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3 integrin antibody inhibit infection by Sin Nombre virus in the deer mouse model
1 Center for Infectious Diseases and Immunity, Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
2 Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108, USA
3 Departments of Biology and Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
Correspondence
Brian Hjelle
bhjelle{at}salud.unm.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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3 antibody (ReoPro) and a polyclonal rabbit anti-recombinant nucleocapsid (N) antibody against SNV was investigated. Concurrent intraperitoneal administration of 100 mg ribavirin kg–1 prevented seroconversion in all mice at day 15 post-inoculation (p.i.). No evidence of infection was detectable by immunohistochemical staining or by quantitative RT-PCR in two of these six mice. Lower doses of ribavirin, between 5 and 50 mg kg–1, were much less effective at inhibiting infection. Mice given 200 µl aliquots of dilutions as high as 1 : 20 of HIP (neutralizing-antibody titre 800) failed to seroconvert by day 15 p.i. SNV N antigen staining and viral S genome were undetectable in these mice. A subset of mice given higher dilutions of HIP became infected. Treatment with 6 mg ReoPro kg–1 did not prevent seroconversion, but was able to reduce viral load. Mice treated with 200 µl anti-N antibody or negative human plasma seroconverted when challenged with SNV, and antigen staining and viral loads were comparable to those seen in untreated controls. These results show that ReoPro can lower viral loads and that ribavirin and HIP, but not anti-N antibody, inhibit seroconversion and reduce viral loads in a dose-dependent manner. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The mRNA for the S segment encodes the nucleocapsid (N) protein, that for the M segment encodes the envelope glycoproteins G1 and G2, and the L segment mRNA encodes the RNA-dependent RNA polymerase. Hantaviruses produce two types of acute febrile illness: haemorrhagic fever with renal syndrome (HFRS), a disease that occurs mainly in Asia and eastern Europe, and hantavirus (cardio)pulmonary syndrome (HCPS or HPS), which has only been found in the western hemisphere (Schmaljohn & Hjelle, 1997). The prototypical and epidemiologically most important aetiological agents of HCPS are Sin Nombre virus (SNV) and Andes virus (ANDV) in North and South America, respectively.
Over 1000 cases of HCPS have been reported, with case-fatality ratios of between 30 and 50 % (Mertz et al., 1997). Patients with HCPS experience thrombocytopenia, increased vascular permeability, interstitial pneumonia, non-cardiogenic pulmonary oedema and cardiac insufficiency (Nolte et al., 1995; Zaki et al., 1995). Death is usually a consequence of cardiac insufficiency rather than pulmonary oedema, despite autopsy studies indicating that the heart is spared pathologically (Nolte et al., 1995; Zaki et al., 1995).
Ribavirin (1--D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a broad-spectrum nucleoside analogue antiviral drug that is especially noted for its actions against RNA viruses. Whilst clinical trials have shown that ribavirin reduces mortality and morbidity in Chinese patients with HFRS, a trial evaluating its efficacy in HCPS was ended prematurely due to low enrolment, and the trial was never completed (Mertz et al., 2004). Thus, the in vivo activity of ribavirin against SNV or other New World viruses remains unknown. Supportive-care options, especially extracorporeal mechanical oxygenation, a form of cardiopulmonary bypass, have become the mainstay treatment for the most gravely ill patients (Crowley et al., 1998; Lee et al., 1999; Ramos et al., 2001; M. Crowley, personal communication).
Whilst it has been suggested that antiviral therapies might be ineffective because the presumed pathogenic immune cascade is already in progress at the time of diagnosis, there are in fact reasons to suspect that reduction in the viral load or of the ability of the virus to enter cells could reduce the likelihood of fatal outcome. First, virtually all patients examined during the course of acute disease have detectable viral RNA in the peripheral blood mononuclear cells and about 70 % have detectable viral RNA in the plasma or serum (Terajima et al., 1999). Additionally, our studies on the association between viral load in plasma and disease severity indicate that those patients with high viral loads on admission are more likely to have a severe course of disease (Xiao et al., 2006). Furthermore, plasma samples from patients convalescing from HCPS contain variable amounts of antibodies capable of neutralizing SNV in vitro (Bharadwaj et al., 2000; Ye et al., 2004) and there is an inverse relationship between the titre of neutralizing antibodies on hospital admission and disease severity (Bharadwaj et al., 2000). This finding suggests that antiviral molecules (antibodies) are modulating the severity of disease and raises the possibility that convalescent plasma and other compounds that attack the virus directly could have therapeutic potential.
