Soluble coxsackie- and adenovirus receptor (sCAR-Fc); a highly efficient compound against laboratory and clinical strains of coxsackie-B-virus

Antiviral Research 136 (2016) 1e8

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Soluble coxsackie- and adenovirus receptor (sCAR-Fc); a highly efficient compound against laboratory and clinical strains of coxsackie-B-virus Sandra Pinkert a, *, Babette Dieringer a, Sabine Diedrich b, Heinz Zeichhardt c, Jens Kurreck a, Henry Fechner a a b c

€t Berlin, Berlin, Germany Department of Applied Biochemistry, Institute of Biotechnology, Technische Universita National Reference Centre for Poliomyelitis and Enteroviruses, Robert Koch Institute, Berlin, Germany €tssicherung in der Virusdiagnostik, Berlin, Germany IQVD e Institut für Qualita

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 July 2016 Received in revised form 6 October 2016 Accepted 19 October 2016 Available online 20 October 2016

Coxsackie-B-viruses (CVB) cause a wide variety of diseases, ranging from mild syndromes to lifethreatening conditions such as pancreatitis, myocarditis, meningitis and encephalitis. Especially newborns and young infants develop severe diseases and long-term sequelae may occur among survivors. Due to lack of specific antiviral therapy the current treatment of CVB infection is limited to symptomatic treatment. Here we analyzed the antiviral activity of a soluble receptor fusion protein, containing the extracellular part of the coxsackievirus and adenovirus receptor (CAR) fused to the constant domain of the human IgG - sCAR-Fc - against laboratory and clinical CVB strains. We found a high overall antiviral activity of sCAR-Fc against various prototypic laboratory strains of CVB, with an inhibition of viral replication up to 3 orders of magnitude (99.9%) at a concentration of 2.5 mg/ml. These include isolates that are not dependent on CAR for infection and isolates that are resistant against pleconaril, the currently most promising anti-CVB therapeutic. A complete inhibition was observed using higher concentration of sCAR-Fc. Further analysis of 23 clinical CVB isolates revealed overall high antiviral efficiency (up to 99.99%) of sCAR-Fc. In accordance with previous data, our results confirm the strong antiviral activity of sCAR-Fc against laboratory CVB strains and demonstrate for the first time that sCAR-Fc is also highly efficient at neutralizing clinical CVB isolates. Importantly, during the sCAR-Fc inhibition experiments, no naturally occurring resistant mutants were observed. © 2016 Elsevier B.V. All rights reserved.

Keywords: Coxsackievirus Soluble receptor Antiviral therapy Clinical isolates

1. Introduction Coxsackie-B-viruses (CVB) belong to the genus enterovirus of the family of picornaviruses and cause a wide range of diseases. The clinical manifestations vary widely, from asymptomatic infection to mild respiratory syndromes, herpangina and hand, foot, and mouth syndrome to more severe, sometimes life-threatening, conditions

Abbreviations: CVB, coxsackie-B-virus; CAR, coxsackievirus- and adenovirus receptor; sCAR-Fc, fusion protein of the extracellular domain of CAR and the carboxyl terminus of human IgG1 Fc domain. * Corresponding author. Department of Applied Biochemistry, Institute of Biotechnology, Technische Universit€ at Berlin, Gustav-Meyer Allee 25, 13355, Berlin, Germany. E-mail address: [email protected] (S. Pinkert). http://dx.doi.org/10.1016/j.antiviral.2016.10.010 0166-3542/© 2016 Elsevier B.V. All rights reserved.

including myocarditis, pancreatitis, meningitis and encephalitis (Melnick, 1996). Furthermore, acute CVB infections can transmit into chronic stage and induce chronic myocarditis and dilated cardiomyopathy and they have also been linked to the develop€ ty et al., 1998; Kandolf et al., 1993). ment of type 1 diabetes (Hyo Currently, there is no approved antiviral therapy available to treat or prevent CVB infections. An assumed antiviral or immunomodulatory potential of immune serum globulin remains unproven and the mainstay of treatment for severe disease is supportive care (Abzug, 2004; Abzug et al., 2015). Many molecules that block CVB replication in vitro, attacking different points of the viral life-cycle, have been reported in the past few decades but none of them received final market approval because of adverse side effects or unsatisfactory antiviral activity (Abzug et al., 2015; Desmond et al., 2006; Fechner et al., 2011; Thibaut et al., 2012; Webster, 2005). One

