Journal of General Virology (2007), 88, 493–505

DOI 10.1099/vir.0.82459-0

Ribavirin, human convalescent plasma and anti-b3 integrin antibody inhibit infection by Sin Nombre virus in the deer mouse model Rafael A. Medina,1 Katy Mirowsky-Garcia,1 Julie Hutt2 and Brian Hjelle1,3 1

Center for Infectious Diseases and Immunity, Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA

Correspondence Brian Hjelle

2

[email protected]

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

Received 11 August 2006 Accepted 29 September 2006

The New World hantavirus Sin Nombre virus (SNV) is an aetiological agent for the often-fatal hantavirus cardiopulmonary syndrome (HCPS). There is no disease model for SNV and specific treatments for HCPS do not exist. By using the deer mouse infectious model, the in vivo inhibitory potential of ribavirin, human anti-SNV immune plasma (HIP), an anti-b3 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 ml 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 ml 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 The genus Hantavirus of the family Bunyaviridae is composed of enveloped, negative-sense RNA viruses. The genomes of hantaviruses are segmented and consist of the small (S), medium (M) and large (L) segments (Jonsson & Schmaljohn, 2001). 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. 0008-2459 G 2007 SGM

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Over 1000 cases of HCPS have been reported, with casefatality 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-b-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 493

R. A. Medina and others

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 b3 subunit of the avb3 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 b3 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–b3 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 494

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 b3 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.

METHODS Animal handling, virus inoculation and drug administration.

All animal protocols used in this study were approved by the Institutional Animal Care and Use Committee of the University of New Mexico (UNM). Outbred 5–6-week-old deer mice (Peromyscus maniculatus rufinus) were bred at the UNM Animal Resource Facility and used at generations F4–8 (Botten et al., 2001). 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 kg21, 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 280 uC, 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 kg21 day21 for 9 days. ReoPro (Centocor Inc.) was administered every other day (days 1, 3, 5, 7 and 9) at 6 mg kg21 (Sassoli et al., 2001). HIP (neutralizing-antibody titre 800) obtained from a convalescent patient, polyclonal rabbit antirecombinant N and negative-control human plasma were administered neat or diluted in a total volume of 200 ml every other day until day 9. Journal of General Virology 88

In vivo inhibition of Sin Nombre hantavirus 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 ml 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 ml 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 mg ml21. We allowed cells to adsorb virus for 1 h at 37 uC before replacing the virus with 500 ml 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 mg penicillin/streptomycin ml21, 0.5 mg fungizone ml21 and 50 mg gentamicin ml21) and varying concentrations of ribavirin. We examined the cells visually for viability and collected supernatants (500 ml) 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 (1022 to 1026) of the supernatants collected. We allowed the virus to adsorb onto the cells for 1 h at 37 uC and then aspirated the inocula and washed the cell monolayer once with PBS. Each well was then layered with 500 ml viral overlay medium (equal volumes of 2.4 % methylcellulose diluted in sterile water and 262.5 % 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 ml, 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 http://vir.sgmjournals.org

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 660 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 ml) was subjected to reverse transcription using an S-segment sense primer (coordinate 167), 59-AGCACATTACAGAGCAGACGGGC39. 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), 59-GCAGACGGGCAGCTGTG-39, an antisense primer (coordinate 245), 59AGATCAGCCAGTTCCCGCT-39, and the positive-sense fluorescent probe (coordinate 198), 59-TGCATTGGAGACCAAACTCGGAGAACTT-39, at a concentration of 200 nM each. A standard curve containing dilutions ranging from 10 to 16107 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 kg21 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 cm61 mm deep incision in the tails (1 cm from the tip of the tail, at approx. 45u from the dorsal vein) of mice anaesthetized i.m. with 100 mg ketamine kg21. The tails were immersed in 45 ml normal saline at 37 uC 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. 495

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RESULTS Treatment regimes and study design Previous experiments in our laboratory have shown that seroconversion to N antigen, detection of neutralizing antibodies and detection of viral antigen in tissues by IHC are seen consistently by 15 days p.i. (Botten et al., 2000a; 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 mg ml21 (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

Fig. 1. Ribavirin SNV in vitro IC50. Number of foci detectable in supernatants at day 7 (original m.o.i. 0.01) is plotted against the concentration of ribavirin used. Results are means of two independent experiments. 496

