy number of the transgene in ICR-PVRTg21 is approximately threefold that in HeLa S3 cells (Koike et al., 1991; 1994a).
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All mice used were free from specific pathogens. ICR mice were purchased from Charles River Japan (Tokyo) and used as non-Tg mice. Viruses and cells The virulent Mahoney virus (PV1(M)OM) and the attenuated Sabin 1 virus (PV1(Sab)IC-0) were derived from infectious cDNA clones pOM1 (Shiroki et al., 1995) and pVS(1)IC-0(T) (Kohara et al., 1988), respectively. Viruses PV1(M)OM and PV1(Sab)IC-0 are termed MMM and SSS, respectively, in this study. A cDNA segment of pOM1 corresponding to a coding sequence for the viral capsid proteins (P1 region) was replaced by the corresponding segment of pVS(1)IC-0(T). A recombinant cDNA clone thus obtained was used for transfection to prepare virus MSM. A similar experiment was carried out to produce virus SMS, whose genome had a reciprocal structure of the MSM genome. Suspension-cultured HeLa S3 cells were maintained in RPMI 1640 medium supplemented with 5% newborn calf serum (NCS) and used for preparation of viruses. African green monkey kidney (AGMK) cells were grown in Dulbecco modified Eagle medium supplemented with 5% NCS and used for the transfection experiment with infectious cDNA clones and for plaque assays. Purification of viruses Viruses were grown in suspension-cultured HeLa cells as described previously (Kajigaya et al., 1985). Viruses in cytoplasmic extracts of infected cells were purified by chromatography on DEAE-Sepharose CL-6B (Li et al., 1992), followed by two cycles of CsCl equilibrium centrifugation in a buffer containing 10 mM Tris–HCl (pH 7.4), 10 mM NaCl, 20 mM EDTA, and 1% Brij 58. For preparation of [35S]methionine-labeled viruses, viruses were grown in methionine-free medium in the presence of [35S]methionine. Calculated specific radioactivities of purified radiolabeled Mahoney and Sabin 1 viruses were 148 and 27 mCi/nmol, respectively. Clinical neurovirulence tests Into the brain or tail vein of each Tg mouse, 30 or 200 ml of a virus suspension was inoculated. More than 10 Tg mice were used for injection with each dose of viruses except for experiments involving IC inoculation of SMS in which 5 Tg mice were used. The mice inoculated with viruses were observed for clinical signs at 12-hr intervals up to 14 days. The 50% lethal doses (LD50) were calculated according to the Dragstehdt–Behrens method, a modified Reed–Muench method (Finny, 1959). Approximate minimum 100% lethal doses (LD100) were also estimated. Distribution of viruses in mice Male Tg mice and non-Tg mice weighing 28–35 g were used. The animals had free access of food and
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water. Each mouse was inoculated with 2.5 1 1002 pmol of viruses (approximately 6.5 1 106 PFU) containing 0.4 mCi of labeled viruses via tail vein under mild ether anesthesia. [125I]albumin was used as a control compound that was known to exhibit significantly restricted BBB permeability. Three mice were dissected 5 hr after the inoculation. The tissues were weighed, minced, and dissolved with a tissue solubilizer, Solvable (Dupont, U.S.A.), at 507 for 3hr, and then 10 ml of liquid scintillation cocktail, Atomlight (Dupont), was added. Radioactivities were measured in a liquid scintillation counter (Aloka LSC1000). As an index of tissue distribution characteristics of poliovirus, the apparent tissue-to-plasma concentration ratio, Kp,app , defined by amount of virus per grams of tissue divided by that per microliter of plasma, was calculated over the time period of the experiment (Terasaki et al., 1984). Least squares regression analysis was performed with the program of Excel (Microsoft, U.S.A.). In some cases, 2 nmol of monoclonal antibody (mAb) against human PVR, p286, that had been prepared and characterized by J. Aoki and found to have very similar characteristics to mAb p403 (Aoki et al., 1994), was intravenously administered to Tg mice 5 hr before the virus inoculation. Kd value of p286 to human PVR on cell surface was calculated to be 8 nM. According to the calculated elimination rate constant of p286 in the mouse plasma, concentration of p286 5 hr after the virus inoculation should be about 80 nM and therefore still enough to inhibit virus binding to PVR. To know accumulation rate of viruses in various tissues of the mouse CNS, Kp,app values in these tissues were measured by the method of integration plot (Smith et al., 1987; Kakee et al., 1996) up to 7.5 hr after the virus inoculation. Briefly, the accumulation rate of poliovirus can be described by the following differential equation: dXbr Å PS 1 Cp dt
(1)
where Xbr is the amount of 35S-labeled poliovirus in the brain at time t, PS is the brain accumulation rate, and Cp is the plasma concentration of 35S-labeled poliovirus. Integration of equation (1) gives Xbr(t) Å PS 1 AUC(0-t) / Cp(t) 1 V0
(2)
where AUC(0-t) represents the area under the plasma concentration time curve of 35S-labeled poliovirus from time 0 to t, and V0 is the rapidly equilibrated distribution volume of 35S-labeled poliovirus. Equation (2) divided by Cp(t) 1 Vbr gives
S D
PS V0 AUC(0-t) Cbr(t) Å / 1 Cp(t) Vbr Cp(t) Vbr
(3)
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FIG. 1. Genome structures of recombinant type 1 polioviruses. The genome structures of the recombinant viruses are shown as a combination of the Mahoney (shadowed boxes) and the Sabin 1 (open boxes) sequences. VPg at the 5* end and poly(A) at the 3* end are indicated by closed circles and wavy lines. Numbers shown above the Mahoney genome represent nucleotide numbers from the 5* terminus.
brain, PS/Vbr , can be obtained from the initial slope of a plot of Cbr(t)/Cp(t), i.e., Kp,app(t) vs AUC(0-t)/Cp(t), as integration plot. Separation of parenchyma and capillary fractions of the brain Parenchyma and capillary fractions of the brain were separated by a modified method of Triguero et al. (1990). Briefly, the brain was minced after removal of meninges and connective tissues, added to 4 vol of physiologic buffer containing 10 mM HEPES (pH 7.4), 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2 , 1 mM MgSO4 , 1 mM NaH2PO4 , and 10 mM D-glucose, and then homogenized in a teflon homogenizer with 10 strokes. The homogenate was added to an equal volume of 32% dextran solution and homogenized with 3 more strokes. An aliquot of 0.2 ml was used to estimate radioactivity and virus titer in a whole brain. The remaining homogenates were centrifuged at 5400 g for 15 min at 47 in a Beckman rotor SW55 to precipitate capillary fraction. To monitor the separation, the activity of alkaline phosphatase, a marker enzyme for capillary fraction, was measured (Triguero et al., 1990). For the same purpose, 125 I-labeled acetylated low density lipoprotein was also employed as a marker material that accumulated only in capillary fraction (Triguero et al., 1990). Sucrose density gradient centrifugation 35
S-labeled materials in parenchyma fraction of the brain was analyzed by centrifugation on 15–30% sucrose density gradient in a buffer containing 0.1% bovine serum albumin and phosphate-buffered saline. The centrifugation was performed at 200,000 g for 2.5 hr at 47 in a Beckman rotor SW41. RESULTS Mouse neurovirulence tests on viruses
where Vbr represents the brain weight, and Cbr(t) is the brain concentration of 35S-labeled poliovirus. The brain accumulation rate of 35S-labeled poliovirus per grams of
The virulent Mahoney strain (MMM), attenuated Sabin 1 strain (SSS), and their recombinant viruses (MSM and SMS) were prepared as described under Materials and Methods. The genome structures of these viruses are shown in Fig. 1. The viruses were tested for their neurovi-
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YANG ET AL. TABLE 1
LD50 and Approximate Minimum LD100 of Poliovirus in Tg Mouse LD50 (PFU/mouse) Virus
IV a
MMM SSS MSM SMS
8.5 1 104 — 3.2 1 105 —
a b
Minimum LD100 (PFU/mouse)
ICb 2.1 7.0 4.2 1.3
1 1 1 1
102 105 102 105
IV a 4.0 1 106 — 6.0 1 106 —
ICb 1.0 7.0 4.0 #8.4
1 1 1 1
104 107 104 107
Intravenous inoculation. Intracerebral inoculation.