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of the most promising and extensively tested low molecular weight drugs is pleconaril, which binds to the canyon pocket of CVB3 thereby inhibiting the binding of the virus to the cellular receptor CAR and subsequent uncoating of viral RNA (Abzug et al., 2015; Desmond et al., 2006; Pevear et al., 1999; Schmidtke et al., 2005; Webster, 2005). Several clinical studies analyzed the antienterovirus activity of pleconaril but it has not been approved by the Food and Drug Administration as antiviral therapy, because of safety issues, unsatisfactory efficiency and rapid emergence of resistant mutants (Desmond et al., 2006; Pevear et al., 1999; Schmidtke et al., 2009, 2005; Thibaut et al., 2012). The usage of soluble receptors as a decoy has been found to efficiently inhibit infection of a broad range of viruses such as measles virus, adenoviruses, HIV and several members of the picornavirus family (Christiansen et al., 2000; Gardner et al., 2015; €ger et al., 2015). Competitive inhibition, as Pinkert et al., 2009; Ro well as steric problems during the entry or uncoating steps, seem to be the main mechanisms responsible for their antiviral activity. The soluble receptors bind to the virus before the virus interacts with the cellular receptor and thus prevent binding of the virus and subsequent uptake into the target cells. The use of soluble receptors is particularly efficient in the case of picornaviruses due to a specific structural feature. In analogy to the physiological mechanism occurring during the natural virus binding to the cell receptor, it has been shown that the exposure to a soluble receptor can induce irreversible conformational changes of the virus capsid. The resulting altered (A)-particles are characterized by loss of their infectivity and were confirmed for poliovirus (PV), rhinovirus (HRV), hepatitis A virus, coxsackieviruses and several other enteroviruses (Goodfellow et al., 2005; Greve et al., 1991; Milstone et al., 2005a; Silberstein et al., 2003; Yamayoshi et al., 2014). The six serotypes of CVB infect cells via interaction with the coxsackievirus- and adenovirus receptor (CAR). CVB1, CVB3, and CVB5 can also use the decay accelerating receptor (DAF) for attachment to the cell surface (Bergelson et al., 1997; Shafren et al., 1995). The antiviral activity of a soluble receptor fusion protein, containing the extracellular part of CAR fused to the constant domain of the human IgG - sCAR-Fc - was shown to be highly efficient against CVB3 infections in vitro and in vivo (Goodfellow et al., 2005; Lim et al., 2006; Pinkert et al., 2009; Stein et al., 2015; Yanagawa et al., 2004). Treatment with recombinant sCARFc via intravenous (i.v.) application three days before or concurrent with CVB3 infection leads to a complete inhibition of cardiac CVB3 infection of the heart and pancreas, but a therapeutic application remained unsuccessful (Yanagawa et al., 2004). In another study, sCAR-Fc expressed from an intramuscular injected plasmid three days before infection protected mice from a lethal CVB3 dose in a three weeks long term investigation (Lim et al., 2006). In this regard, we have shown that sCAR-Fc, delivered in vivo after systemic adenovector application, completely blocked CVB3 infection in mice in a prophylactic manner, and even concomitant and postinfection employment strongly reduced cardiac infection, myocardial injury and inflammation and improved the resulting impaired cardiac function (Pinkert et al., 2009; Stein et al., 2015). We have also shown that sCAR-Fc was able to cure a cardiac cell line persistently infected with CVB3 (Pinkert et al., 2011). In the present study we analyzed the inhibitory efficiency of sCAR-Fc against different laboratory CVB strains and CVB isolates from patients with neurological and non-neurological symptoms. We found that sCAR-Fc is highly effective against a broad spectrum of laboratory CVB strains, including virus isolates resistant to pleconaril and CVB3 strains which can infect cells via CARindependent uptake mechanisms. We further show that sCAR-Fc is highly efficient against various clinical CVB isolates, without observed naturally resistant mutants.

