Recombinant porcine surfactant protein D inhibits influenza A virus replication ex vivo

Accepted Manuscript Title: Recombinant porcine surfactant protein D inhibits influenza A virus replication ex vivo Author: Marine L.B. Hillaire Martin van Eijk Stella E. Vogelzang-van Trierum Ron A.M. Fouchier Albert D.M. E Osterhaus Henk P. Haagsman Guus F. Rimmelzwaan PII: DOI: Reference:

S0168-1702(13)00478-4 http://dx.doi.org/doi:10.1016/j.virusres.2013.12.032 VIRUS 96180

To appear in:

Virus Research

Received date: Accepted date:

24-9-2013 7-12-2013

Please cite this article as: Hillaire, M.L.B., van Eijk, M., Trierum, S.E.V.-v., Fouchier, R.A.M., Osterhaus, A.D.M.E., Haagsman, H.P., Rimmelzwaan, G.F.,Recombinant porcine surfactant protein D inhibits influenza A virus replication ex vivo, Virus Research (2014), http://dx.doi.org/10.1016/j.virusres.2013.12.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Recombinant porcine surfactant protein D inhibits influenza A virus replication ex vivo

2 Marine L. B. Hillaire(1), Martin van Eijk(2), Stella E. Vogelzang-van Trierum(1), Ron A.M.

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Fouchier(1), Albert D. M. E Osterhaus(1, 3), Henk P. Haagsman(2), and Guus F. Rimmelzwaan(1,

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3*)

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Department of Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands

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(2)

Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine,

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Utrecht University, Utrecht, the Netherlands (3)

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Viroclinics Biosciences BV, Rotterdam, the Netherlands

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G. F. Rimmelzwaan, Department of Viroscience, Erasmus Medical Center, PO Box 2040,

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3000 CA Rotterdam, The Netherlands, tel +31 10 704 4066, fax +31 10 704 4760.

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Abstract

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Influenza is a major burden to public health. Due to high mutation rates and selection

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pressure, mutant viruses emerge which are resistant to currently used antiviral drugs.

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Therefore, there is a need for the development of novel classes of antiviral drugs that suffer

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less from the emergence of resistant viruses. Antiviral drugs based on collectin-like surfactant

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protein D (SP-D) may fulfil these requirements. Especially porcine SP-D displays strong

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antiviral activity to influenza A viruses. In the present study the antiviral activity of

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recombinant porcine SP-D was investigated in ex vivo cultures of respiratory tract tissue

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infected with human influenza A virus of the H3N2 subtype. Porcine SP-D has antiviral

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activity in these test systems. It is suggested that porcine SP-D may be used as a venue to

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develop a novel class of antiviral drugs.

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Keywords, porcine surfactant protein D, influenza A virus, antiviral activity

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Influenza A viruses (IAVs) cause seasonal epidemics that are associated with excess

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morbidity and mortality. Two surface proteins, the hemagglutinin (HA) and the

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neuraminidase (NA), determine influenza A virus subtype. So far 17 subtypes of HA and 10

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types of NA have been identified (Fouchier et al., 2005; Li et al., 2012; Tong et al., 2012).

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Influenza A/H3N2 and A/H1N1 viruses are the predominant cause of seasonal epidemics. In

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addition to seasonal epidemics, IAVs can cause pandemics after introduction of novel

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antigenically distinct viruses. The pandemics of 1918, 1957 and 1968 have been caused by

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influenza viruses H1N1, H2N2 and H3N2, respectively. Recently, a H1N1 virus of swine

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origin caused the first pandemic of the 21st century. Although this virus was not of a novel

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subtype, it was antigenically distinct, spread rapidly and caused numerous deaths worldwide

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(Dawood et al., 2012).

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Although vaccination remains the most important countermeasure against influenza, vaccines

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may not be efficacious when they do not match the epidemic strain antigenically. In the case

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of a pandemic outbreak vaccines may become available too late. Under these circumstances

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antiviral drugs may be used therapeutically or prophylactically. For the treatment of influenza

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two classes of antiviral drugs are available, neuraminidase inhibitors and matrix 2 protein

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inhibitors (amantadane and rimantadine). These two classes of antiviral drugs that act at

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different steps of the virus replication cycle, can inhibit influenza replication efficiently and

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shorten the duration of the illness (Aoki et al., 2003; Reuman et al., 1989; Younkin et al.,

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1983). However, resistance to these two classes of antivirals drugs is rapidly acquired and