All hantaviruses that are known to be pathogenic and that have been tested have been shown to enter susceptible cells through interaction with the 3 subunit of the 
v3 integrin receptor in vitro (Gavrilovskaya et al., 1998, 1999; Mackow et al., 1999; Raymond et al., 2005). This integrin subunit is expressed on the surface of vascular endothelial cells, which are the major target cells for SNV in both humans and its native reservoir species (Botten et al., 2000a, 2003; Nolte et al., 1995; Zaki et al., 1995). Recently, phage bearing specific, cyclic, nonamer peptides that were selected on the basis of their binding to the 3 integrin protein were found to be capable of as much as 90 % inhibition of infection of susceptible cells by Hantaan virus (HTNV) and SNV (Larson et al., 2005). Collectively, these data indicate that therapies directed at blocking hantavirus–3 interactions may be successful in inhibiting SNV infection in vivo.
The RNA-binding N protein is the major structural protein of the viral capsid. N is highly immunogenic in vivo, but antibodies against it are not neutralizing. However, in some studies, N antigen or genes encoding N antigen have been shown to confer partial protection against hantavirus challenge (Bharadwaj et al., 2002; Kamrud et al., 1999; Schmaljohn et al., 1990).
SNV, like other aetiological agents of haemorrhagic fevers, is a potential agent for biological terrorism due to its high lethality, known aerosol route of transmission and the lack of specific therapies. Hence, there is a particular need for efficient antiviral therapies, with known in vivo activity, that could be administered prophylactically or therapeutically against SNV or other hantaviruses during a bioweapon attack. Currently, a disease model for SNV infection does not exist, despite efforts to identify such a model (J. Botten, K. Mirowsky-Garcia & B. Hjelle, unpublished data; Hooper et al., 2001). In this study, we therefore used the deer mouse infection model to test four different reagents for their ability to inhibit SNV infection and/or replication in vivo: ribavirin, human convalescent anti-SNV immune plasma (HIP), a humanized antibody directed against the human 3 integrin (ReoPro) and a polyclonal rabbit anti-recombinant N antibody. By using the prevention of seroconversion and/or the reduction in viral loads, as assessed by immunohistochemical stains and quantitative RT-PCR (qRT-PCR), as end points for efficacy, we show that human convalescent plasma, ribavirin and ReoPro all display in vivo anti-SNV activity.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We conducted all procedures that utilized infected mice at our outdoor experimental facility, located at the Sevilleta National Wildlife Refuge, Socorro, NM, USA, whilst adhering strictly to the biosafety recommendations of the US Centers for Disease Control and Prevention (Botten et al., 2000b; Mills et al., 1995). Full-body personal protective equipment, full-face hoods and powered air-purifying respirators were worn by animal handlers at all times during experimental procedures involving potential exposure to infectious virus. Virus inoculations were given intramuscularly (i.m.) by administering 50–100 AID50 (animal infectious doses at which 50 % of the animals become infected) to five to ten mice per experimental group, using exclusively animal-passaged SNV strain SN77734, as described previously (Botten et al., 2000a). We administered drugs, serum, plasma or PBS intraperitoneally (i.p.). Mice were euthanized at day 15 post-inoculation (p.i.) by i.p. injection of 250 mg tribromoethanol kg–1, followed by exsanguination via cardiac puncture and cervical dislocation. Mice were necropsied on site, and we collected blood and excised the lung, heart, liver, spleen and kidneys from each individual. A piece of each tissue sample was either placed immediately in liquid nitrogen and later stored at –80 °C, or placed in formalin until it could be processed further.