2. Material and methods 2.1. Cells and viruses The human cervix carcinoma cell line HeLa was cultured in Dulbeco's modified Eagle's medium (DMEM e Gibco®, Life Technologies, Grand Island, NY, USA) supplemented with 5% fetal calf serum (FCS), 1% penicillin/streptomycin and 1% nonessential amino acids (NEAA e Gibco®), 2% HEPES buffer (Gibco®). The CAR- and DAF-negative chinese hamster ovary cell line (CHO-K1) (Pinkert et al., 2016; Schmidtke et al., 2000) and the human embryonic kidney cell line HEK293T were cultured in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin. Cells were grown at 37
C in a humidified atmosphere. CVB3 (Nancy strain; VR-30), CVB1 (VR-28) and CVB6 (VR-155) were obtained from ATCC and propagated in HeLa cells. CVB2, CVB4, CVB5 was a kind gift from Karin Klingel (Institut für Patho€tsklinikum Tübingen). Virus expansion, virus logie, Universita infection and plaque assays were performed on HeLa cells. CVB3PD variant was derived by serial passages of CVB P on HuFi cells and propagated in CHO (CVB3-PDCHO) or HuFi (CVB3-PDHuFi) cells (Schmidtke et al., 2000). The myocarditics variant CVB3 31-1-93 was isolated from heart tissue after four heart passages of CVB3-PD in outbreed NMRI mice (Merkle et al., 1999). The CVB3 pleconaril resistant isolate 97-927R3 was previously established by growth of the clinical isolate CVB3 97927 in presence of pleconaril and three rounds of plaque purification in the presence of a high pleconaril dose (Schmidtke et al., 2009). CVB3-HL1 was obtained from the cell culture supernatant of the persistently infected cardiac cell line HL1CVB3 (Pinkert et al., 2011). The strain CVB3 H3 was generated by transfection of the cDNA containing plasmid pBK-CMV-H3 into HEK293T cells using PEI (polyethylenimine) Max (Polyciences, Warrington, PA, USA). Completely lysed cells were harvested 48 h post transfection (p.t.) and the virus was stored in aliquots after three freeze/thaw cycles and removal of the cell debris by centrifugation for 20 min at 2000  g and 4
C. The clinical isolates of CVB3 were obtained from patients suffering from meningitis or respiratory diseases and passaged one time in HEp-2 cells. Clinical isolates of the CVB serotypes 1, 2, 4 and 5 were isolated from patients with neurological symptoms (meningitis or encephalitis) and passaged one time in HEp-2 or the rhabdomyosarcoma RD cells. Viral titers were determined on HeLa cell monolayer by plaque assay. Aliquots were stored at 80
C. 2.2. Production of sCAR-Fc To produce the soluble fusion protein of the extracellular domain of human CAR and the carboxyl terminus of the human IgG-Fc domain (sCAR-Fc), HEK293T cells at a density of 90% were transfected with the previously described sCAR-Fc-expressing €ger et al., 2015) using PEI Max. Meplasmid pscAAVsCAR-Fc (Ro dium was replaced 16 h p.t. with fresh medium containing 1% FCS and supernatant containing sCAR-Fc was harvested after additional 3 days. The concentration of sCAR-Fc was measured by ELISA detecting human IgG-Fc (Bethyl Laboratories, Montgomery, TX, USA) and sCAR-Fc containing supernatants were stored at 20
C until use. 2.3. Virus infection and plaque assay Viruses were pre-incubated in cell culture supernatant containing sCAR-Fc at the indicated concentration or, as the negative control, in medium of mock-transfected cells for 30 min at 4
C or 37
C. After removing the culture medium, HeLa or CHO-K1 cells were inoculated with the pre-incubated virus suspension (moi 0.01,

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0.1, 0.5 or moi 1 as indicated in the respective figure legends) for 30 min at 37
C. Thus, the sCAR-Fc concentrations stayed equal during the pre-incubation and inoculation step. After incubation, the virus-containing medium was removed and cells were washed twice with PBS to remove unbound virus. Thereafter, fresh medium was added to the cells and, after additional incubation for 8 h (HeLa cells) or 24 h (CHO-K1 cells) at 37
C, the cells were frozen. After three freeze/thaw cycles, samples were centrifuged to remove the cell debris and infectious virus particles were quantified using plaque assay as described previously (Pinkert et al., 2011). 2.4. Virus neutralization assay Confluent HeLa cells were incubated for 30 min at 37
C with CVB pre-incubated as described above and directly overlaid with agar-containing Eagle's minimal essential medium (MEM). Two to three days later, cells were stained with 0.025% neutral red/PBS and plaque forming units (pfu) were counted 3 h after staining. The incubation for about two/three days ensured that the viruses efficiently replicated. 2.5. Statistical analysis For statistical analysis, Prism software 5.0 (Graph-Pad, San Diego, CA) was used. To compare two groups, Student t-test or the t-test with Welch's correction were performed as indicated in the legends. Asterisks are used in the table indicate statistically significant differences (*, P < 0.05).

Fig. 1. Antiviral activity of sCAR-Fc in a dose-dependent manner. Different amounts of CVB3 were incubated with an increasing concentration of sCAR-Fc (0.5, 1, 2 and 5 mg/ ml) for 30 min at 4
C. HeLa cells were then incubated with the sCAR-Fc/CVB3 samples for 30 min at 37
C. After removing the virus inoculates, cells were incubated for additional 8 h at 37
C and viral replication was determined by standard plaque assay. The results represent means ± SD of three independent experiments each with two parallels.