rulence in the Tg mice via IC and IV inoculation routes. The results are shown by values of LD50 and approximate values of minimum LD100 in Table 1. Viruses MMM and SSS showed the strongest and weakest neurovirulence among the viruses, respectively, when the mice were IC-inoculated (Table 1). Neurovirulence levels of viruses MSM and SMS are similar to those of MMM and SSS, respectively, although MSM appears to be slightly weaker than MMM and SMS slightly stronger than SSS. Thus, virion surface characteristics had little or no correlation with mouse neurovirulence. This observation is compatible with our previous results obtained from monkey tests (Omata et al., 1986; Kohara et al., 1988) and Tg mouse tests (Horie et al., 1994). Similar results were obtained when the mice were IV-inoculated with MMM and MSM although much larger amounts of viruses were required to cause paralysis and death (Table 1). In the case of IV inoculation, however, it took at least one more day for the mice to be killed as compared with the case of IC inoculation. LD50 values of SSS and SMS could not be calculated because no mice were killed even by IV inoculating 1 1 108 PFU of these viruses. Approximate values of minimum LD100 were estimated and are shown in Table 1. The LD100 values obtained from the experiments involving IV inoculation of MMM and MSM were 150 to 400 times more than those involving IC inoculation of these viruses. Main dissemination route of poliovirus from blood to the CNS is not known at present. However, if circulating virus invades the brain at first as suggested (Koike et al., 1994b; Nomoto et al., 1994), the data suggest that 0.25–0.6% of IV-inoculated virus has to invade the brain to kill all the mice. Considering the fact that approximately 0.3% of a total amount of IV-inoculated virus is distributed to the cerebrum, most of the virus distributed should move into the brain.
know the amount of virus delivered to individual tissues including the brain tissues, we took advantage of physiological pharmacokinetic analysis as described under Materials and Methods (Terasaki et al., 1984). Tissue distribution of MMM, SSS, or MSM in Tg mice or non-Tg mice was examined 5 hr after the IV inoculation. The result of MSM distribution is shown in Fig. 2A. Similar results were obtained for MMM and SSS distributions (data not shown). Amounts of poliovirus delivered to the CNS including the cerebrum and cerebellum are the smallest among all tissues tested. However, Kp,app values in the cerebrum and cerebellum are more than 200 ml/g tissue, and are significantly greater than the vascular volume of the brain, i.e., about 10 ml/g tissue (Pardridge et al., 1990; 1991). The data may suggest that polioviruses cross the blood–brain barrier. A similar in vivo distribution characteristic of circulating poliovirus was obtained when non-Tg mice was used. The data shown in Fig. 2A suggest that the Kp,app values obtained from experiments with Tg mice and non-Tg mice have a good correlation with each other on a level of P õ 0.01 (correlation coefficient: r 2 Å 0.903; n Å 9). This suggests that specific distributions of poliovirus to the brain tissues are not due to specific expression of human PVR on the brain capillary. To confirm this possibility, Tg mice were IV-injected with anti-PVR mAb p286 5 hr before the virus inoculation as described under Materials and Methods. This mAb has an ability to block human PVR-mediated poliovirus binding and infection to susceptible cells. As shown in Fig. 2B, preadministration of p286 appears not to affect the differential tissue distribution of poliovirus. The Kp,app values again have a good correlation with each other on a level of P õ 0.01 (correlation coefficient: r 2 Å 0.981; n Å 9). A similar result was obtained from the experiment involving MMM. These results indicate that human PVR expressed in the mouse tissues does not play any significant role for the differential tissue distribution of poliovirus.
It is possible that IV-inoculated poliovirus is delivered selectively to the CNS. Expression of human PVR may contribute to the virus delivery after IV inoculation. To
FIG. 2. Tissue distribution of poliovirus in mice after IV inoculation. (A) Kp,app values of [35S]methionine-labeled MSM in various tissues were calculated in Tg mice (vertical axis) and non-Tg mice (horizontal axis) 5 hr after the IV inoculation. (B) Tg mice were IV-injected with mAb p286 5 hr before the IV inoculation of [35S]methionine-labeled MSM as described under Materials and Methods. Kp,app values of MSM in various tissues were calculated in Tg mice (vertical axis) and Tg mice pretreated with the mAb (horizontal axis) 5 hr after the IV inoculation.
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Tissue distribution of IV-inoculated virus
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FIG. 3. Integration plot of poliovirus Mahoney strain in the CNS of Tg mice after IV inoculation. Tg mice were IV-inoculated with [35S]methionine-labeled MMM as described under Materials and Methods. Average Kp,app values in the cerebrum (A), cerebellum (B), brain stem (C), and spinal cord (D) were calculated at indicated AUC/Cp values. Three Tg mice were used for injection at each time. Calculation of AUC/Cp values followed that reported by Smith et al. (1987).
Rate of poliovirus accumulation in the CNS To know the rate of poliovirus accumulation from the circulating blood to the CNS, Tg mice were IV-inoculated with 35S-labeled MMM, and the time course of virus accumulation in individual tissues of the CNS followed up to 7.5 hr after the inoculation when the virus replication was not yet detected in any of these tissues. Experiments were repeated three times for MMM and SSS and twice for MSM. The results are shown in Fig. 3 by integration plot (Smith et al., 1987; Kakee et al., 1996). Standard errors were too small to show in Fig. 3. Slopes shown in Fig. 3 represent virus accumulation rates in individual tissues. Linear accumulation of the virus was observed in any these tissues up to 7.5 hr after the inoculation. The rates were estimated to be 0.164, 0.171, 0.151, and 0.117 ml/min/g tissue in the cerebrum, cerebellum, brain stem, and spinal cord, respectively. In other tissues, linear accumulation was not seen, and the rates are coming to plateaus during the time used (data not shown). Similar experiments were carried out on the cerebrum by using non-Tg mice and/or viruses carrying the Sabin 1 capsid to know the contribution of human PVR and virion surface characteristics to accumulation rates of viruses. As shown in Table 2, the rates observed in Tg mice resemble those in non-Tg mice. Thus, human PVR seems not to play an important role in delivery of circulating poliovirus to the cerebrum. The accumulation rates of SSS and MSM were estimated to be 0.223 and 0.268 ml/min/g tissue in Tg mice and are slightly higher than that (0.164 ml/min/g tissue) of MMM (Table 2). A similar observation was seen in
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non-Tg mice. It is possible that the Sabin 1 capsid has a slightly higher efficiency in the virus accumulation in the cerebrum as compared with the virulent Mahoney capsid. It should be noted that the accumulation rates observed for polioviruses are more than 100 times higher compared with that of albumin (Table 2), which is known to exhibit significantly restricted permeability across the BBB and are only three times less than that of anti-transferrin receptor mAb, OX-26, which has been considered as a potential candidate of the drug delivery vehicle specific to the CNS (Pardridge et al., 1991) (Table 2). Accumulation rate of cationized rat serum albumin, which is known to have an efficient BBB permeability (Pardridge et al., 1990), is similar to those of polioviruses (Table 2). These data strongly suggest that polioviruses permeate the BBB at a fairly high rate, independently of PVR and virus strains. State of poliovirus in the CNS Since the possible permeation of poliovirus through the BBB is not mediated by human PVR, which is considered to be the essential cellular molecule for establishment of the virus infection (Mendelsohn et al., 1989; Koike et al., 1990), it is possible that intact infectious poliovirus particles exist in parenchyma fraction of the brain. Accordingly, a modified method of Triguero et al. (1990) was employed to separate the parenchyma fraction from capillary fraction as described under Materials and Methods. As shown in Table 3, about 10% of a total activity of alkaline phosphatase (a marker enzyme for capillary) was still detected in parenchyma fraction. A similar purity was estimated when distribution of acetylated low density lipoprotein was examined after the IV administration. Tg mice or non-Tg mice were inoculated with 35S-labeled MMM or 35S-labeled SSS. The brain homogenates were prepared at times indicated in Fig. 4 and separated into parenchyma and capillary fractions. After the separation, about 90% of a total 35S-labeled materials in the brain were recovered in parenchyma fraction (Fig. 4).