To address the efficacy of antiviral treatments given concurrently with virus inoculation, we first treated five to ten mice with the drugs at the highest concentration to identify strong inhibitors of SNV in vivo, and then administered the drug at increasingly dilute concentrations to groups of five to eight mice to establish the concentrations at which the drug remained efficacious. We administered drugs or plasma 1 h before virus inoculation on day 1. Different regimes were then followed for each treatment. Ribavirin (MP Biomedicals Inc.) was administered daily at 100, 50, 12.5 or 5 mg kg–1 day–1 for 9 days. ReoPro (Centocor Inc.) was administered every other day (days 1, 3, 5, 7 and 9) at 6 mg kg–1 (Sassoli et al., 2001). HIP (neutralizing-antibody titre 800) obtained from a convalescent patient, polyclonal rabbit anti-recombinant N and negative-control human plasma were administered neat or diluted in a total volume of 200 µl every other day until day 9. HIP contains antibodies to neutralizing epitopes of the virus glycoproteins, as well as antibodies to N antigen. To adjust the dosage of rabbit anti-N antiserum so as to deliver dosages comparable to that administered in the neat human plasma, we titrated the human and rabbit plasma samples by end-point dilution using strip immunoblot assay (SIA) and determined that the rabbit plasma had to be diluted 20-fold (Bharadwaj et al., 2000). Passive-immunization experiments were done by treating mice with HIP at dilutions of 1 : 5, 1 : 20, 1 : 80 and 1 : 320. We weighed all mice at each time of drug administration and on the day that they were sacrificed. Mouse weight was used to adjust the amount of drug before each administration to maintain consistent delivery throughout the procedure and to detect any gain or loss of weight. We inoculated control animals with virus and gave them 100–200 µl PBS at the same times that drugs or plasma were administered to treated animals. These mice were grouped as a final group, which is referred to as ‘untreated control’ in the remainder of this study. Uninfected-control animals were mock-inoculated i.m. with 50 µl PBS.
Ribavirin inhibitory concentration 50 (IC50) and focus assay.
We seeded 48-well plates with Vero E6 cells 1 day before inoculation with SNV at an m.o.i. of 0.01. We then inoculated cells in duplicate wells with SNV diluted in medium containing ribavirin at concentrations of 0, 1, 3, 6, 10, 13, 16, 20, 23, 26, 30, 35, 50 or 100 µg ml–1. We allowed cells to adsorb virus for 1 h at 37 °C before replacing the virus with 500 µl Dulbecco's modified Eagle's medium (DMEM) containing 2.5 % fetal bovine serum (FBS, supplemented with 20 mM HEPES, 10 mM non-essential amino acids solution, 4 mM glutamine, 40 µg penicillin/streptomycin ml–1, 0.5 µg fungizone ml–1 and 50 µg gentamicin ml–1) and varying concentrations of ribavirin. We examined the cells visually for viability and collected supernatants (500 µl) every 2 days, whilst replacing the medium with fresh medium containing ribavirin until day 9. To evaluate the in vitro efficacy of ribavirin treatments, we used focus assays. Forty-eight-well plates were seeded with Vero E6 cells on the day before inoculation with serial 10-fold dilutions (10–2 to 10–6) of the supernatants collected. We allowed the virus to adsorb onto the cells for 1 h at 37 °C and then aspirated the inocula and washed the cell monolayer once with PBS. Each well was then layered with 500 µl viral overlay medium (equal volumes of 2.4 % methylcellulose diluted in sterile water and 2x2.5 % FBS/DMEM). After culturing the cells for 7 days, we fixed them with cold methanol containing 0.5 % H2O2. We labelled the cells with a primary rabbit anti-recombinant N antibody (1 : 1000) followed by a peroxidase-conjugated goat anti-rabbit IgG (1 : 1000) (Jackson ImmunoResearch Laboratories), and developed with a diaminobenzene/metal peroxidase substrate. We counted brown foci on each well under an inverted light microscope and determined the SNV titre present in supernatants of infected cells treated with different concentrations of ribavirin.
SIA.
We examined deer mouse blood samples for antibodies to SNV N antigen by SIA on day 15 p.i. at a 1 : 200 dilution as described previously (Yee et al., 2003). A 1 : 1000 concentration of alkaline phosphatase-conjugated goat anti-Peromyscus leucopus antibody (Kierkegaard & Perry Laboratories) was used to detect deer mouse antibodies against SNV N. Parallel SIAs were conducted by using a goat anti-rabbit or a goat anti-human IgG to rule out the detection of human or rabbit antibodies in the blood of mice that had been treated with human plasma or rabbit serum, respectively (data not shown).
Focus-reduction neutralization test (FRNT) using total mouse blood.