3. Results 3.1. Dose dependency of the antiviral activity of sCAR-Fc Previous experiments have shown that sCAR-Fc completely inhibited CVB3 replication at high concentration (Pinkert et al., 2009). Complete inhibition is not an effective approach to quantitatively compare the ability of sCAR-Fc to inhibit different CVB strains and to detect differences in inhibition between CVB strains. Thus, we first determined the concentration of sCAR-Fc at which viral inhibition is highly efficient but not complete. For this investigation we used the prototypic CVB3 Nancy strain, a well characterized laboratory isolate. Cell culture supernatants containing sCAR-Fc at different concentrations were pre-incubated with CVB3 and analyzed for their virus inhibition activity. As shown in Fig. 1, the viral replication of CVB3 was inhibited in a distinctly dose-dependent manner. Inhibition ranged from 79% at the lowest concentration of 0.5 mg/ml sCAR-Fc to 99.9% at a concentration of 2.5 mg/ml sCAR-Fc. A complete inhibition of viral replication was observed at a sCAR-Fc concentration of 5 mg/ml. Interestingly, no differences in inhibition efficiency were observed for an increased virus load; for example, inhibition efficiency at 2.5 mg/ml sCAR-Fc and CVB3 moi 0.01 was about 99.9%, similar to 99.95% at an CVB3 dose of moi 1. Based on these findings, we used a concentration of 2.5 mg/ml sCAR-Fc in the following experiments. 3.2. Susceptibility of different CVB-serotypes to sCAR-Fc In a long-time surveillance study of neonatal enterovirus infections, five of the six CVB serotypes (CVB1 e CVB5) were among the ten most common enteroviruses detected in neonates, accounting for about 35% of all cases (Khetsuriani et al., 2006). Therefore, we analyzed the direct virus neutralization and the replication inhibition induced by sCAR-Fc for all six CVB serotypes. The results revealed a high neutralization capability of sCAR-Fc for all serotypes (Fig. 2A). For this experiment, 5000 pfu of each virus

serotypes were incubated with sCAR-Fc and neutralization efficiencies were quantified as described in the material and methods section. Complete inhibition was observed for CVB2, CVB4 and CVB6, whereas residual plaques were counted for CVB1 (5 ± 3.55 pfu; 99.9% neutralization efficiency) CVB3 (1.5 ± 0.7 pfu; 99.97% neutralization efficiency) and CVB5 (58.5 ± 29.63 pfu; 98.8% neutralization efficiency). Consistently, the inhibition of viral replication was highly efficient, with a complete prevention of new emerging viruses for CVB2, CVB4 and CVB6 whereas CVB1, CVB3 and CVB5 showed virus replication but with an overall decrease of at least 99% by sCAR-Fc (Fig. 2B). In conclusion, our data demonstrate that sCAR-Fc is highly efficient against all six serotypes of CVB. 3.3. Susceptibility of CAR-dependent and CAR-independent CVB3 laboratory strains to sCAR-Fc During the last few decades, a broad panel of laboratory CVB3 variants based on the Nancy strain was established to investigate virus receptor interaction, host cell tropism and pathogenicity. Analyses of these virus variants demonstrate that phenotypic host range and cell-type specificity correlate with amino acid polymorphisms of the capsid region (Schmidtke et al., 2000). Alterations in virus receptor affinity are mainly dependent on amino acid changes within the canyon structure of the viral capsid, which also seems to be a factor influencing the interaction between the virus and the soluble receptor and subsequently antiviral activity. Therefore, we analyzed whether the antiviral efficiency of sCAR-Fc varied in a panel of eight CVB3 laboratory variants possessing a wide range of different receptor affinities. Five variants are dependent on CAR for infection (CVB3, CVB3-P, CVB3 31-1-93, CVB3-HA, CVB3-H3 (Schmidtke et al., 2000; Zautner et al., 2006)) and three variants are able to infect cells independently from CAR. These included CVB3-PD derived in CHO (CVB3-PDCHO) and HuFi

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Fig. 2. Antiviral activity of sCAR-Fc against different CVB serotypes. (A) sCAR-Fc neutralization assay; 5000 pfu of each CVB serotype were incubated with sCAR-Fc (2.5 mg/ml) for 30 min at 4
C. After pre-incubation, HeLa cells were inoculated with virus/sCAR-Fc samples for an additional 30 min at 37
C and directly overlaid with agar. Remaining plaques were counted after 3 days. (B) CVB serotypes 1 to 6 (moi 0.01) were incubated with sCAR-Fc (2.5 mg/ml) containing medium (þ) or control medium () for 30 min at 4
C and then added to HeLa cells for additional 30 min at 37
C. Cells were incubated for 8 h and viral replication was determined by plaque assay. Values are given as means ± SD of three independent experiments each with two parallels.