TABLE 2 Accumulation Rate of Poliovirus into the Mouse Cerebrum Accumulation rate (ml/min/g tissue) Virus or control material
Tg mouse
Non-Tg mouse
MMM SSS MSM Albumin OX-26a Cationized RSAb
0.164 0.223 0.268 —
0.123 0.246 — 0.001 0.65 (Rat) 0.123 (Rat)
a Monoclonal antibody against transferrin receptor (Pardridge et al., 1991). b Cationized rat serum albumin (Pardridge et al., 1990).
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YANG ET AL. TABLE 3 Comparison of Alkaline Phosphatase Activity and Kp,app of I-Acetylated-LDL between Capillary and Parenchyma Fraction
125
Supernatant (parenchyma rich) Pellet (capillary rich) Supernatant/total
Activity of alkaline phosphatase
Kp,app of acetylated LDL
(nmol/min/mg-protein)
(ml/g brain)
10.5 { 0.1 69.1 { 2.5 0.132 { 0.007
1.67 { 0.01 14.4 { 0.02 0.104 { 0.002
Note. Four mice were used for each experiment.
These suggest that most polioviruses in the brain exist in parenchyma fraction, although it is possible that some viruses associated to the capillary fraction are detached from capillary during centrifugation and recovered in parenchyma fraction. To know whether 35S-labeled materials recovered in the parenchyma fraction at 7.5 hr after the inoculation are intact virion particles, the radiolabeled materials were analyzed by centrifugation on sucrose density gradients. As shown in Fig. 5, the majority of radioactivities was associated to materials at a sedimentation constant of 160S, a position of intact virion particle. The data indicate that 35S-labeled materials in parenchyma fraction of the brain tissue contain intact poliovirus particles. Indeed, this fraction showed a plaque forming activity (data not shown).
FIG. 5. Analysis of radioactive materials by sucrose density gradient centrifugation. Radioactive materials recovered from the cerebrum of Tg mice (l—l) or non-Tg mice (s---s) 7.5 hr after the IV inoculation of 35 S-labeled MMM were analyzed by centrifugation on sucrose density gradients as described under Materials and Methods. Positions of intact virion (160S) and poliovirus-related particles (135S and 80S) during the uncoating process are indicated by arrows.
FIG. 4. Radioactivities in parenchyma and capillary fractions of the cerebrum. Five Tg mice (A and C) or five non-Tg mice (B and D) were inoculated with [35S]methionine-labeled MMM (A and B) or SSS (C and D), and the cerebrum were separated into parenchyma and capillary fractions as described under Materials and Methods. Average Kp,app values with standard errors of radioactivities recovered in parenchyma (l—l) and capillary (s---s) fractions were plotted at indicated times.
pharmacokinetic analysis in addition to virological techniques. Amount of virus in plasma is changing with time after the virus inoculation because of trapping viruses by tissues and degradation of virion particles. Furthermore, in Tg mice, inoculated virus may multiplicate in some susceptible tissues and leak to the blood stream, although this phenomenon was not seen in the mice during the time period used in this study. These factors must affect virus concentration in plasma, resulting in alteration on pressure for virus transport from the blood stream to individual tissues. This difficulty in studying virus distribution was overcome in this study by employing Kp,app value, apparent tissue-to-plasma concentration ratio (Terasaki et al., 1984), which is also termed as tissue distribution volume (Pardridge et al., 1990). Instability of poliovirus differs in virus strains. Viruses carrying the Sabin 1 capsid (SSS and MSM) are less stable than those carrying the Mahoney capsid (MMM and SMS) in the brain of non-Tg mice (data not shown). This observation is compatible with previous results reported by Koike et al. (1991). Difference in virion stabilities must be one of determinants for neurovirulence phenotype. It was shown that poliovirus accumulated and probably permeated into various tissues of the mouse CNS such as the cerebrum, cerebellum, brain stem, and spinal cord (Fig. 3). The main dissemination route for causing the disease, however, is not clarified yet. To the cerebrum and spinal cord, approximately 0.3 and 0.09% of a total amount of inoculated virus were distributed, respectively, at 5 hr after the inoculation when the absolute quantity of virus almost plateaued. Although the amount of delivered virus was smaller in the spinal cord than the cerebrum, virus replication was detected in the spinal cord earlier than the cerebrum (data not shown). This might be explainable by different sensitivities of neurons to poliovirus between the two tissues. Indeed, the Tg
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DISCUSSION Distribution and dissemination of IV-inoculated poliovirus in Tg and non-Tg mice were studied by means of
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mice is more sensitive to IS-inoculated poliovirus than IC-inoculated poliovirus. It has been reported that the first tissue of the CNS where poliovirus replicates is the brain after the oral administration of the virus to ICRPVRTg1 (Koike et al., 1994b; Nomoto et al., 1994). The data appear not to be compatible with the observations described above. Neurons of the brain of ICR-PVRTg1 mice might be sensitive enough even to the small amount of poliovirus that permeates to the cerebrum at a very early stage, whereas those of ICR-PVRTg21 mice require a relatively larger amount of poliovirus for establishment of infection. It has been well-documented that ICR-PVR Tg1 is more sensitive to poliovirus than ICR-PVRTg21 (Koike et al., 1994a). Thus, sensitivity of neurons and/or some other cells of the CNS tissues may determine which dissemination route is important for causing disease. In this study, clinical signs of the mice appear to be caused mainly by invasion of the virus into the spinal cord. Ren and Racaniello (1992b) have reported that poliovirus invades the CNS by neural pathway after inoculation of the virus into the leg muscle of TgPVR1-17 mice. Therefore, it is possible that IV-inoculated poliovirus replicates in the muscle and is transported to the CNS through axons. Although MMM distributed to the skeletal muscle multiplicated to some extent (data not shown), maximum level of the virus in the muscle was approximately 104 PFU/muscles of mouse after IV inoculation of 6.5 1 106 PFU/mouse that was more than minimum LD100 (Table 1). On the other hand, lethal doses of MMM inoculated intramuscularly were more than 106 (data not shown). These observations indicate that the axonal pathway from the skeletal muscle is not mainly involved in the dissemination of the virus to the CNS. It has been reported that about 3000 monocytes move into the CNS per day (Hickey et al., 1991) and that human monocytes are sensitive to poliovirus infection (Freistadt and Eberle, 1996). Therefore, it is possible that monocytes are carriers of the virus from the blood stream to the CNS. Accordingly, association of poliovirus with monocytes and susceptibility of Tg mouse monocytes to poliovirus were examined. The results suggest that the monocytes do not associate with poliovirus and are not susceptible to poliovirus (data not shown). Monocytes, thus, appear not to be carriers of poliovirus. The most striking observation in this study was that tissue distribution and the CNS accumulation (probably permeation) of poliovirus occurs independently of human PVR. PVR may not be expressed on vascular endothelial cells. Since PVR is essential for poliovirus infection, these processes appear not to involve poliovirus replication. Indeed, most of virus present in the cerebrum at 7.5 hr after the inoculation was intact and infectious particles (Fig. 5). Furthermore, IV-inoculation of MMM to Tg mice did not cause any significant difference of the apparent permeability across the BBB of [14C]inulin, which was
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used as an essentially BBB impermeable marker molecule (Kakee et al., 1996), at 2, 48, and 72 hr after the inoculation (data not shown). This indicates that the BBB is not damaged by lytic replication of poliovirus at indicated times above. These data support the notion that poliovirus permeates the BBB at a fairly high rate without the virus replication. If it is also true in humans, circulating poliovirus including the Sabin vaccine strains easily invade the human CNS. Some host cellular molecules other than PVR must be involved in the BBB permeability of poliovirus. Molecular mechanisms for this dissemination process of poliovirus are now being investigated. ACKNOWLEDGMENTS We are grateful to H. Toyoda and S. Kuge for helpful suggestions and discussions. We thank K. Iwasaki and Y. Sasaki for expert technical assistance and E. Suzuki and M. Watanabe for help in preparation of the manuscript. This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan, the Ministry of Health and Welfare of Japan, and the Science and Technology Agency of the Japanese Government.