The focus assay was carried out as described above, except that, for the FRNT variant, we pre-treated 100 focus-forming units of SNV in a volume of 200 µl, containing mouse blood at final dilutions of 1 : 20 and 1 : 40 in 2.5 % FBS/DMEM for 1 h. The application of viral overlay medium and all subsequent steps were as described above. Neutralization activity was determined as the ability to reduce the number of foci by 80 % or more compared with inocula that were not treated with serum or blood (Bharadwaj et al., 1999).
Immunohistochemistry (IHC).
We fixed five mouse tissues (heart, lung, kidney, liver and spleen) in formalin for at least 24 h before embedding them in paraffin. We used a polyclonal rabbit anti-N antiserum (1 : 10 000) to detect the presence of N antigen in the paraffin-blocked tissues mounted on glass slides as described previously (Botten et al., 2000a). Specific stain appeared as red, punctate, cytoplasmic granules. To quantify infection in mouse tissues, we used an antigen-expression scoring system on a scale of 0+ to 4+ as described previously (Botten et al., 2003).
Further quantification of IHC staining data, restricted to heart and lung tissues of the untreated control and the ReoPro treatment groups, was performed blindly by a veterinary pathologist. Results were reported as the total number of infected cells per 10 random fields as seen with the x60 magnification objective.
qRT-PCR assay.
We carried out qRT-PCR to quantify viral loads in heart tissues of mice given the different treatments as described previously, using primers in the viral S genome (Botten et al., 2000a). We obtained total-tissue RNA from frozen heart tissues by using an RNeasy mini kit (Qiagen). Total tissue RNA (5 or 10 µl) was subjected to reverse transcription using an S-segment sense primer (coordinate 167), 5'-AGCACATTACAGAGCAGACGGGC-3'. We then amplified each of the cDNAs produced in triplicate by using an ABI Prism 7000 TaqMan machine (Applied Biosystems), with an S-segment sense primer (coordinate 179), 5'-GCAGACGGGCAGCTGTG-3', an antisense primer (coordinate 245), 5'-AGATCAGCCAGTTCCCGCT-3', and the positive-sense fluorescent probe (coordinate 198), 5'-TGCATTGGAGACCAAACTCGGAGAACTT-3', at a concentration of 200 nM each. A standard curve containing dilutions ranging from 10 to 1x107 copies of template was used for each reaction plate (Botten et al., 2000a). Copy numbers obtained were normalized to the mass of RNA that was estimated by reading A260.
Bleeding-time assay and determination of the presence of ReoPro in mouse blood samples.
To determine whether ReoPro was bioactive in the blood of deer mice to which it had been administered and was capable of interacting with the deer mouse integrin on platelets, we determined bleeding times by using a method similar to that described by Hansen & Balthasar (2001). Four mice were given ReoPro i.p. at 6 mg kg–1 on days 1, 3 and 5. In parallel, we administered control PBS treatments to a group of five mice. One hour after the third administration of the drug (day 5), we determined bleeding times. We made a 1 cmx1 mm deep incision in the tails (1 cm from the tip of the tail, at approx. 45° from the dorsal vein) of mice anaesthetized i.m. with 100 mg ketamine kg–1. The tails were immersed in 45 ml normal saline at 37 °C and the time until bleeding stopped was measured. We then collected post-treatment bloods immediately by cardiac exsanguination and euthanized the mice by cervical dislocation. The bloods collected were used to determine the presence of ReoPro in the mouse circulation by using a Western blot-based SIA test (data not shown).
Statistical analysis.
We used the heart viral titres obtained by qRT-PCR to perform a Welch ANOVA test to examine statistical difference between each treatment group compared with the ‘untreated-control’ group. The same test was used to determine whether the values of blinded total IHC scores for the heart tissues of the ReoPro and untreated-control treatment groups were significantly different and to determine significance of the results of the tail-bleeding experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; J. Botten, K. Mirowsky-Garcia & B. Hjelle, unpublished data). Therefore, to assess the effectiveness of each treatment administered, we examined seroconversion (by SIA and FRNT) and determined the presence of viral antigens in different tissues by IHC at day 15 after virus inoculation. In order to determine the infectious virus titre contained in the lung tissues treated with the different drugs or plasma, we started with a TCID50 assay; however, when we carried out this assay with the untreated controls and the human negative plasma-treated group (a group that consistently showed seroconversion and extensive IHC staining in the tissues), virus was detectable in only 60 % of cultures, even after 6 weeks blind passage (data not shown). As a result, we used a qRT-PCR approach to measure viral RNA loads in the heart tissues of these mice.