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 kg21 and evaluated its ability to prevent seroconversion and reduce viral loads in mouse tissues. By using SIA, we found that a treatment dose of 100 mg kg21 prevented seroconversion in all mice (Table 1). However, ribavirin’s ability to prevent seroconversion was reduced considerably when administered at lower concentrations. At 50 mg kg21, only one of seven mice failed to seroconvert and, at 12.5 and 5 mg kg21, 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 kg21 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). To determine whether viral tissue loads were reduced by ribavirin treatment, we used IHC to detect N protein in five different tissues, which were scored by using a semiquantitative visual-scoring system (Botten et al., 2003). 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 kg21, 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). qRTPCR 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 kg21 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 kg21 showed abundant antigen staining, whilst treatment with 5 mg ribavirin kg21 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 kg21) 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). Journal of General Virology 88

In vivo inhibition of Sin Nombre hantavirus

Table 1. Viral N-antigen staining, seroconversion, neutralizing activity of bloods and detection of viral RNA in ribavirin-treated mice Ribavirin treatment

100 mg kg

21

50 mg kg21

12.5 mg kg21

5 mg kg21

Animal

4712 4713 4714 4715 4716 4743 5524 5525 5526 5527 5533 5535 5537 5529 5532 5543 5545 5546 5534 5548 5549 5550 5551 5552 5553

IHC*

SIAD

Heart

Kidney

Liver

Lung

Spleen

0+ 4+ 4+ 3+ 0+ 0+ 4+ 0+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+

0+ 2+ 1+ 0+ 0+

0+ 4+ 0+ 2+ 0+ 0+ 2+ 0+

0+ 4+ 4+ 3+ 0+ 0+ 3+ 0+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 3+ 4+ 4+ 4+ 4+ 3+ 4+ 4+

0+ 3+ 1+ 0+ 0+ 0+

ND ND

0+ 2+ 2+ 2+ 3+ 1+ 2+ 1+ 0+ 2+ 1+ 3+ 2+ 1+ 2+ 0+ 2+ 3+

ND

4+ 4+ 4+ 4+ 3+ 1+ ND

4+ 2+ 4+ 4+ 2+ 2+ 2+ 2+ 4+

ND

0+ 1+ 3+ 2+ 4+ 2+ 1+ 1+ 0+ 2+ 4+ 4+ 2+ 0+ 1+ 0+ ND

3+

2 2 2 2 2 2 + 2 + + + + + + + + + + + + + + + + +

Neutralizing test 1 : 20

1 : 40

No Yes No No No No Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes

No Yes No No No No Yes No Yes Yes Yes Yes No Yes Yes Yes Yes

ND

ND

Yes Yes Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes Yes Yes

qRT-PCRd

2 + + + + 2 + 2 + + + + + + + + + + + + + + + + +

*Antigen scoring: 0+, no positive cells; 1+, one to five positive cells; 2+, six to 25 positive cells; 3+, 26–50 positive cells; 4+, ¢51 positive cells; ND, not done. D+, Detection of anti-N antibodies; 2, no detection of anti-N antibodies. d+, Detection of viral RNA; 2, no detection of viral RNA.

Treatment with SNV convalescent HIP prevents seroconversion and reduces infection and replication of SNV in vivo When we administered 200 ml neat or 1 : 5- or 1 : 20-diluted HIP, we found that seroconversion was prevented in all SNVchallenged mice (Table 2). 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. http://vir.sgmjournals.org

No viral N antigen staining could be detected in mice given dilutions of HIP of 1 : 20 or lower (Table 2; Fig. 2b). qRTPCR 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). 497

L

H

L

(c)

100 mg kg–1

Untreated control

50 mg kg–1

ReoPro 6 mg kg–1

12.5 mg kg–1

Anti-N

H

(b)

H

L

H

L

L Human negative plasma

1 : 20

H

L

1 : 80

1: 320

Journal of General Virology 88

Fig. 2. Ribavirin and HIP inhibit SNV infection, whereas ReoPro and polyclonal anti-N antibody fail to reduce the SNV N antigen stains in deer mouse tissues consistently. Photomicrographs are representative IHC stains for SNV N antigen of deer mouse heart (H) and lung (L) tissues shown at day 15 p.i. after concurrent treatment with: (a) 100, 50 or 12.5 mg ribavirin kg”1; (b) 200 ml aliquots of dilutions of 1 : 20, 1 : 80 or 1 : 320 of HIP; or (c) 6 mg ReoPro kg”1, a 1 : 20 dilution of rabbit anti-recombinant N antibodies, control human negative plasma or PBS, as described in Methods. Positive N-antigen expression in cells is seen as reddish, punctate staining. Scores shown correspond to semiquantitative analysis of SNV N antigen expressed in the tissues. The scale is as follows: 0+, no positive cells; 1+, one to five positive cells; 2+, six to 25 positive cells; 3+, 26–50 positive cells; 4+, ¢51 positive cells, as seen at 660 magnification. Arrows highlight representative positive stains.