REFERENCES Abe, S., Ota, Y., Koike, S., Kurata, T., Horie, H., Nomura, T., Hashizume, S., and Nomoto, A. (1995a). Neurovirulence tests for oral live poliovaccines using poliovirus-sensitive transgenic mice. Virology 206, 1075–1083. Abe, S., Ota, Y., Doi, Y., Nomoto, A., Nomura, T., Chumakov, K. M., and Hashizume, S. (1995b). Studies on neurovirulence in poliovirussensitive transgenic mice and cynomolgus monkeys for the different temperature-sensitive viruses derived from the Sabin type 3 virus. Virology 210, 160–166. Aoki, J., Koike, S., Ise, I., Sato-Yoshida, Y., and Nomoto, A. (1994). Amino acid residues on human poliovirus receptor involved in interaction with poliovirus. J. Biol. Chem. 269, 8431–8438. Bodian, D. (1955). Emerging concept of poliomyelitis infection. Science 122, 105–108. Finny, D. J. (1959). The design and analysis of an immunological assay. Acta Microbiol. Hung. VI, 341–368. Freistadt, M. S., and Eberle, K. E. (1996). Correlation between poliovirus type 1 Mahoney replication in blood cells and neurovirulence. J. Virol. 70, 6486–6492. Hickey, W. F., Hsu, B. L., and Kimura, H. (1991). T-lymphocyte entry into the central nervous system. J. Neurosci. Res. 28, 254–260. Hogle, J. M., Chow, M., and Filman, D. J. (1985). Three-dimensional structure of poliovirus at 2.9 a˚ngstrom resolution. Science 229, 1358– 1363. Horie, H., Koike, S., Kurata, T., Sato-Yoshida, Y., Ise, I., Ota, Y., Abe, S., Hioki, K., Kato, H., Taya, C., Nomura, T., Hashizume, S., Yonekawa, H., and Nomoto, A. (1994). Transgenic mice carrying the human poliovirus receptor: new animal model for study of poliovirus neurovirulence. J. Virol. 68, 681–688. Kajigaya, S., Arakawa, H., Kuge, S., Koi, T., Imura, N., and Nomoto, A. (1985). Isolation and characterization of defective-interfering particles of poliovirus Sabin 1 strain. Virology 142, 307–316. Kakee, A., Terasaki, T., and Sugiyama, Y. (1996). Brain efflux index as a novel method of analyzing efflux transport at the blood-brain barrier. J. Pharmacol. Exp. Ther. 277, 1550–1559. Kitamura, N., Semler, B. L., Rothberg, P. G., Larsen, G. R., Adler, C. J., Dorner, A. J., Emini, E. A., Hanecak, R., Lee, J. J., van der Werf, S., Anderson, C. W., and Wimmer, E. (1981). Primary structure, gene
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organization and polypeptide expression of poliovirus RNA. Nature 291, 547–553. Kohara, M., Abe, S., Komatsu, T., Tago, K., Arita, M., and Nomoto, A. (1988). A recombinant virus between the Sabin 1 and Sabin 3 vaccine strains of poliovirus as a possible candidate for a new type 3 poliovirus live vaccine strain. J. Virol. 62, 2828–2835. Koike, S., Taya, C., Aoki, J., Matsuda, Y., Ise, I., Takeda, H., Matsuzaki, T., Amanuma, H., Yonekawa, H., and Nomoto, A. (1994a). Characterization of three different transgenic mouse lines that carry human poliovirus receptor gene: influence of the transgene expression on pathogenesis. Arch. Virol. 139, 351–363. Koike, S., Taya, C., Kurata, T., Abe, S., Ise, I., Yonekawa, H., and Nomoto, A. (1991). Transgenic mice susceptible to poliovirus. Proc. Natl. Acad. Sci. USA 88, 951–955. Koike, S., Horie, H., Ise, I., Okitsu, A., Yoshida, M., Iizuka, N., Takeuchi, K., Takegami, T., and Nomoto, A. (1990). The poliovirus receptor protein is produced both as membrane-bound and secreted forms. EMBO J. 9, 3217–3224. Koike, S., Aoki, J., and Nomoto, A. (1994b). Transgenic mouse for the study of poliovirus pathogenicity, In ‘‘Cellular Receptors for Animal Viruses’’ (E. Wimmer, Ed.), pp. 463–480. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Li, T., Zang, A., Iizuka, N., Nomoto, A., and Arnold, E. (1992). Crystallization and preliminary X-ray diffraction studies of coxsackievirus B1. J. Mol. Biol. 223, 1171–1175. Mendelsohn, C. L., Wimmer, E., and Racaniello, V. R. (1989). Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily. Cell 56, 855–865. Nomoto, A., Koike, S., and Aoki, J. (1994). Tissue tropism and species specificity of poliovirus infection. Trends in Microbiol. 2, 47–51. Omata, T., Kohara, M., Kuge, S., Komatsu, T., Abe, S., Semler, B. L., Kameda, A., Itoh, H., Arita, M., Wimmer, E., and Nomoto, A. (1986). Genetic analysis of the attenuation phenotype of poliovirus type 1. J. Virol. 58, 348–358.
Pardridge, W. M., Triguero, D., Buciak, J., and Yang, J. (1990). Evaluation of cationized rat albumin as a potential blood-brain barrier drug transport vector. J. Pharmacol. Exp. Ther. 255, 893–899. Pardridge, W. M., Buciak, J. L., and Friden, P. M. (1991). Selective transport of an anti-transferrin receptor antibody through the blood-brain barrier in vivo. J. Pharmacol. Exp. Ther. 259, 66–70. Racaniello, V. R., and Baltimore, D. (1981). Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc. Natl. Acad. Sci. USA 78, 4887–4891. Ren, R., Costantini, F., Gorgacz, E. J., Lee, J. J., and Racaniello, V. R. (1990). Transgenic mice expressing a human poliovirus receptor: a new model for poliomyelitis. Cell 63, 353–362. Ren, R., and Racaniello, V. R. (1992a). Human poliovirus receptor gene expression and poliovirus tissue tropism in transgenic mice. J. Virol. 66, 296–304. Ren, R., and Racaniello, V. R. (1992b). Poliovirus spreads from muscle to the central nervous system by neural pathways. J. Infect. Dis. 166, 747–752. Sabin, A. B. (1956). Pathogenesis of poliomyelitis: Reappraisal in the light of new data. Science 123, 1151–1157. Sabin, A. B., and Boulger, L. R. (1973). History of Sabin attenuated poliovirus oral live vaccine strains. J. Biol. Stand. 1, 115–118. Shiroki, K., Ishii, T., Aoki, T., Kobashi, M., Ohka, S., and Nomoto, A. (1995). A new cis-acting element for RNA replication within the 5* noncoding region of poliovirus type 1 RNA. J. Virol. 69, 6825–6832. Smith, Q. R., Momma, S., Aoyagi, M., and Rapoport, S. I. (1987). Kinetics of neural amino acid transport across the blood-brain barrier. J. Neurochem. 49, 1651–1658. Terasaki, T., Iga, T., Sugiyama, Y., and Hanano, M. (1984). Pharmacokinetic study on the mechanism of tissue distribution of dexorubicin: interorgan and interspecies variation of tissue-to-plasma partition coefficients in rats, rabbits, and guinea pigs. J. Pharm. Sci. 73, 1359– 1363. Triguero, D., Buciak, J., and Pardridge, W. M. (1990). Capillary depletion method for quantification of blood-brain barrier transport of circulating peptides and plasma proteins. J. Neurochem. 54, 1882–1888.