cells (CVB3-PDHuFi), which are able to infect cells via heparan sulfate in the absence of CAR (Zautner et al., 2006), and CVB3-HL1 isolated from a persistently infected cardiac cell line that infect cells by an unknown entry mechanism (Pinkert et al., 2011). At first, we evaluated the reduction of viral replication after pre-incubation of the virus with sCAR-Fc before infection. In general, all CVB3 laboratory strains were efficiently inhibited by sCAR-Fc, regardless of their receptor specificity. The inhibition of the CVB3 variants that are strictly dependent on CAR (Fig. 3A) reached an efficiency of well over two orders of magnitude; ranging from 99.84% (CVB3-H3) to complete inhibition for CVB3-P. The CAR-independent CVB3 variants differed from each other in their susceptibility to sCAR-Fc (Fig. 3B). Although CVB3-HL1 replication decreased in the same range like the previous analyzed variants (more than three orders of magnitude), the CVB3-PD variant derived from CHO-K1 or HuFi cells displayed a diminished susceptibility to sCAR-Fc, with an inhibition value of only about 94%. In CAR expressing cells, such as HeLa cells, CVB3-PD and CVB3HL1 infect the cells mainly via interaction with CAR (Pinkert et al., 2011). To analyze whether the absence of cellular CAR, and thereby the interaction with the alternative receptor, has an influence on sCAR-Fc antiviral activity, we repeated the inhibition experiments with CVB3-PD in the CAR- and DAF-negative CHO-K1 cell line. The results show that the viral replication was strongly inhibited by sCAR-Fc (Fig. 3C). Thus, the antiviral efficiency of sCAR-Fc against CVB3-PDCHO in CHO-K1 cells is about 92% compared to 94% inhibition efficiency when infection experiments were performed in HeLa (Fig. 3B) cells. This demonstrates that the CVB3-PD variant, although not strictly dependent on CAR, remains susceptible to sCAR-Fc. In general, the formation of A-particles by cellular receptors or soluble receptor proteins takes place at 37
C (Milstone et al., 2005b). In order to figure out whether the incubation temperature has an influence on the reduced inhibition of CVB3-PD by sCAR-Fc, we increased the pre-incubation temperature from 4
C to 37
C for CVB3-PD and CVB3 as control. The increase in the incubation temperature led to an increased antiviral efficiency in both strains but, interestingly, with a higher impact on the CVB3-PD variant (Fig. 3D). In detail, for CVB3 the higher temperature leads to a 6.3-fold improved inhibition of virus replication by sCAR-Fc compared to a 322-fold higher inhibition of CVB3-PDCHO. This

indicates that the temperature is a significant factor influencing coxsackievirus neutralization by sCAR-Fc. In conclusion sCAR-Fc is able to inhibit a broad panel of CVB3 laboratory variants with a similar and high efficiency, independent of their receptor specificity. 3.4. Inhibition of clinical isolates from various CVB-serotypes by sCAR-Fc As mentioned before, CVB infections lead to a wide range of clinical manifestations, with often fatal outcomes in neonates and young children. Especially aseptic meningitis and encephalitis are commonly associated with neonatal infections and no approved anti-viral therapy is currently available. Thus, we decided to evaluate the potential applicability of sCAR-Fc as anti-viral therapy, especially for CVB-induced neurological infections and quantified the inhibition efficiency of sCAR-Fc against 23 clinical isolates of all clinical relevant CVB serotypes. All isolates were associated with clinical manifestation, 8 of them (all CVB3) with non-neurological symptoms and 15 (3 of each serotype but CVB6) were isolated from patients with meningitis or encephalitis. Each isolate was tested in plaque neutralization assays for its sensitivity to sCAR-Fc. In general, sCAR-Fc exhibited potent antiviral activity against all clinical isolates ranging from 99% to more than 99,997% (2 - >4 orders of magnitude) (Table 1) and no isolates resistant against sCAR-Fc were observed. Thereby, the inhibition efficiency was comparable with the antiviral activity of sCAR-Fc against the prototypic CVB strains (Fig. 2) and the CVB3 derived laboratory variants (Fig. 3). As shown for the laboratory strains CVB3 and CVB3-PD, the antiviral efficiency of sCAR-Fc increased after the incubation temperature was raised from 4
C to 37
C for almost all clinical isolates. Interestingly, after raising the incubation temperature, the clinical isolates based on the CVB3 serotype showed a higher increase in neutralization compared to the other serotypes. For example, sCARFc neutralized 94.2% (1.24 orders of magnitude) of the clinical isolate CVB3 10e414 when pre-incubation was proceeded under 4
C. This inhibition increased 580-fold to 99.99% (4 orders of magnitude) when sCAR-Fc incubation was performed at 37
C. In contrast, the CVB5 isolate 14e697 was neutralized about 99.98% (3.77 orders of magnitude) at 4
C and after incubation at 37
C the