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positively selected for. During past influenza seasons a high proportion of resistant viruses

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was circulating (Bright et al., 2005; Deyde et al., 2007). For example, in the 2007-2008

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season most of the circulating H1N1 IAV were resistant to the neuraminidase inhibitor

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oseltamivir (WHO, 2009). Also influenza H3N2 viruses emerged between 1995 and 2005 that

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were resistant to adamantanes (Bright et al., 2005). Therefore, there is a need for new classes

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of antiviral drugs against IAVs that do not induce resistance. In this respect, collectin-like

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surfactant protein D (SP-D) are of interest since they display antiviral properties against a

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variety of viruses (Hartshorn, 2010; Hillaire et al., 2013).

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SP-D is a C-type lectin that binds to glycans on the surface of pathogens via its carbohydrate

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recognition domain (CRD) and this way can neutralize a broad range of bacteria and viruses

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(Hartshorn, 2010). Only a few studies have addressed the potential of SP-D as an antiviral

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drug in vivo. In one of these studies the antiviral potential of a trimeric neck CRD fragment of

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human SP-D (hSP-D) was investigated, which was administered before, or at different time

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points after inoculation of mice with respiratory syncytial virus. This treatment reduced viral

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loads in the lungs of infected mice (Hickling et al., 1999). Recently, the antiviral potential of

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truncated hSP-D with two mutations in its CRD (D325A/R343V was assessed in a murine

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model (Crouch et al., 2011). Co-administration of this recombinant mutant hSP-D with a

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mouse adapted H1N1 influenza strain (A/WSN/33) decreased morbidity caused by the

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infection.

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We have previously shown that recombinant porcine SP-D (RpSP-D) has stronger antiviral

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activity against IAVs than human SP-D (hSP-D) in vitro subtype which could be related to

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unique structural features of the CRD of pSP-D (Hillaire et al., 2011). These involve the

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presence of a sialylated N-linked glycosylation sites and a loop of three amino acids near the

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lectin binding site (Hillaire et al., 2011; van Eijk et al., 2012; van Eijk et al., 2002; van Eijk et

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al., 2003). Especially higher-order oligomeric forms of RpSP-D can effectively neutralize

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various IAVs of different subtypes, especially those of the H3N2. Pandemic 2009 H1N1

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viruses and A/H5N1 viruses are both resistant to inhibition by hSP-D (Hartshorn et al., 2008;

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Job et al., 2010). These viruses were neutralized by RpSP-D, although relatively high doses

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were required to block virus binding and to prevent infection of MDCK cells (Hillaire et al.,

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2011). This is probably due to a low degree of glycosylation of their hemagglutinins since it

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was also shown that the neutralization activity of RpSP-D is largely dependent on the number

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and position of glycans on HA (Hillaire et al., 2012). So far, the potential of RpSP-D as an

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antiviral drug against influenza has not been tested ex vivo.

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The therapeutic potential of RpSP-D was investigated in an ex vivo lung culture system. .

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Since the ultimate aim was to develop an antiviral drug based on the use of collectins we

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argued that the production of a well-defined and highly purified recombinant protein was

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essential (van Eijk et al., 2011). RpSP-D was expressed, purified and characterized as

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described (van Eijk et al., 2011). Briefly, SP-D sequence-containing pUPE 101.01 expression

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plasmids were transfected into HEK293-EBNA1 cells using the protocol previously described

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(Durocher et al., 2002). Culture supernatant from transfected cells was harvested after 5 days,

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a solution of CaCl2 was added (10 mM final concentration) and SP-D was affinity purified

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with mannan-agarose. After elution with EDTA containing buffer (5 mM Hepes, 0.9% NaCl,

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5 mM EDTA, pH 7.4), the eluted SP-D was separated by size exclusion chromatography into

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differently assembled forms (trimers, dodecamers). Only highly oligomerized RpSP-D was

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used since we demonstrated previously that dodecameric RpSP-D inhibits IAV replication in

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vitro more efficiently than trimers.