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Efficient Delivery of Circulating Poliovirus to the Central Nervous System Independently of Poliovirus Receptor WEI-XING YANG,* TETSUYA TERASAKI,*,1 KAZUKO SHIROKI,† SEII OHKA,† JUNKEN AOKI,* SHUICHI TANABE,* TATSUJI NOMURA,‡ EIJI TERADA,§ YUICHI SUGIYAMA,* and AKIO NOMOTO†,2 *Faculty of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan; †The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan; ‡Central Institute for Experimental Animals, 1430 Nogawa, Miyamae-ku, Kawasaki, Kanagawa 216, Japan; and §Institute of Laboratory Animal Sciences, School of Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228, Japan Received October 28, 1996; returned to author for revision December 16, 1996; accepted January 10, 1997 The transgenic (Tg) mice carrying the human gene for poliovirus receptor (PVR) are susceptible to poliovirus intravenously (IV) inoculated as well as intracerebrally or intraspinally inoculated. Thus, IV-inoculated poliovirus may invade the central nervous system (CNS) through the blood–brain barrier (BBB). To know the contribution of PVR to tissue distribution and BBB permeability of IV-inoculated polioviruses, these dissemination processes were investigated and compared between the Tg mice and non-Tg mice. Distribution profile of IV-inoculated poliovirus in various tissues of the Tg mice is similar to that in non-Tg mice. The data suggest that tissue distribution of the virus occurs independently of the transgene for PVR. The amount of poliovirus delivered to the CNS suggested the existence of a specific delivery system of the virus to the CNS. Virus accumulation in the CNS of the Tg mice was measured up to 7.5 hr after the IV inoculation. The viruses, regardless of whether the virulent or attenuated strain, seem to accumulate at a constant rate of approximately 0.2 ml/ min/g tissue. Similar phenomena were observed when the viruses were inoculated into non-Tg mice. The rates of the virus accumulation in the CNS are more than 100 times higher than that of albumin, which is considered not to permeate through the BBB via a specific transport system, and only three times lower than that of monoclonal antibody against transferrin receptor (OX-26), which is a potential candidate as a drug delivery vehicle specific to the CNS. These data suggest that polioviruses permeate through the BBB at a fairly high rate, independently of PVR and virus strains. q 1997 Academic Press
INTRODUCTION
by primary multiplication in the alimentary mucosa. After viral multiplication in tissues such as the tonsils and Peyer’s patches, the virus moves into the deep cervical and mesenteric lymph nodes and then into the blood. If viremia is established, the virus invades the central nervous system (CNS) and paralytic poliomyelitis occurs as a result of the destruction of neurons in the CNS, especially the motor neurons in the anterior horn of the spinal cord and neurons of the motor cortex and brain stem. Although many tissues are exposed to the virus during the viremic phase, most tissues do not support poliovirus replication. Thus poliovirus seems to have a distinct tropism with regard to tissues and cell types. Little is known, however, about mechanisms for the virus dissemination characteristic of poliovirus. Humans are the only natural host of poliovirus, although poliovirus can be experimentally transferred to monkeys, in which it also causes a paralytic disease. Other animal species are not susceptible to most poliovirus strains. Because of the characteristic species specificity of poliovirus, monkeys have been used as the only animal model for poliomyelitis, and inoculation of the virus into the monkey CNS has been the only method to evaluate neurovirulence levels of poliovirus. The virus-specific tropism must be controlled by host
Poliovirus, known to be the causative agent of poliomyelitis, is a human enterovirus that belongs to the family Picornaviridae and is classified into three stable serotypes, 1, 2, and 3. The poliovirion is an icosahedral, nonenveloped particle that is composed of 60 copies of each of four capsid proteins, VP1, VP2, VP3, and VP4, and a single-stranded RNA genome of positive polarity. The three-dimensional structure of the virion particle (Hogle et al., 1985) as well as the primary structures of the RNA genome and the viral proteins (Kitamura et al., 1981; Racaniello and Baltimore, 1981) have been elucidated. The three-dimensional structure has revealed depressions called ‘‘canyon’’ on the surface of the virion, which have been suggested to be attachment sites for poliovirus receptor (PVR) on the surface of permissive cells. The life cycle of poliovirus was first described in the 1950s (Bodian, 1955; Sabin, 1956). Poliovirus infection is initiated by oral ingestion of the virus, which is followed 1 Present address: Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki-aza, Aoba-ku, Sendai 980-77, Japan. 2 To whom correspondence and reprint requests should be addressed. Fax: 81-3-5449-5408. E-mail: [email protected].
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factors that support the virus replications. Among these host factors, a specific cell surface receptor is considered to be one of the most important factors that determine host range of the virus. Indeed, the transgenic (Tg) mice carrying the human PVR gene were shown to be susceptible to all three poliovirus serotypes, and the infected mice showed clinical signs resemble those of humans and monkeys (Ren et al., 1990; Koike et al., 1991). In the CNS of the infected Tg mice, poliovirus antigens were detected only in neurons, and neuronal damages were observed (Koike et al., 1991, 1994b; Ren and Racaniello, 1992a; Horie et al., 1994). Clinical and pathological similarities of poliovirus infection in the Tg mice to those in monkeys suggest that the Tg mice become an additional animal model for poliomyelitis (Horie et al., 1994). To control poliomyelitis, attenuated poliovirus strains of all three serotypes have been developed and effectively used as oral live poliovirus vaccines, that is, Sabin 1 (serotype 1), Sabin 2 (serotype 2), and Sabin 3 (serotype 3) (Sabin and Boulger, 1973). They have a very poor capacity to replicate in the CNS of monkeys and show a very weak monkey neurovirulence, whereas the virulent strains replicate well in the CNS and show a strong monkey neurovirulence. The virulent and attenuated phenotypes of poliovirus observed in monkeys appeared to be preserved in the Tg mice, since much larger doses of virus were required for the attenuated strains to cause paralysis compared with those of the virulent strains. In fact, relative neurovirulence levels of polioviruses estimated in the Tg mice showed a good correlation with those in monkeys (Horie et al., 1994). The mouse neurovirulence tests involving intracerebral (IC) and intraspinal (IS) inoculation routes have been established to date (Horie et al., 1994; Abe et al., 1995a; 1995b). Different neurovirulence levels are also observed between the virulent and attenuated poliovirus strains when the viruses are intravenously (IV)-inoculated into the Tg mice (Koike et al., 1994b). Using this inoculation route, we investigate dissemination of poliovirus from the blood stream to the CNS of the Tg and non-Tg mice. Our data indicate that tissue distribution of the virus occurs independently of human PVR. Furthermore, we show that IVinoculated poliovirus accumulates in various tissues of the CNS, independently of human PVR, at a fairly high rate. The results suggest that poliovirus is able to permeate through the blood–brain barrier (BBB) of mice without the aid of human PVR. MATERIALS AND METHODS Transgenic mice The Tg mouse line ICR-PVRTg21 (Koike et al., 1991), in the hemizygous stage of 8 to 10 weeks of age, was used as an animal model for poliomyelitis. The copy number of the transgene in ICR-PVRTg21 is approximately threefold that in HeLa S3 cells (Koike et al., 1991; 1994a).