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Fig. 3. Antiviral activity of sCAR-Fc against different laboratory CAR-dependent and -independent CVB3 variants. Different laboratory CVB3-Nancy (moi 0.01) variants that infect cells dependent on CAR (A) or independent of CAR (B) were incubated with sCAR-Fc (2.5 mg/ml,þ) or control medium (¡) for 30 min at 4
C and then added to HeLa cells for additional 30 min at 37
C. After incubation, HeLa cells were cultured for additional 8 h at 37
C and virus replication was quantified by plaque assay. (C) The CAR-independent variant CVB3-PDCHO (moi 0.1 and moi 1) was pre-incubated with or without sCAR-Fc (2.5 mg/ml) for 30 min at 4
C and than added to the CAR-negative cell line CHO-K1 for an additional 30 min at 37
C. Quantification of viral replication was carried out 24 h after infection by detecting infectious particles using plaque assay. (D) Differences in virus inhibition by sCAR-Fc are dependent on the pre-incubation temperature. CVB3 and the CAR independent variant CVB3-PDCHO (moi 0.5) were pre-incubated with sCAR-Fc for 4
C or 37
C and cells were subsequently infected as described. The results represent means ± SD of at least two independent experiments each with two parallels.

neutralization efficiency of sCAR-Fc increased only 6.7-fold to 99.997% (4.64 orders of magnitude). Otherwise, there were no differences in the virus neutralization efficiency of sCAR-Fc regarding different clinical symptoms (neurological symptoms vs. non-neurological symptoms) caused by the isolates. We also did not observe significant differences in the inhibition efficiency associated with varying serotypes as described for the prototypic viruses (Fig. 2). In conclusion, sCAR-Fc showed an overall highly efficient antiviral activity against various clinical CVB isolated from patients with neurological and non-neurological symptoms.

4. Discussion Soluble receptors used as virus decoys were analyzed regarding their antiviral efficiency and safety in vitro and in vivo for a wide range of virus infections (Lim et al., 2006; Pinkert et al., 2009; Stein et al., 2015; Yanagawa et al., 2003). During the last decade, we and other groups have extensively studied the antiviral activity of the soluble receptor fusion protein sCAR-Fc against CVB3 infections. These studies showed that sCAR-Fc strongly inhibits CVB3 infection in vitro and completely prevents or significantly decreases pathological alterations induced by CVB3 in mice (Lim et al., 2006;

Pinkert et al., 2011, 2009; Stein et al., 2015; Werk et al., 2009; Yanagawa et al., 2004). Other than these studies, which were focused on the therapeutic efficiency in general, as a proof-ofconcept using the well-established laboratory strain CVB3 Nancy, here, we investigated the antiviral activity of sCAR-Fc against a broad range of laboratory CVB3 strains and investigated for the first time the antiviral efficiency of sCAR-Fc against clinical CVB isolates from patients. The most important finding of our study is that sCAR-Fc is not only efficient against laboratory CVB strains but also very effective against clinical CVB3 isolates that currently circulate among the patient population. In fact, all of the 23 clinical CVB isolates investigated in this study were efficiently inhibited by sCAR-Fc. Moreover, no clinical isolate as well as none of the investigated laboratory-adapted strains were found to be resistant to treatment with sCAR-Fc. This also includes isolates, which are resistant to pleconaril representing the most effective antiviral low molecular compound that has been clinically tested against CVB infections and isolates which can infect cells via CAR-independent mechanisms such as PD (Pevear et al., 1999; Zautner et al., 2006). These results are of specific importance for possible clinical applications of sCAR-Fc, as an essential requirement for use as an antiviral compound for the treatment of patients is its ability to neutralize

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S. Pinkert et al. / Antiviral Research 136 (2016) 1e8 Table 1 Summary of antiviral activity of sCAR-Fc against clinical CVB isolates in percent and [orders of magnitude]. The results are given as means of at least three independent experiments each in duplicates. 4
C Non-neurological symptoms CVB3

Neurological symptoms CVB1

CVB2

CVB3

CVB4

07-59 10-242 10-413 06-191 07-320 10-414 10-1315 97-927R3

a

12-425 12-506 08-409 12-198 13-457 14-1158 05-348 02-180 01-1072 12-87 13-686 14-120

CVB3 Nancy

90.43 91.67 92.82 93.76 92.88 94.20 88.90 99.45

37
C [1.01log] [1.07log] [1.14log] [1.2log] [1.15log] [1.24log] [0.95log] [2.26log]

99.79 [2.68log] 99.96 [3.42log] 99.95 [3.37log] 99.89 [2.99log] 99.98 [3.71log] 99.97 [3.69log] 89.90 [0.99log] 94.03 [1.22log] 97.30 [1.56log] 99.993 [4.19log] 99.98 [3.97log] 99.995 [4.3log] 99.95 [3.30log]

99.78 99.59 99.98 99.91 99.88 99.99 99.95 99.99

[2.66log]* [2.39log]* [3.69log]* [3.05log]* [2.92log]* [4log]* [3.30log]* [4log]*