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C57BL/6 mice were sacrificed and lungs and trachea were excised for ex vivo cultures. The

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lungs were inflated with 2 ml of IMDM (Lonza) containing 100 U/ml penicillin and 100

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μg/ml streptomycin and TPCK trypsine (Sigma Aldricht) (infection medium). Trachea and

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right or left lung were incubated in 1 or 0.5 ml of infection medium respectively. Lungs were

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inoculated with 104 or 105 TCID50 of influenza virus A/Hong Kong/2/68 (A/H3N2), trachea

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were inoculated with 103 TCID50 of the same virus on a rocker for one hour at room

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temperature. Inocula were aspirated and the tissues were washed and subsequently incubated

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at 37 °C for 24 or 48 hours in an atmosphere of 95% O2 and 5% CO2. These inoculated tissues

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were treated with 6.5 or 13 µg of RpSP-D added one hour or 24 hours post inoculation or

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were left untreated. Organs and supernatants were harvested to determine virus titer or

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inflated with formalin (4%) for immunohistochemistry. Lungs were homogenized and

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quadruplicate ten-fold serial dilutions of these samples were used to inoculate Madin-Darby

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Canine Kidney (MDCK) cells as described previously (Bodewes et al., 2009). Seven days

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later, the HA activity of the culture supernatants was used as indicator for virus replication.

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The virus titers were calculated according to the Spearman–Kärber method (Kärber, 1931).

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Untreated control lung tissue cultures reached a mean virus titer of 105.1 TCID50/gr and 106.4

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TCID50/gr after inoculation with 104 and 105 TCID50 of the A/H3N2 virus, respectively

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(figure 1A and B). Lung cultures that were inoculated with 104 TCID50 of IAV A/Hong

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Kong/2/68 were treated with 6.5 µg or 13 µg of RpSP-D and reduced the mean titer to 103

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TCID50/gr of tissue (figure 1A). Repeated administration at 24 hours post inoculation of

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RpSP-D resulted in a similar reduction of virus titer (103.5 TCID50/gr or 101.5 TCID50/gr of

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tissue for 2x6.5 and 2x13 µg of RpSP-D respectively) (figure 1A). Similar results were

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obtained after inoculating the lung tissue cultures with a dose of 105 TCID50/ml of A/Hong

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Kong/2/68 (figure 1B). There results show that although RpSP-D inhibited virus replication in

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ex vivo lungs cultures, the infection was not fully blocked and in some of the individual tissue

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cultures the infection was not inhibited at all. Furthermore, a single high dose or repeated

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doses of RpSP-D were required to obtain an appreciable inhibition of virus replication. More

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promising results were obtained with trachea cultures as described below.

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Untreated trachea tissue cultures reached mean virus titer of 109,8 TCID50/gr. The addition of

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6.5 µg of RpSP-D reduced virus replication considerably resulting in a 30,000 fold reduction

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of virus titer. Addition of 13 µg of RpSP-D reduced virus titer even further (103,8 TCID50/gr)

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(figure 1C).

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No statistically significant differences were observed between the respective experimental

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groups. However, when the virus titers in all treatment groups were lumped and compared

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with those of the untreated control group, the differences were statistically significant

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(p<0.05, Mann-Whitney test) for infection of lung tissue with 105 TCID50 and for infection of

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trachea tissue with 103 TCID50.

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The extent of viral spread in the lungs and trachea tissues was assessed by

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immunohistochemistry. After fixation and embedding in paraffin, lungs and trachea were

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sectioned at 3 µm and incubated with a monoclonal antibody directed against the

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nucleoprotein of IAV as described previously to visualize IAV antigen expression and used

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to estimate the percentage of infected cells (Rimmelzwaan et al., 2001). Four to seven lungs

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were included per condition and four longitudinal sections of each lung were used. Three to

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five trachea were included per condition and four cross-sections of each trachea were

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analyzed.

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Representative images are depicted in figure 2A and B for all conditions tested. The average

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percentage of infected cells was calculated and is displayed in figure 2C and D.

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A low percentage of infected cells was found in untreated lungs (2.3 %) at 24 and 48 hours

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post inoculation. A single administration of 6.5 µg of RpSP-D one hour post inoculation

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reduced the percentage of infected cells to 1.6 % and 1.8 % at 24 and 48 hours post

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inoculation respectively. Increasing the dose to 13 µg of RpSP-D significantly decreased the

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percentage of infected cells further to 1.2 % and 0.9 % at 24 and 48 hours post inoculation

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with 105 TCID50 respectively (p<0.05, Mann-Whitney test). Two doses of 6.5 µg or two doses

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of 13 µg of RpSP-D, administered at one hour and 24 hours post inoculation, significantly

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reduced the percentage of infected cells to 0.9% and 0.7% respectively compared to untreated

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controls (p<0.05, Mann-Whitney test).

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In untreated control trachea, 0.6 % and 25.9 % of infected cells were observed at 24 hours and

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48 hours post inoculation with 103 TCID50, respectively. After a single dose of 6.5 µg of

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RpSP-D, the percentage of infected cells at 48 hours post inoculation dropped to 18.4 %.