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All mice used were free from specific pathogens. ICR mice were purchased from Charles River Japan (Tokyo) and used as non-Tg mice. Viruses and cells The virulent Mahoney virus (PV1(M)OM) and the attenuated Sabin 1 virus (PV1(Sab)IC-0) were derived from infectious cDNA clones pOM1 (Shiroki et al., 1995) and pVS(1)IC-0(T) (Kohara et al., 1988), respectively. Viruses PV1(M)OM and PV1(Sab)IC-0 are termed MMM and SSS, respectively, in this study. A cDNA segment of pOM1 corresponding to a coding sequence for the viral capsid proteins (P1 region) was replaced by the corresponding segment of pVS(1)IC-0(T). A recombinant cDNA clone thus obtained was used for transfection to prepare virus MSM. A similar experiment was carried out to produce virus SMS, whose genome had a reciprocal structure of the MSM genome. Suspension-cultured HeLa S3 cells were maintained in RPMI 1640 medium supplemented with 5% newborn calf serum (NCS) and used for preparation of viruses. African green monkey kidney (AGMK) cells were grown in Dulbecco modified Eagle medium supplemented with 5% NCS and used for the transfection experiment with infectious cDNA clones and for plaque assays. Purification of viruses Viruses were grown in suspension-cultured HeLa cells as described previously (Kajigaya et al., 1985). Viruses in cytoplasmic extracts of infected cells were purified by chromatography on DEAE-Sepharose CL-6B (Li et al., 1992), followed by two cycles of CsCl equilibrium centrifugation in a buffer containing 10 mM Tris–HCl (pH 7.4), 10 mM NaCl, 20 mM EDTA, and 1% Brij 58. For preparation of [35S]methionine-labeled viruses, viruses were grown in methionine-free medium in the presence of [35S]methionine. Calculated specific radioactivities of purified radiolabeled Mahoney and Sabin 1 viruses were 148 and 27 mCi/nmol, respectively. Clinical neurovirulence tests Into the brain or tail vein of each Tg mouse, 30 or 200 ml of a virus suspension was inoculated. More than 10 Tg mice were used for injection with each dose of viruses except for experiments involving IC inoculation of SMS in which 5 Tg mice were used. The mice inoculated with viruses were observed for clinical signs at 12-hr intervals up to 14 days. The 50% lethal doses (LD50) were calculated according to the Dragstehdt–Behrens method, a modified Reed–Muench method (Finny, 1959). Approximate minimum 100% lethal doses (LD100) were also estimated. Distribution of viruses in mice Male Tg mice and non-Tg mice weighing 28–35 g were used. The animals had free access of food and
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water. Each mouse was inoculated with 2.5 1 1002 pmol of viruses (approximately 6.5 1 106 PFU) containing 0.4 mCi of labeled viruses via tail vein under mild ether anesthesia. [125I]albumin was used as a control compound that was known to exhibit significantly restricted BBB permeability. Three mice were dissected 5 hr after the inoculation. The tissues were weighed, minced, and dissolved with a tissue solubilizer, Solvable (Dupont, U.S.A.), at 507 for 3hr, and then 10 ml of liquid scintillation cocktail, Atomlight (Dupont), was added. Radioactivities were measured in a liquid scintillation counter (Aloka LSC1000). As an index of tissue distribution characteristics of poliovirus, the apparent tissue-to-plasma concentration ratio, Kp,app , defined by amount of virus per grams of tissue divided by that per microliter of plasma, was calculated over the time period of the experiment (Terasaki et al., 1984). Least squares regression analysis was performed with the program of Excel (Microsoft, U.S.A.). In some cases, 2 nmol of monoclonal antibody (mAb) against human PVR, p286, that had been prepared and characterized by J. Aoki and found to have very similar characteristics to mAb p403 (Aoki et al., 1994), was intravenously administered to Tg mice 5 hr before the virus inoculation. Kd value of p286 to human PVR on cell surface was calculated to be 8 nM. According to the calculated elimination rate constant of p286 in the mouse plasma, concentration of p286 5 hr after the virus inoculation should be about 80 nM and therefore still enough to inhibit virus binding to PVR. To know accumulation rate of viruses in various tissues of the mouse CNS, Kp,app values in these tissues were measured by the method of integration plot (Smith et al., 1987; Kakee et al., 1996) up to 7.5 hr after the virus inoculation. Briefly, the accumulation rate of poliovirus can be described by the following differential equation: dXbr Å PS 1 Cp dt
(1)
where Xbr is the amount of 35S-labeled poliovirus in the brain at time t, PS is the brain accumulation rate, and Cp is the plasma concentration of 35S-labeled poliovirus. Integration of equation (1) gives Xbr(t) Å PS 1 AUC(0-t) / Cp(t) 1 V0
(2)
where AUC(0-t) represents the area under the plasma concentration time curve of 35S-labeled poliovirus from time 0 to t, and V0 is the rapidly equilibrated distribution volume of 35S-labeled poliovirus. Equation (2) divided by Cp(t) 1 Vbr gives
S D
PS V0 AUC(0-t) Cbr(t) Å / 1 Cp(t) Vbr Cp(t) Vbr
(3)
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FIG. 1. Genome structures of recombinant type 1 polioviruses. The genome structures of the recombinant viruses are shown as a combination of the Mahoney (shadowed boxes) and the Sabin 1 (open boxes) sequences. VPg at the 5* end and poly(A) at the 3* end are indicated by closed circles and wavy lines. Numbers shown above the Mahoney genome represent nucleotide numbers from the 5* terminus.
brain, PS/Vbr , can be obtained from the initial slope of a plot of Cbr(t)/Cp(t), i.e., Kp,app(t) vs AUC(0-t)/Cp(t), as integration plot. Separation of parenchyma and capillary fractions of the brain Parenchyma and capillary fractions of the brain were separated by a modified method of Triguero et al. (1990). Briefly, the brain was minced after removal of meninges and connective tissues, added to 4 vol of physiologic buffer containing 10 mM HEPES (pH 7.4), 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2 , 1 mM MgSO4 , 1 mM NaH2PO4 , and 10 mM D-glucose, and then homogenized in a teflon homogenizer with 10 strokes. The homogenate was added to an equal volume of 32% dextran solution and homogenized with 3 more strokes. An aliquot of 0.2 ml was used to estimate radioactivity and virus titer in a whole brain. The remaining homogenates were centrifuged at 5400 g for 15 min at 47 in a Beckman rotor SW55 to precipitate capillary fraction. To monitor the separation, the activity of alkaline phosphatase, a marker enzyme for capillary fraction, was measured (Triguero et al., 1990). For the same purpose, 125 I-labeled acetylated low density lipoprotein was also employed as a marker material that accumulated only in capillary fraction (Triguero et al., 1990). Sucrose density gradient centrifugation 35
S-labeled materials in parenchyma fraction of the brain was analyzed by centrifugation on 15–30% sucrose density gradient in a buffer containing 0.1% bovine serum albumin and phosphate-buffered saline. The centrifugation was performed at 200,000 g for 2.5 hr at 47 in a Beckman rotor SW41. RESULTS Mouse neurovirulence tests on viruses
where Vbr represents the brain weight, and Cbr(t) is the brain concentration of 35S-labeled poliovirus. The brain accumulation rate of 35S-labeled poliovirus per grams of
The virulent Mahoney strain (MMM), attenuated Sabin 1 strain (SSS), and their recombinant viruses (MSM and SMS) were prepared as described under Materials and Methods. The genome structures of these viruses are shown in Fig. 1. The viruses were tested for their neurovi-
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LD50 and Approximate Minimum LD100 of Poliovirus in Tg Mouse LD50 (PFU/mouse) Virus
IV a
MMM SSS MSM SMS
8.5 1 104 — 3.2 1 105 —
a b
Minimum LD100 (PFU/mouse)
ICb 2.1 7.0 4.2 1.3
1 1 1 1
102 105 102 105
IV a 4.0 1 106 — 6.0 1 106 —
ICb 1.0 7.0 4.0 #8.4
1 1 1 1
104 107 104 107
Intravenous inoculation. Intracerebral inoculation.