Ribavirin inhibits SNV infection in vitro, and prevents seroconversion and reduces viral titres in the deer mouse model
Ribavirin's ability to inhibit SNV has not been addressed in either in vitro or in vivo experimental models. To address this, we infected Vero E6 cells with SNV at an m.o.i. of 0.01 whilst exposing the cells to different concentrations of ribavirin. Our results showed that the in vitro SNV IC50 of ribavirin is between 1 and 6 µg ml–1 (Fig. 1). To assess whether ribavirin is also capable of inhibiting SNV in vivo, we infected and treated deer mice concurrently, and carried out titration experiments to determine the concentrations at which ribavirin remained effective. Following experimental designs similar to those of Huggins et al. (1986) and recent human clinical trials (Huggins et al., 1991; Mertz et al., 2004), we treated deer mice with 100, 50, 12.5 or 5 mg ribavirin kg–1 and evaluated its ability to prevent seroconversion and reduce viral loads in mouse tissues.
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). However, ribavirin's ability to prevent seroconversion was reduced considerably when administered at lower concentrations. At 50 mg kg–1, only one of seven mice failed to seroconvert and, at 12.5 and 5 mg kg–1, seroconversion occurred in all subjects (five of five and seven of seven mice, respectively; Table 1). Similarly, we found that most mice treated with 100 mg ribavirin kg–1 lacked neutralizing activity in their blood. Blood samples of mice treated with lower concentrations of ribavirin were able to neutralize SNV in vitro (Table 1).
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). These tissues included heart and lung (Fig. 2a), which have previously been shown to be the major sites of replication during acute and persistent SNV infection (Botten et al., 2000a, 2003), as well as kidney, liver and spleen. We also used qRT-PCR for the SNV S segment to quantify viral loads in heart tissues. In mice treated with 100 mg ribavirin kg–1, we found that three animals (4712, 4716 and 4743) showed no antigen staining in the tissues analysed. Additionally, animal 4715 had reduced levels of staining and mice 4713 and 4714 had viral antigen-staining levels comparable to those of the untreated-control mice (Fig. 2a; Table 1
). qRT-PCR analysis of heart tissues of these mice showed that, in two animals (4712 and 4743), the titre of the S genome was below the limit of detection, but that the other mice had detectable titres of viral RNA (Fig. 3a). At lower treatment doses, ribavirin was unable to inhibit virus replication consistently. IHC analysis revealed that mice treated with 50 mg ribavirin kg–1 had extensive antigen staining in the tissues, with the exception of animal 5225, which had no antigen staining in the tissues (Table 1; Fig. 2a). Most mice treated with 12.5 mg ribavirin kg–1 showed abundant antigen staining, whilst treatment with 5 mg ribavirin kg–1 did not have measurable effects on viral antigen staining (Table 1). A concentration-dependent inhibition of viral load was also seen after qRT-PCR analysis of the heart tissues of these mice. This technique was found to be the most sensitive assay for detecting antiviral effects (Fig. 3a). None of the ribavirin concentrations used in the treatments resulted in evident adverse toxic effects. This was first monitored in a toxicity study group (at 100 mg kg–1) and also throughout the different treatment procedures by examining weight loss and appearance and observing for signs of illness or alterations in grooming behaviour (data not shown).
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). However, when we treated mice at dilutions of 1 : 80 and 1 : 320, seroconversion was prevented in only a subset of the virus-inoculated mice. FRNT using the blood of SNV-challenged mice that received undiluted or 1 : 5-diluted HIP was able to neutralize virus in vitro. It was unclear whether this neutralizing activity was due to residual HIP or had been provoked by the SNV challenge. However, only a subset of mice that received HIP at dilutions of 1 : 20 and 1 : 80 retained neutralizing activity in their serum. In contrast, those mouse sera that retained neutralizing activity in the groups that received 1 : 80 and 1 : 320 dilutions of HIP showed seroconversion to N antigen, suggesting that this neutralization was in response to viral challenge.