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After one dose of 13 µg of RpSP-D, the percentage of infected cells at 48 hours post

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inoculation decreased significantly to 4.2 % (p<0.05, Mann-Whitney test).

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Treatment of infected trachea with 13 µg RpSP-D reduced virus titers a million fold. This

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reduction in virus replication was also reflected by a reduction in the number of virus infected

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tracheal epithelial cells. These results indicate that administration of RpSP-D to the infected

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trachea culture efficiently inhibits viral spread in this tissue.

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In lung tissue, only a modest reduction of viral spread was observed, which could be related

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to a better access of SP-D to trachea epithelial cells ex vivo and/or stronger tropism of the

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A/H3N2 virus for cells of the upper respiratory tract (van Riel et al., 2007). In contrast to in

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vitro experiments that we have performed previously (Hillaire et al., 2011), RpSP-D was

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administered after infection of the tissue with IAV in order to mimic treatment of infections.

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Although we wished to treat mice after infection as well, we were unable to execute this type

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of experiment since treatment of mice with buffer, containing SP-D or not, increased lung

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virus titers and clinical signs in these mice. This is probably due to enhanced viral spread in

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the lungs caused by excess fluid. In order to evenly deliver RpSP-D to the lungs more

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sophisticated techniques are required, for example, delivery as aerosols, which may not

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increase viral spread in the lungs.

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For use in humans, RpSP-D is most likely not suitable since it might be immunogenic in a

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xenogeneic host. We therefore speculate that expanding our knowledge on structure of pSP-D

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may aid to develop hSP-D based antiviral drug. In contrast to existing classes of antiviral

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drugs like the neuraminidase inhibitors and inhibitors of the M2 protein it is unlikely that SP-

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D based antiviral drugs would similarly select for resistant IAV strains easily since they target

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relatively conserved glycan motifs on HA. However, it cannot be excluded that the

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widespread use of such drugs may drive the selection of viruses with a lower number of N-

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linked glycosylation sites. We speculate that these viruses may be less sensitive to the

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neutralizing activity of SP-D, but still may be sensitive to a certain extent. Of interest, since it

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has been shown that SP-D also has strong antibacterial activity, an SP-D-based antiviral drug

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may not only inhibit IAV replication, but also may be beneficial for treating secondary

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bacterial infections, a common complication of influenza.

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This study demonstrates that RpSP-D has antiviral activity in ex vivo cultures of respiratory

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tract tissue in particular trachea cultures. Treatment of these cultures with RpSP-D reduced

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viral replication and viral spread. Further research in this area seems warranted and should

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focus on better methods of administration and the development of derivatives of human SP-D

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with the strong antiviral properties against a broad range of IAVs known for RpSP-D.

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Figure legends

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Figure 1. RpSP-D inhibits IAVs replication in respiratory tract tissues ex vivo

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Mouse lungs (A and B) and trachea (C) were inoculated with influenza virus A/Hong

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Kong/2/68 (A/H3N2) ex vivo at the indicated virus dose, 104 TCID50/ml (A), 105 TCID50/ml

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(B) and 103 TCID50/ml (C) and treated with various doses RpSP-D one hour post inoculation

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or not. The data represent the virus titers at 48 hours post inoculation of individual tissue

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cultures. The bars indicate the mean virus titer of each of the treatment group.

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Figure 2. RpSP-D limits viral spread in the lungs and trachea ex vivo

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Mouse lungs (A) and trachea (B) were inoculated with influenza virus A/Hong Kong/2/68

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A/H3N2 ex vivo and treated with various doses RpSP-D one hour post inoculation as

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indicated. Administration of RpSP-D was repeated for some organs at 24 hours post

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inoculation and is indicated as 2x. At 24 and 48 hours post inoculation, organs were fixed in

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formalin, embedded in paraffin and sections of the organs were stained for the presence of

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virus-infected cells to assess the percentage of infected cells in the respective organs (C and

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D). * indicates statistically significant differences (p<0.05, Mann-Whitney test)

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Acknowledgments

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The authors thank Rogier Bodewes for support and advice. This work was supported by grant

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10388 from the Technology Foundation STW.