99.77 [2.64log]ns 99.995 [4.35log]* 99.99 [4.19log]* 99.97 [3.53log]* 99.98 [3.89log]ns 99.99 [4.08log]* 99.12 [2.05log]* 99.90 [3log]* 99.97 [3.52log]* 99.994 [4.26log]ns 99.994 [4.25log]* 99.996 [4.38log]ns 99.995 [4.30log]*

*Significant increase in inhibition efficiency after raise of the pre-incubation temperature from 4
C to 37
C (Welch's t-test). ns Not significant increase in inhibition efficiency after raise of the pre-incubation temperature from 4
C to 37
C (Welch's t-test). a CVB3 pleconaril resistant isolate, previously established by growth of the clinical isolate CVB3 97927 in presence of pleconaril and three rounds of plaque purification (Schmidtke et al., 2009).

viruses that actually circulate in the patient population. For quantitative comparison of the antiviral activity of sCAR-Fc, a dose of 2.5 mg/ml sCAR-Fc causing high, but not complete, inhibition of the laboratory prototype CVB3 Nancy was used. At higher concentration, sCAR-Fc was able to completely neutralize all laboratory strains and clinical CVB isolates in our experimental setup. A further increase of the neutralization activity of sCAR-Fc was observed after increasing the pre-incubation temperature from 4
C to 37
C. The latter indicate that the inhibition of CVB by sCAR-Fc is not only due to competitive inhibition but also to the formation of A-particles that appears mainly at 37
C (Goodfellow et al., 2005; Milstone et al., 2005b). An interesting observation was the difference in inhibition between the various prototypic CVB serotypes (Fig. 2) that are able to use DAF as a co-receptor for virus attachment (CVB1, CVB3, CVB5) and those that only use CAR for attachment and internalization (CVB2, CVB4, CVB6). At a dose of 2.5 mg/ml, sCAR-Fc strongly inhibited CVB1, 3 and 5, whereas the serotypes 2, 4 and 6 were completely neutralized. DAF serves as a further attachment receptor that escorts the virus to CAR which is difficult to access. We and others observed that additional expression of DAF in CARtransfected CHO-K1 cells significantly increased CVB3 infection compared to CHO-K1 cells which solely expressed CAR (Coyne and Bergelson, 2006; Pinkert et al., 2016). Thus, our data suggest that DAF as an additional attachment receptor increases the ability of CVB1, 3 and 5 to infect the cells before sCAR-Fc interacts with the virus and prevent the virus-receptor interaction. However, we surprisingly did not observe these differences in sCAR-Fc neutralization efficiency for the various serotypes of clinical isolates. Different adaptation of the clinical and laboratory strains during virus propagation may be an explanation. In comparison to other antiviral compounds that have been tested in the last two decades against coxsackievirus infections, sCAR-Fc displayed a high overall efficiency. Most of all established antivirals had a lower efficiency, independent whether they interfere with virus uptake or with viral replication by impairing viral

enzymes or cellular processes involved in the viral replication (Albulescu et al., 2015; Fechner et al., 2011; Si et al., 2007; Zell et al., 2004). Peconaril, which inhibits virus uptake and uncoating by interacting with the virion, also showed high antiviral efficiency in vitro (Pevear et al., 1999). At a concentration of 2600 nM (1 mg/ ml) pleconaril inhibited a clinical CVB3 isolate of about four orders of magnitude (Braun et al., 2015). Direct comparison of antiviral activity in relation to the used concentration revealed the higher efficiency of sCAR-Fc. In fact we observed a comparable antiviral efficiency for sCAR-Fc and clinical CVB isolates (Table 1) at a distinct (130-fold) lower molar concentration (19.5 nM ≙ 2.5 mg/ml for the 130 kDa dimer). Higher efficiency of sCAR-Fc was also shown in in vivo studies with comparable experimental setups. Prophylactic administration of sCAR-Fc completely protected mice against CVB3 infection in pancreas and heart, whereas protective efficiency of pleconaril was limited to reduction of viral titer in both tissues (Pevear et al., 1999; Pinkert et al., 2009). In terms of utility it should be noted that pleconaril inhibits various picornaviruses, including members of coxsackie A viruses (CVA), enteroviruses and rhinoviruses (HRV) whereas the antiviral effect of sCAR-Fc is specific for group B coxsackieviruses (Pevear et al., 1999). In addition to the efficiency of an antiviral approach, the existence of resistant viruses or the emergence of escape mutants during treatment and possible side effects induced by the antiviral compound are the most important issues. Rapidly emerging resistant viruses were observed during treatment with most of the anticoxsackievirus drugs, including protease inhibitors, nucleoside analogues, siRNAs or drugs that interfere with cellular proteins necessary for viral replication (Fechner et al., 2011). Different analyses also determined that use of pleconaril might be limited by naturally existing and rapidly emerging resistant mutants (Groarke and Pevear, 1999; Hayden et al., 2003; Pevear et al., 1999; Thibaut et al., 2012). Thus the prototypic strain CVB3 Nancy and further laboratory strains based on CVB3 Nancy are pleconaril-resistant. In addition, in different studies, drug-resistant CVB3 mutants were isolated from an initially pleconaril-susceptible wild-type CVB3