rulence in the Tg mice via IC and IV inoculation routes. The results are shown by values of LD50 and approximate values of minimum LD100 in Table 1. Viruses MMM and SSS showed the strongest and weakest neurovirulence among the viruses, respectively, when the mice were IC-inoculated (Table 1). Neurovirulence levels of viruses MSM and SMS are similar to those of MMM and SSS, respectively, although MSM appears to be slightly weaker than MMM and SMS slightly stronger than SSS. Thus, virion surface characteristics had little or no correlation with mouse neurovirulence. This observation is compatible with our previous results obtained from monkey tests (Omata et al., 1986; Kohara et al., 1988) and Tg mouse tests (Horie et al., 1994). Similar results were obtained when the mice were IV-inoculated with MMM and MSM although much larger amounts of viruses were required to cause paralysis and death (Table 1). In the case of IV inoculation, however, it took at least one more day for the mice to be killed as compared with the case of IC inoculation. LD50 values of SSS and SMS could not be calculated because no mice were killed even by IV inoculating 1 1 108 PFU of these viruses. Approximate values of minimum LD100 were estimated and are shown in Table 1. The LD100 values obtained from the experiments involving IV inoculation of MMM and MSM were 150 to 400 times more than those involving IC inoculation of these viruses. Main dissemination route of poliovirus from blood to the CNS is not known at present. However, if circulating virus invades the brain at first as suggested (Koike et al., 1994b; Nomoto et al., 1994), the data suggest that 0.25–0.6% of IV-inoculated virus has to invade the brain to kill all the mice. Considering the fact that approximately 0.3% of a total amount of IV-inoculated virus is distributed to the cerebrum, most of the virus distributed should move into the brain.
know the amount of virus delivered to individual tissues including the brain tissues, we took advantage of physiological pharmacokinetic analysis as described under Materials and Methods (Terasaki et al., 1984). Tissue distribution of MMM, SSS, or MSM in Tg mice or non-Tg mice was examined 5 hr after the IV inoculation. The result of MSM distribution is shown in Fig. 2A. Similar results were obtained for MMM and SSS distributions (data not shown). Amounts of poliovirus delivered to the CNS including the cerebrum and cerebellum are the smallest among all tissues tested. However, Kp,app values in the cerebrum and cerebellum are more than 200 ml/g tissue, and are significantly greater than the vascular volume of the brain, i.e., about 10 ml/g tissue (Pardridge et al., 1990; 1991). The data may suggest that polioviruses cross the blood–brain barrier. A similar in vivo distribution characteristic of circulating poliovirus was obtained when non-Tg mice was used. The data shown in Fig. 2A suggest that the Kp,app values obtained from experiments with Tg mice and non-Tg mice have a good correlation with each other on a level of P õ 0.01 (correlation coefficient: r 2 Å 0.903; n Å 9). This suggests that specific distributions of poliovirus to the brain tissues are not due to specific expression of human PVR on the brain capillary. To confirm this possibility, Tg mice were IV-injected with anti-PVR mAb p286 5 hr before the virus inoculation as described under Materials and Methods. This mAb has an ability to block human PVR-mediated poliovirus binding and infection to susceptible cells. As shown in Fig. 2B, preadministration of p286 appears not to affect the differential tissue distribution of poliovirus. The Kp,app values again have a good correlation with each other on a level of P õ 0.01 (correlation coefficient: r 2 Å 0.981; n Å 9). A similar result was obtained from the experiment involving MMM. These results indicate that human PVR expressed in the mouse tissues does not play any significant role for the differential tissue distribution of poliovirus.
It is possible that IV-inoculated poliovirus is delivered selectively to the CNS. Expression of human PVR may contribute to the virus delivery after IV inoculation. To
FIG. 2. Tissue distribution of poliovirus in mice after IV inoculation. (A) Kp,app values of [35S]methionine-labeled MSM in various tissues were calculated in Tg mice (vertical axis) and non-Tg mice (horizontal axis) 5 hr after the IV inoculation. (B) Tg mice were IV-injected with mAb p286 5 hr before the IV inoculation of [35S]methionine-labeled MSM as described under Materials and Methods. Kp,app values of MSM in various tissues were calculated in Tg mice (vertical axis) and Tg mice pretreated with the mAb (horizontal axis) 5 hr after the IV inoculation.
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FIG. 3. Integration plot of poliovirus Mahoney strain in the CNS of Tg mice after IV inoculation. Tg mice were IV-inoculated with [35S]methionine-labeled MMM as described under Materials and Methods. Average Kp,app values in the cerebrum (A), cerebellum (B), brain stem (C), and spinal cord (D) were calculated at indicated AUC/Cp values. Three Tg mice were used for injection at each time. Calculation of AUC/Cp values followed that reported by Smith et al. (1987).
Rate of poliovirus accumulation in the CNS To know the rate of poliovirus accumulation from the circulating blood to the CNS, Tg mice were IV-inoculated with 35S-labeled MMM, and the time course of virus accumulation in individual tissues of the CNS followed up to 7.5 hr after the inoculation when the virus replication was not yet detected in any of these tissues. Experiments were repeated three times for MMM and SSS and twice for MSM. The results are shown in Fig. 3 by integration plot (Smith et al., 1987; Kakee et al., 1996). Standard errors were too small to show in Fig. 3. Slopes shown in Fig. 3 represent virus accumulation rates in individual tissues. Linear accumulation of the virus was observed in any these tissues up to 7.5 hr after the inoculation. The rates were estimated to be 0.164, 0.171, 0.151, and 0.117 ml/min/g tissue in the cerebrum, cerebellum, brain stem, and spinal cord, respectively. In other tissues, linear accumulation was not seen, and the rates are coming to plateaus during the time used (data not shown). Similar experiments were carried out on the cerebrum by using non-Tg mice and/or viruses carrying the Sabin 1 capsid to know the contribution of human PVR and virion surface characteristics to accumulation rates of viruses. As shown in Table 2, the rates observed in Tg mice resemble those in non-Tg mice. Thus, human PVR seems not to play an important role in delivery of circulating poliovirus to the cerebrum. The accumulation rates of SSS and MSM were estimated to be 0.223 and 0.268 ml/min/g tissue in Tg mice and are slightly higher than that (0.164 ml/min/g tissue) of MMM (Table 2). A similar observation was seen in
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non-Tg mice. It is possible that the Sabin 1 capsid has a slightly higher efficiency in the virus accumulation in the cerebrum as compared with the virulent Mahoney capsid. It should be noted that the accumulation rates observed for polioviruses are more than 100 times higher compared with that of albumin (Table 2), which is known to exhibit significantly restricted permeability across the BBB and are only three times less than that of anti-transferrin receptor mAb, OX-26, which has been considered as a potential candidate of the drug delivery vehicle specific to the CNS (Pardridge et al., 1991) (Table 2). Accumulation rate of cationized rat serum albumin, which is known to have an efficient BBB permeability (Pardridge et al., 1990), is similar to those of polioviruses (Table 2). These data strongly suggest that polioviruses permeate the BBB at a fairly high rate, independently of PVR and virus strains. State of poliovirus in the CNS Since the possible permeation of poliovirus through the BBB is not mediated by human PVR, which is considered to be the essential cellular molecule for establishment of the virus infection (Mendelsohn et al., 1989; Koike et al., 1990), it is possible that intact infectious poliovirus particles exist in parenchyma fraction of the brain. Accordingly, a modified method of Triguero et al. (1990) was employed to separate the parenchyma fraction from capillary fraction as described under Materials and Methods. As shown in Table 3, about 10% of a total activity of alkaline phosphatase (a marker enzyme for capillary) was still detected in parenchyma fraction. A similar purity was estimated when distribution of acetylated low density lipoprotein was examined after the IV administration. Tg mice or non-Tg mice were inoculated with 35S-labeled MMM or 35S-labeled SSS. The brain homogenates were prepared at times indicated in Fig. 4 and separated into parenchyma and capillary fractions. After the separation, about 90% of a total 35S-labeled materials in the brain were recovered in parenchyma fraction (Fig. 4).