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; Fig. 2b). qRT-PCR analysis of these treatment groups also showed that the S genome was undetectable in the heart tissues (Fig. 3b). Mice treated with higher dilutions of HIP (1 : 80 and 1 : 320) showed variable levels of antigen staining in the tissues, with increasing viral staining seen in the 1 : 320 treatment group. This pattern was also observed after qRT-PCR analysis (Table 2; Fig. 3b). Statistical analysis of the heart viral RNA load of the 1 : 80 and 1 : 320 treatment groups showed P values of 0.0535 and 0.0692, respectively (Fig. 3b).
In contrast, when we treated mice with an undiluted-control plasma sample, all mice (eight of eight) seroconverted and showed extensive levels of antigen staining in the tissues (Table 3; Fig. 2c). This was also consistent with the high viral loads observed in their heart samples, which were comparable to those of the untreated controls (Fig. 3c).
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; Table 3). However, qRT-PCR analysis of these heart tissues showed moderate viral loads that differed statistically significantly from those of untreated-control mice (Fig. 3c). Similarly, after blinded evaluation of the total number of cells staining positive for viral N antigen in heart tissues by a veterinary pathologist, a difference (P<0.05) was also observed between mice that received ReoPro and untreated-control mice (Fig. 4). Similar results were obtained for the quantification of lung tissues (data not shown), suggesting a modest inhibitory effect by the anti-integrin antibody compared with ribavirin and HIP.
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). Uninfected mice given three doses of ReoPro at 6 mg kg–1 every other day showed significantly longer bleeding times than PBS-treated control mice (Fig. 4, inset). Furthermore, Western blot analyses using deer mouse serum as the primary antibody and an anti-human Fab conjugate as detection reagent showed that serum samples from all mice treated with ReoPro contained a detectable amount of ReoPro after the third dose (data not shown).
Administration of 200 µl of a 1 : 20 dilution of a polyclonal rabbit anti-recombinant N antibody did not prevent seroconversion in any mouse challenged with SNV; also, neither viral RNA load nor immunostaining pattern was affected by the treatment (Table 3; Figs 2c, 3c).
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DISCUSSION |
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). Nonetheless, the requirement of Biosafety Level 4 laboratories to conduct in vivo studies has greatly hampered research in this area. We have shown herein that an infection model that does not result in clinical disease can nevertheless be used to distinguish, through quantitative and semiquantitative methods, the in vivo efficacies of different antiviral treatments. We used four different approaches aimed at inhibiting SNV infection in an in vivo model at different stages of the virus life cycle. We show that SNV infection can be inhibited in vivo by administering either ribavirin or convalescent anti-SNV HIP concurrently, that this effect is dose-dependent, and that concurrent administration of ReoPro had a subtle inhibitory effect on viral loads in the tissues of SNV-challenged mice. By contrast, anti-N antibodies showed no such inhibition.
Ribavirin has been shown to inhibit virus replication of a variety of viruses effectively (Canonico et al., 1984; Huggins et al., 1986; Johnson, 1993; McKee et al., 1988; Severson et al., 2003). Two efforts made in the USA to evaluate the efficacy of ribavirin in treating HCPS have led to disappointment. One trial was not able to recruit enough subjects and the other lacked a placebo control arm and, as a result, the studies lacked sufficient statistical power to detect a therapeutic effect (Chapman et al., 1999; Mertz et al., 2004). Our in vitro studies carried out with this drug showed a significant inhibitory effect against SNV, with an IC50 of 1–6 µg ml–1 (Fig. 1). This inhibitory activity agrees with that seen against other hantaviruses in vitro (Canonico et al., 1984; Kirsi et al., 1983; Severson et al., 2003), lending support for renewed efforts to evaluate the drug in treatment protocols. One disadvantage of ribavirin treatment is its toxic effect at high concentrations, which has been reported in human trials (Booth et al., 2003; Chapman et al., 1999; McKee et al., 1988) and other animal models (Huggins et al., 1986). In our study, however, we found that this drug had no gross toxic effects on juvenile deer mice despite a strong antiviral effect at higher doses (Table 1; Figs 2a, 3a). The decreased inhibitory activity observed at doses below 100 mg kg–1 indicates that ribavirin inhibits SNV replication in vivo in a dose-dependent manner (Figs 2a, 3a; Table 1).