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498 H

(a)

In vivo inhibition of Sin Nombre hantavirus

Fig. 3. Concurrent treatment with ribavirin, HIP or ReoPro is capable of inhibiting replication and/or reducing viral loads in the hearts of deer mice infected with SNV. (a) Ribavirin. S-segment titres obtained after concurrent treatment with 100, 50, 12.5 and 5 mg kg”1. (b) Anti-SNV HIP. S-segment titres obtained after concurrent treatment with undiluted or 1 : 5, 1 : 20, 1 : 80 or 1 : 320 dilutions of HIP (FRNT titre 800). (c) Untreated controls and treatments with ReoPro, anti-SNV N antibodies and human negative plasma. Values are reported as the no. copies SNV S segment (mg total heart RNA)”1, determined by qRTPCR at day 15 p.i. Treatment group and animal identification numbers are shown on the x axis. Asterisks indicate groups that were found to have significantly different S-segment titres (when a=0.05) compared with the untreated-control group. P values are also shown for treatment groups given the 1 : 80 and 1 : 320 dilutions of HIP. Arrows indicate low-level-positive heart tissues not seen in the graph scale.

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499

R. A. Medina and others

Table 2. Viral N-antigen staining, seroconversion, neutralizing activity of bloods and detection of viral RNA in convalescent human immune plasma (HIP)-treated mice HIP treatment

Neat

1 : 5 dilution

1 : 20 dilution

1 : 80 dilution

1 : 320 dilution

Animal

5275 5276 5277 5278 5279 5280 5281 5580 5587 5588 5589 5590 5591 5593 5594 5581 5582 5583 5584 5586 5605 5607 5685 5691 5692 5693 5696 5704 5708 5688 5689 5690 5702 5703 5706 5707

IHC*

SIAD

Heart

Kidney

Liver

Lung

Spleen

0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 3+ 4+ 3+ 2+ 4+ 4+ 1+ 4+ 4+ 4+ 4+ 4+

0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 1+ 2+ 0+ 0+ 2+ 1+ 0+ 1+ 4+ 2+ 2+ 1+

0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 4+ 0+ 0+ 4+ 0+ 0+ 2+ 4+ 3+ 2+ 1+

0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 1+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 1+ 4+ 0+ 0+ 4+ 1+ 0+

0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 0+ 1+ 0+ 0+ 2+ 0+ 0+ 1+ 4+ 3+ 4+ 0+

ND

4+ 3+ 4+ 2+

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 + + 2 2 + + + + 2 + +

Neutralizing test 1 : 20

1 : 40

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No Yes Yes Yes No No Yes Yes No No No Yes Yes Yes No Yes Yes

Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes No Yes No No No No Yes Yes No No No Yes Yes Yes No Yes Yes

qRT-PCRd

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 + + + + + + + + + + + + + +

*Antigen scoring: 0+, no positive cells; 1+, one to five positive cells; 2+, six to 25 positive cells; 3+, 26–50 positive cells; 4+, ¢51 positive cells; ND, not done. D+, Detection of anti-N antibodies; 2, no detection of anti-N antibodies. d+, Detection of viral RNA; 2, no detection of viral RNA.

Treatment with ReoPro reduces viral load, whilst polyclonal rabbit anti-N antibody fails to prevent seroconversion or reduce viral loads in the deer mouse model We investigated whether ReoPro or antibodies specific to the SNV N antigen can inhibit SNV infection in vivo when administered concomitantly with a viral challenge. We found that all mice treated with 6 mg ReoPro kg21 500

seroconverted by 15 days p.i. and that viral antigen was present in the tissues at levels comparable to those of untreated controls (Fig. 2c; 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 Journal of General Virology 88

In vivo inhibition of Sin Nombre hantavirus

Table 3. Viral N-antigen staining, seroconversion, neutralizing activity of bloods and detection of viral RNA in ReoPro- or antiN antibody-treated mice and treated- and untreated-control mice Treatment

Untreated controls

ReoPro 6 mg kg21

Anti-N antibody

Human negative plasma

Animal

4741 4867 4869 4870 5293 5294 5528 5544 5547 5597 5598 5705 5710 5738 5739 5741 4866 4868 4875 5683 5684 5687 5289 5290 5295 5296 5298 5273 5274 5283 5286 5291 5695 5697 5743 5749 5752