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under treatment (Braun et al., 2015; Groarke and Pevear, 1999; Schmidtke et al., 2009). The high frequency of resistant mutants (~5  105) (Groarke and Pevear, 1999) is due to the high error rate of the viral polymerase and to the fact that one specific amino acid exchange in the VP1 protein confers coxsackievirus resistance against pleconaril (Schmidtke et al., 2005). In contrast, we and others did not observe any sCAR-Fc resistant mutants (Lim et al., 2006; Pinkert et al., 2009; Yanagawa et al., 2004), neither naturally existing (no resistant CVB3 under 107 pfu e data not shown) nor emerging during treatment (Pinkert et al., 2011). Moreover, we further revealed in this study that sCAR-Fc worked very efficiently against strains that are pleconaril resistant; e.g. CVB3 Nancy and related strains and the clinical isolate CVB3 97-927R3. Safety and possible side effects of an antiviral compound are further important issues, in addition to the pure efficiency. Many successfully tested therapeutic agents were not translated into the clinic, discontinued during early phase of clinical trials or finally not approved because of safety issues (Albulescu et al., 2015; De Palma et al., 2007; Schmidtke et al., 2009; Senior, 2002; Thibaut et al., 2012). In contrast, during several years of analysis of sCAR-Fc as an antiviral therapeutic, we have not observed any cytotoxicity or side effects in vivo (Pinkert et al., 2009; Stein et al., 2015; Werk et al., 2009). This is even true for the highest sCAR-Fc dose of 50 mg/ml € ger et al., 2015). Also during repeated administered in vitro (Ro treatment over two weeks, we did not observe any negative effect on cell culture (Pinkert et al., 2011) and four week sCAR-Fc treatment in mice did not result in any obvious adverse effects (not shown), indicating the safety of this compound. But contrary to the high oral bioavailability of pleconaril, that made it attractive for clinical application, sCAR-Fc is currently need to be administered by i.v. injection (Webster, 2005). In conclusion, the present study demonstrates, in accordance with previous data, the high antiviral activity of sCAR-Fc. In particular, our data indicate, that sCAR-Fc is not only effective against all prototypic CVB serotypes but also neutralizes clinical CVB strains with high efficiency. Furthermore, no naturally occurring viral sCAR-Fc resistant mutants were found. Thus sCAR-Fc represents a promising candidate for potential use in treatment of human CVB infections. Acknowledgements We gratefully thank Michaela Schmidtke for providing the CVB3 variants CVB3-PDCHO, CVB3-PDHuFi, CVB3 31-1-93 and CVB3 97927R3, for critical reading of the manuscript and the helpful discussion. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) trough Grant FE785/2-2 to H.F. References Abzug, M.J., 2004. Presentation, diagnosis, and management of enterovirus infections in neonates. Paediatr. Drugs 6, 1e10. Abzug, M.J., Michaels, M.G., Wald, E., Jacobs, R.F., Romero, J.R., S anchez, P.J., Wilson, G., Krogstad, P., Storch, G.A., Lawrence, R., Shelton, M., Palmer, A., Robinson, J., Dennehy, P., Sood, S.K., Cloud, G., Jester, P., Acosta, E.P., Whitley, R., Kimberlin, D., 2015. A randomized, double-blind, placebo-controlled trial of pleconaril for the treatment of neonates with enterovirus sepsis. J. Pediatr. Infect. Dis. Soc. http://dx.doi.org/10.1093/jpids/piv015. Albulescu, L., Strating, J.R.P.M., Thibaut, H.J., van der Linden, L., Shair, M.D., Neyts, J., van Kuppeveld, F.J.M., 2015. Broad-range inhibition of enterovirus replication by OSW-1, a natural compound targeting OSBP. Antivir. Res. 117, 110e114. http:// dx.doi.org/10.1016/j.antiviral.2015.02.013. Bergelson, J.M., Cunningham, J.A., Droguett, G., Kurt-Jones, E.A., Krithivas, A., Hong, J.S., Horwitz, M.S., Crowell, R.L., Finberg, R.W., 1997. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275, 1320e1323. Braun, H., Kirchmair, J., Williamson, M.J., Makarov, V.A., Riabova, O.B., Glen, R.C., Sauerbrei, A., Schmidtke, M., 2015. Molecular mechanism of a specific capsid binder resistance caused by mutations outside the binding pocket. Antivir. Res.

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