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References

209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255

Aoki, F.Y., Macleod, M.D., Paggiaro, P., Carewicz, O., El Sawy, A., Wat, C., Griffiths, M., Waalberg, E., Ward, P., Group, I.S., 2003. Early administration of oral oseltamivir increases the benefits of influenza treatment. J Antimicrob Chemother 51(1), 123-129. Bodewes, R., Kreijtz, J.H., Baas, C., Geelhoed-Mieras, M.M., de Mutsert, G., van Amerongen, G., van den Brand, J.M., Fouchier, R.A., Osterhaus, A.D., Rimmelzwaan, G.F., 2009. Vaccination against human influenza A/H3N2 virus prevents the induction of heterosubtypic immunity against lethal infection with avian influenza A/H5N1 virus. PLoS One 4(5), e5538. Bright, R.A., Medina, M.J., Xu, X., Perez-Oronoz, G., Wallis, T.R., Davis, X.M., Povinelli, L., Cox, N.J., Klimov, A.I., 2005. Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern. Lancet 366(9492), 1175-1181. Crouch, E., Nikolaidis, N., McCormack, F.X., McDonald, B., Allen, K., Rynkiewicz, M.J., Cafarella, T.M., White, M., Lewnard, K., Leymarie, N., Zaia, J., Seaton, B.A., Hartshorn, K.L., 2011. Mutagenesis of surfactant protein D informed by evolution and x-ray crystallography enhances defenses against influenza A virus in vivo. J Biol Chem 286(47), 40681-40692. Dawood, F.S., Iuliano, A.D., Reed, C., Meltzer, M.I., Shay, D.K., Cheng, P.Y., Bandaranayake, D., Breiman, R.F., Brooks, W.A., Buchy, P., Feikin, D.R., Fowler, K.B., Gordon, A., Hien, N.T., Horby, P., Huang, Q.S., Katz, M.A., Krishnan, A., Lal, R., Montgomery, J.M., Molbak, K., Pebody, R., Presanis, A.M., Razuri, H., Steens, A., Tinoco, Y.O., Wallinga, J., Yu, H., Vong, S., Bresee, J., Widdowson, M.A., 2012. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis. Deyde, V.M., Xu, X., Bright, R.A., Shaw, M., Smith, C.B., Zhang, Y., Shu, Y., Gubareva, L.V., Cox, N.J., Klimov, A.I., 2007. Surveillance of resistance to adamantanes among influenza A(H3N2) and A(H1N1) viruses isolated worldwide. J Infect Dis 196(2), 249-257. Durocher, Y., Perret, S., Kamen, A., 2002. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293EBNA1 cells. Nucleic Acids Res 30(2), E9. Fouchier, R.A., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., Rimmelzwaan, G.F., Olsen, B., Osterhaus, A.D., 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 79(5), 2814-2822. Hartshorn, K.L., 2010. Role of surfactant protein A and D (SP-A and SP-D) in human antiviral host defense. Front Biosci (Schol Ed) 2, 527-546. Hartshorn, K.L., Webby, R., White, M.R., Tecle, T., Pan, C., Boucher, S., Moreland, R.J., Crouch, E.C., Scheule, R.K., 2008. Role of viral hemagglutinin glycosylation in antiinfluenza activities of recombinant surfactant protein D. Respir Res 9, 65. Hickling, T.P., Bright, H., Wing, K., Gower, D., Martin, S.L., Sim, R.B., Malhotra, R., 1999. A recombinant trimeric surfactant protein D carbohydrate recognition domain inhibits respiratory syncytial virus infection in vitro and in vivo. Eur J Immunol 29(11), 34783484. Hillaire, M.L., Haagsman, H.P., Osterhaus, A.D., Rimmelzwaan, G.F., van Eijk, M., 2013. Pulmonary Surfactant Protein D in First-Line Innate Defence against Influenza A Virus Infections. J Innate Immun.