IHC*

SIAD

Heart

Kidney

Liver

Lung

Spleen

4+ 4+ 4+ 2+ 4+ 4+ 3+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+

4+ 1+ 2+ 0+ 4+ 4+ 1+ 1+ 3+ 2+ 0+ 4+ 4+ 3+ 4+ 4+ 1+ 2+ 0+ 2+ 2+ 1+ 2+ 2+ 2+ 2+ 3+ 3+ 4+ 3+ 4+ 4+ 4+ 2+ 2+ 4+ 4+

4+ 3+ 2+ 0+ 4+ 4+ 1+ 2+ 4+ 0+ 0+ 4+ 4+ 2+ 4+ 4+ 0+ 0+ 0+ 4+

4+ 4+ 4+ 4+ 4+ 4+ 1+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 3+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+

4+ 0+ 2+ 0+ 4+ 4+ 0+ 2+ 0+ 2+ 0+ 4+ 3+ 4+ 4+ 4+ 0+ 0+ 0+

ND

1+ 2+ 1+ 4+ 4+ 2+ 4+ 4+ 4+ 4+ 4+ 4+ 2+ 2+ 4+ 4+

ND

1+ ND

1+ 2+ 4+ 3+ 2+ 4+ 4+ 3+ 4+ 4+ 4+ 3+ 3+ 2+ 4+

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Neutralizing test 1 : 20

1 : 40

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes

ND

ND

Yes Yes Yes Yes

Yes Yes Yes Yes

ND

ND

Yes

Yes

ND

ND

Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes

qRT-PCRd

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

*Antigen scoring: 0+, no positive cells; 1+, one to five positive cells; 2+, six to 25 positive cells; 3+, 26–50 positive cells; 4+, ¢51 positive cells; ND, not done. D+, Detection of anti-N antibodies; 2, no detection of anti-N antibodies. d+, Detection of viral RNA; 2, no detection of viral RNA.

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. To verify that the deer mice had received a dose of ReoPro sufficient to induce a physiological response, we examined http://vir.sgmjournals.org

the effects of ReoPro on bleeding time, as has been evaluated previously in rats (Coller et al., 1995). Uninfected mice given three doses of ReoPro at 6 mg kg21 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 501

R. A. Medina and others

Fig. 4. Total IHC quantification of heart tissues of infected deer mice after concurrent treatment with 6 mg ReoPro kg”1, scored blindly by a veterinary pathologist. Graph shows the total number of infected cells found in 10 optical fields under 660 magnification. Treatment group and animal identification numbers are shown on the x axis. The inset shows that administration of ReoPro prolongs bleeding of deer mice. Bleeding times of a 1 mm61 cm incision in the tail of deer mice are reported in s, with error bars (grey) representing the SD of the mean (black middle line). Control and treated groups (0 and 6 mg ReoPro kg”1, three doses) consisted of four to five deer mice per group. Asterisks indicate scores or times that differed significantly (when a=0.05) from the untreated-control group.

a detectable amount of ReoPro after the third dose (data not shown). Administration of 200 ml 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).

DISCUSSION SNV causes a severe and often fatal disease for which there is currently no preventive or therapeutic treatment. The need for an expanded array of therapeutic options against haemorrhagic fever viruses has been highlighted previously (Borio et al., 2002). 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 SNVchallenged 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., 502

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 mg ml21 (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 kg21 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 Junı´n 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 Journal of General Virology 88

In vivo inhibition of Sin Nombre hantavirus

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 avb3 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-b3 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 b3 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 avb3 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 http://vir.sgmjournals.org

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.

ACKNOWLEDGEMENTS We thank A. Galvez, R. Hamel and R. Ricci for excellent technical assistance and Dr Diane Goade for kindly providing the human convalescent immune plasma. We are also grateful to the US Fish and Wildlife Service (Sevilleta National Wildlife Refuge, Socorro, NM, USA) for its valuable cooperation in this study. This work was supported by United States Public Health Service grants U01 AI 56618, U01 AI054779, R21 AI053400, R21 AI53334 and U19 AI45452. R. A. M. was supported by Fogarty Actions for Building Capacity award D43 TW01133. Images in this paper were generated in the UNM Cancer Center Fluorescence Microscopy Facility, which received support from NCRR 1 S10 RR14668, NSF MCB9982161, NCRR P20 RR11830, NCI R24 CA88339, NCRR S10 RR19287, NCRR S10 RR016918, the University of New Mexico Health Sciences Center and the University of New Mexico Cancer Center.

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