TABLE 2 Accumulation Rate of Poliovirus into the Mouse Cerebrum Accumulation rate (ml/min/g tissue) Virus or control material
Tg mouse
Non-Tg mouse
MMM SSS MSM Albumin OX-26a Cationized RSAb
0.164 0.223 0.268 —
0.123 0.246 — 0.001 0.65 (Rat) 0.123 (Rat)
a Monoclonal antibody against transferrin receptor (Pardridge et al., 1991). b Cationized rat serum albumin (Pardridge et al., 1990).
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YANG ET AL. TABLE 3 Comparison of Alkaline Phosphatase Activity and Kp,app of I-Acetylated-LDL between Capillary and Parenchyma Fraction
125
Supernatant (parenchyma rich) Pellet (capillary rich) Supernatant/total
Activity of alkaline phosphatase
Kp,app of acetylated LDL
(nmol/min/mg-protein)
(ml/g brain)
10.5 { 0.1 69.1 { 2.5 0.132 { 0.007
1.67 { 0.01 14.4 { 0.02 0.104 { 0.002
Note. Four mice were used for each experiment.
These suggest that most polioviruses in the brain exist in parenchyma fraction, although it is possible that some viruses associated to the capillary fraction are detached from capillary during centrifugation and recovered in parenchyma fraction. To know whether 35S-labeled materials recovered in the parenchyma fraction at 7.5 hr after the inoculation are intact virion particles, the radiolabeled materials were analyzed by centrifugation on sucrose density gradients. As shown in Fig. 5, the majority of radioactivities was associated to materials at a sedimentation constant of 160S, a position of intact virion particle. The data indicate that 35S-labeled materials in parenchyma fraction of the brain tissue contain intact poliovirus particles. Indeed, this fraction showed a plaque forming activity (data not shown).
FIG. 5. Analysis of radioactive materials by sucrose density gradient centrifugation. Radioactive materials recovered from the cerebrum of Tg mice (l—l) or non-Tg mice (s---s) 7.5 hr after the IV inoculation of 35 S-labeled MMM were analyzed by centrifugation on sucrose density gradients as described under Materials and Methods. Positions of intact virion (160S) and poliovirus-related particles (135S and 80S) during the uncoating process are indicated by arrows.
FIG. 4. Radioactivities in parenchyma and capillary fractions of the cerebrum. Five Tg mice (A and C) or five non-Tg mice (B and D) were inoculated with [35S]methionine-labeled MMM (A and B) or SSS (C and D), and the cerebrum were separated into parenchyma and capillary fractions as described under Materials and Methods. Average Kp,app values with standard errors of radioactivities recovered in parenchyma (l—l) and capillary (s---s) fractions were plotted at indicated times.
pharmacokinetic analysis in addition to virological techniques. Amount of virus in plasma is changing with time after the virus inoculation because of trapping viruses by tissues and degradation of virion particles. Furthermore, in Tg mice, inoculated virus may multiplicate in some susceptible tissues and leak to the blood stream, although this phenomenon was not seen in the mice during the time period used in this study. These factors must affect virus concentration in plasma, resulting in alteration on pressure for virus transport from the blood stream to individual tissues. This difficulty in studying virus distribution was overcome in this study by employing Kp,app value, apparent tissue-to-plasma concentration ratio (Terasaki et al., 1984), which is also termed as tissue distribution volume (Pardridge et al., 1990). Instability of poliovirus differs in virus strains. Viruses carrying the Sabin 1 capsid (SSS and MSM) are less stable than those carrying the Mahoney capsid (MMM and SMS) in the brain of non-Tg mice (data not shown). This observation is compatible with previous results reported by Koike et al. (1991). Difference in virion stabilities must be one of determinants for neurovirulence phenotype. It was shown that poliovirus accumulated and probably permeated into various tissues of the mouse CNS such as the cerebrum, cerebellum, brain stem, and spinal cord (Fig. 3). The main dissemination route for causing the disease, however, is not clarified yet. To the cerebrum and spinal cord, approximately 0.3 and 0.09% of a total amount of inoculated virus were distributed, respectively, at 5 hr after the inoculation when the absolute quantity of virus almost plateaued. Although the amount of delivered virus was smaller in the spinal cord than the cerebrum, virus replication was detected in the spinal cord earlier than the cerebrum (data not shown). This might be explainable by different sensitivities of neurons to poliovirus between the two tissues. Indeed, the Tg
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DISCUSSION Distribution and dissemination of IV-inoculated poliovirus in Tg and non-Tg mice were studied by means of
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mice is more sensitive to IS-inoculated poliovirus than IC-inoculated poliovirus. It has been reported that the first tissue of the CNS where poliovirus replicates is the brain after the oral administration of the virus to ICRPVRTg1 (Koike et al., 1994b; Nomoto et al., 1994). The data appear not to be compatible with the observations described above. Neurons of the brain of ICR-PVRTg1 mice might be sensitive enough even to the small amount of poliovirus that permeates to the cerebrum at a very early stage, whereas those of ICR-PVRTg21 mice require a relatively larger amount of poliovirus for establishment of infection. It has been well-documented that ICR-PVR Tg1 is more sensitive to poliovirus than ICR-PVRTg21 (Koike et al., 1994a). Thus, sensitivity of neurons and/or some other cells of the CNS tissues may determine which dissemination route is important for causing disease. In this study, clinical signs of the mice appear to be caused mainly by invasion of the virus into the spinal cord. Ren and Racaniello (1992b) have reported that poliovirus invades the CNS by neural pathway after inoculation of the virus into the leg muscle of TgPVR1-17 mice. Therefore, it is possible that IV-inoculated poliovirus replicates in the muscle and is transported to the CNS through axons. Although MMM distributed to the skeletal muscle multiplicated to some extent (data not shown), maximum level of the virus in the muscle was approximately 104 PFU/muscles of mouse after IV inoculation of 6.5 1 106 PFU/mouse that was more than minimum LD100 (Table 1). On the other hand, lethal doses of MMM inoculated intramuscularly were more than 106 (data not shown). These observations indicate that the axonal pathway from the skeletal muscle is not mainly involved in the dissemination of the virus to the CNS. It has been reported that about 3000 monocytes move into the CNS per day (Hickey et al., 1991) and that human monocytes are sensitive to poliovirus infection (Freistadt and Eberle, 1996). Therefore, it is possible that monocytes are carriers of the virus from the blood stream to the CNS. Accordingly, association of poliovirus with monocytes and susceptibility of Tg mouse monocytes to poliovirus were examined. The results suggest that the monocytes do not associate with poliovirus and are not susceptible to poliovirus (data not shown). Monocytes, thus, appear not to be carriers of poliovirus. The most striking observation in this study was that tissue distribution and the CNS accumulation (probably permeation) of poliovirus occurs independently of human PVR. PVR may not be expressed on vascular endothelial cells. Since PVR is essential for poliovirus infection, these processes appear not to involve poliovirus replication. Indeed, most of virus present in the cerebrum at 7.5 hr after the inoculation was intact and infectious particles (Fig. 5). Furthermore, IV-inoculation of MMM to Tg mice did not cause any significant difference of the apparent permeability across the BBB of [14C]inulin, which was
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used as an essentially BBB impermeable marker molecule (Kakee et al., 1996), at 2, 48, and 72 hr after the inoculation (data not shown). This indicates that the BBB is not damaged by lytic replication of poliovirus at indicated times above. These data support the notion that poliovirus permeates the BBB at a fairly high rate without the virus replication. If it is also true in humans, circulating poliovirus including the Sabin vaccine strains easily invade the human CNS. Some host cellular molecules other than PVR must be involved in the BBB permeability of poliovirus. Molecular mechanisms for this dissemination process of poliovirus are now being investigated. ACKNOWLEDGMENTS We are grateful to H. Toyoda and S. Kuge for helpful suggestions and discussions. We thank K. Iwasaki and Y. Sasaki for expert technical assistance and E. Suzuki and M. Watanabe for help in preparation of the manuscript. This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan, the Ministry of Health and Welfare of Japan, and the Science and Technology Agency of the Japanese Government.
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