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Hillaire, M.L., van Eijck, M., Nieuwkoop, N., Vogelzang-van Trierum, S.E., Fouchier, R., A, M., Osterhaus, A., M, D, E., Haagsman, H.P., Rimmelzwaan, G., F., 2012. The number and position of N-linked glycosylation sites in the hemagglutinin determine differential recognition of seasonal and 2009 pandemic H1N1 influenza virus by porcine surfactant protein D. Virus Research. Hillaire, M.L., van Eijk, M., van Trierum, S.E., van Riel, D., Saelens, X., Romijn, R.A., Hemrika, W., Fouchier, R.A., Kuiken, T., Osterhaus, A.D., Haagsman, H.P., Rimmelzwaan, G.F., 2011. Assessment of the antiviral properties of recombinant porcine SP-D against various influenza A viruses in vitro. PLoS One 6(9), e25005. Job, E.R., Deng, Y.M., Tate, M.D., Bottazzi, B., Crouch, E.C., Dean, M.M., Mantovani, A., Brooks, A.G., Reading, P.C., 2010. Pandemic H1N1 Influenza A Viruses Are Resistant to the Antiviral Activities of Innate Immune Proteins of the Collectin and Pentraxin Superfamilies. J Immunol 185, 4284-4291. Kärber, 1931. Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Naunyn Schmiedebergs Arch Pharmacol 162, 480–483. Li, Q., Sun, X., Li, Z., Liu, Y., Vavricka, C.J., Qi, J., Gao, G.F., 2012. Structural and functional characterization of neuraminidase-like molecule N10 derived from bat influenza A virus. Proc Natl Acad Sci U S A 109(46), 18897-18902. Reuman, P.D., Bernstein, D.I., Keefer, M.C., Young, E.C., Sherwood, J.R., Schiff, G.M., 1989. Efficacy and safety of low dosage amantadine hydrochloride as prophylaxis for influenza A. Antiviral Res 11(1), 27-40. Rimmelzwaan, G.F., Kuiken, T., van Amerongen, G., Bestebroer, T.M., Fouchier, R.A., Osterhaus, A.D., 2001. Pathogenesis of influenza A (H5N1) virus infection in a primate model. J Virol 75(14), 6687-6691. Tong, S., Li, Y., Rivailler, P., Conrardy, C., Castillo, D.A., Chen, L.M., Recuenco, S., Ellison, J.A., Davis, C.T., York, I.A., Turmelle, A.S., Moran, D., Rogers, S., Shi, M., Tao, Y., Weil, M.R., Tang, K., Rowe, L.A., Sammons, S., Xu, X., Frace, M., Lindblade, K.A., Cox, N.J., Anderson, L.J., Rupprecht, C.E., Donis, R.O., 2012. A distinct lineage of influenza A virus from bats. Proc Natl Acad Sci U S A 109(11), 4269-4274. van Eijk, M., Bruinsma, L., Hartshorn, K.L., White, M.R., Rynkiewicz, M.J., Seaton, B.A., Hemrika, W., Romijn, R.A., van Balkom, B.W., Haagsman, H.P., 2011. Introduction of N-linked glycans in the lectin domain of surfactant protein D (SP-D): impact on interactions with influenza A viruses. J Biol Chem 286, 20137–20151. van Eijk, M., Rynkiewicz, M.J., White, M.R., Hartshorn, K.L., Zou, X., Schulten, K., Luo, D., Crouch, E.C., Cafarella, T.R., Head, J.F., Haagsman, H.P., Seaton, B.A., 2012. A Unique Sugar-binding Site Mediates the Distinct Anti-influenza Activity of Pig Surfactant Protein D. J Biol Chem 287(32), 26666-26677. van Eijk, M., van de Lest, C.H., Batenburg, J.J., Vaandrager, A.B., Meschi, J., Hartshorn, K.L., van Golde, L.M., Haagsman, H.P., 2002. Porcine surfactant protein D is Nglycosylated in its carbohydrate recognition domain and is assembled into differently charged oligomers. Am J Respir Cell Mol Biol 26(6), 739-747. van Eijk, M., White, M.R., Crouch, E.C., Batenburg, J.J., Vaandrager, A.B., Van Golde, L.M., Haagsman, H.P., Hartshorn, K.L., 2003. Porcine pulmonary collectins show distinct interactions with influenza A viruses: role of the N-linked oligosaccharides in the carbohydrate recognition domain. J Immunol 171(3), 1431-1440. van Riel, D., Munster, V.J., de Wit, E., Rimmelzwaan, G.F., Fouchier, R.A., Osterhaus, A.D., Kuiken, T., 2007. Human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals. Am J Pathol 171(4), 1215-1223. WHO, 2009. Influenza A(H1N1) virus resistance to oseltamivir - 2008/2009 influenza season, northern hemisphere.

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Younkin, S.W., Betts, R.F., Roth, F.K., Douglas, R.G., Jr., 1983. Reduction in fever and symptoms in young adults with influenza A/Brazil/78 H1N1 infection after treatment with aspirin or amantadine. Antimicrob Agents Chemother 23(4), 577-582.

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• RpSP-D inhibits influenza virus replication in respiratory tract tissue cultures

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• Recombinant SP-D holds promise as an antiviral drug against influenza

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