The effectiveness of passive immunotherapy in treating human arenavirus infections caused by Junín virus has long since been established (Enria et al., 1984; Maiztegui et al., 1979), and positive effects have also been claimed for other animal models (Jahrling & Peters, 1984; Leifer et al., 1970). Encouraging results have been shown in a Seoul virus (SEOV) model and in a lethal animal model of HCPS due to ANDV (Custer et al., 2003; Zhang et al., 1989). These and other data (Bharadwaj et al., 2000) have raised the possibility that passive immunotherapy might be useful in treating HCPS due to SNV infection. Our study showed that passive immunotherapy with convalescent HIP was effective in inhibiting SNV infection in the deer mouse model (Table 2; Figs 2b, 3b). Administration of antibodies in divided doses allowed us to titrate the dose at which HIP remained 100 % efficacious (at a 1 : 20 dilution of an antibody with an FRNT titre of 1 : 800). Collectively, these data indicate that HIP has potent antiviral activities in vivo.
Several previous investigations have demonstrated that passive administration of anti-N monoclonal antibodies or DNA-N vaccination conferred partial protection against challenge by HTNV, SEOV, Puumala virus and SNV, but the mechanism of such protection is not known (Bharadwaj et al., 2002; Kamrud et al., 1999; Lundkvist et al., 1996; Schmaljohn et al., 1990; Yoshimatsu et al., 1993). Our studies show that treatment with anti-N antibodies neither inhibited nor reduced viral infection in vivo (Table 3; Figs 2c, 3c), suggesting that cell-mediated immunity may help to explain the protection seen with hantavirus N vaccines. Additionally, when considered in conjunction with the positive results observed with HIP, our results further support the hypothesis that it is the neutralizing activity in the human plasma aimed at proteins other than N that confers protection against SNV. Nonetheless, further studies will be needed to confirm or refute a role for anti-N antibodies as a potential passive immunotherapeutic for HCPS.
ReoPro, the humanized version of the c7E3 Fab, is licensed (Food and Drug Administration-approved) for use as an anticoagulant due to its antagonism of the platelet GPIIb/IIIa glycoprotein. It also interferes with signalling through the vitronectin receptor, the integrin v3 in endothelial cells (Coller et al., 1995), and c7E3 Fab has been shown to be an effective antagonist of these receptors in vivo in rats (Sassoli et al., 2001). The ability of anti-3 integrin antibodies to block the entry of pathogenic hantaviruses in vitro has long been recognized (Gavrilovskaya et al., 1998). Given the rarity of HCPS worldwide, the demonstration that a licensed pharmaceutical blocks hantavirus infection in vitro raised hopes that it might also be effective in vivo, and therefore motivated our efforts to determine its efficacy in the deer mouse infection model. Our results with ReoPro demonstrated a significant but moderate reduction in viral load as assessed by qRT-PCR and immunostaining studies (Table 3; Figs 3c, 4). The fact that the dosage was adequate to see any antiviral effects was supported by our ability to detect the antibody by Western blot in the blood of treated mice (data not shown) and by its ability to prolong bleeding times (Fig. 4, inset). The prolongation of bleeding times also suggests that ReoPro has at least some ability to engage the deer mouse 3 molecule functionally. Overall, ReoPro demonstrates a slight but measurable antiviral effect in deer mice when administered in therapeutically active doses. These results should be tempered by the absence of any demonstration that v3 integrin is used for virus entry into deer mouse cells and, therefore, the modest response to ReoPro could indicate that SNV uses a different entry molecule in deer mice or that ReoPro does not bind the deer mouse integrin avidly.
The results of this study should help to establish useful methods to monitor the efficacy of antiviral therapies in a hantavirus infection model or any model wherein animals are not rendered moribund by virus challenge. The ability of mice serum samples to neutralize SNV in vitro correlated well with our ability to detect seroconversion (anti-N antibodies) through SIA. Additionally, we found that semiquantitative or total IHC quantification of tissues and the qRT-PCR data obtained were in close agreement with one another, with qRT-PCR being the more sensitive means of detecting low levels of SNV infection, and total IHC quantification being a good technique for corroborating marginal differences. The TCID50 assay, by comparison, was not sensitive enough for use with this model system. Our experimental treatment model should, therefore, serve as a good baseline to assess further the inhibitory activity of these and other therapeutic approaches, as well as addressing treatment against SNV in a post-infection scenario.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 August 2006;
accepted 29 September 2006.
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51 positive cells, as seen at x60 magnification. Arrows highlight representative positive stains.
