Influenza Virus H1N1 inhibition by serine protease inhibitor (serpin) antithrombin III.

NIH Public Access Author Manuscript Int Trends Immun. Author manuscript; available in PMC 2014 May 28.

NIH-PA Author Manuscript

Published in final edited form as: Int Trends Immun. 2014 April 1; 2(2): 83–86.

Influenza Virus H1N1 inhibition by serine protease inhibitor (serpin) antithrombin III Donald F. Smee, PhD1, Brett L. Hurst, MS1, Craig W. Day, PhD1, and Ralf Geiben-Lynn, PhD2,* 1Institute

for Antiviral Research, Utah State University, Logan, Utah, USA

2Acceleration

BioPharmaceuticals, Inc. Woburn, MA, USA

Abstract NIH-PA Author Manuscript

Endogenous serine protease inhibitors (serpins) are anti-inflammatory mediators with multiple biologic functions. Serpins are also part of the early innate immune response to viral infection that includes mannose binding lectins, soluble CD14, defensins and antimicrobial peptides. Recently, serpin antithrombin III (ATIII) was shown to have broad-spectrum antiviral activity against HIV, HSV and HCV. We tested ATIII's antiviral activity against a variety of influenza virus strains. In our studies we found strong in vitro inhibition of influenza virus A H1N1 isolates. Our data also demonstrate that ATIII potency was more than 100-fold that of ribavirin. We also found that inhibition was dependent on viral hemagglutinin with decreasing efficacy in the order of H1N1 > H3N2 > H5N1 >> Flu B. In vivo efficacy is currently still lacking demonstrating need for more advanced delivery methods for this biomolecule. Understanding how ATIII regulates influenza virus inhibition may reveal new avenues for therapeutic interventions.

Keywords Serpin; Antithrombin III; influenza virus H1N1; hemagglutinin dependent


I. Introduction Approximately 500 million people are inflicted by influenza virus infection each year with annual epidemics producing significant morbidity and mortality. Influenza virus infection is usually a self-limiting disease among healthy adults, but can also lead to severe secondary bacterial pneumonia in elderly individuals and infants. The possibility exists that a highly virulent strain of influenza virus, such as the H5N1 avian influenza strain which emerged originally in Hong Kong in 1997 [1] or the ‘Spanish flu’ outbreak of 1918 [2], may adapt to become easily transmissible among humans and cause serious pandemics. At the present time, options for controlling influenza infection are limited. Protection through vaccination is limited due to the antigenic variation of the influenza virus. Currently four antiviral therapies have been approved for the prevention and treatment of influenza virus infection. *

Correspondence to R. Geiben-Lynn ([email protected]).. VI. CONFLICT OF INTEREST STATEMENT We have no conflict of interest to declare.

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The drugs amantadine and rimantadine, which inhibit influenza A as M2 ion channel blockers, are limited by adverse side effects, their lack of effectiveness against influenza B viruses, and the rapid development of viral resistance (>90% in some studies) [3]. Inhibitors of neuraminidase (NA), zanamivir and oseltamivir, are able to reduce virus replication of both influenza A and B but this class of inhibitor is also limited by the rapid development of viral resistance. The continued development of new agents with enhanced potency, reduced toxicity, and a greater genetic barrier to resistances, as well as targeting other steps in the influenza replication cycle is a critical need for the effectiveness of influenza therapy. As is the case with HIV, the development of highly active combination therapeutic strategies, perhaps used in parallel with available prophylactic measures, will yield an optimal strategy for controlling influenza virus and it is imperative that these strategies be in place prior to the appearance and spread of any pandemic human strain which is expected to result from cross-species transmission from the avian and swine influenza populations.


The course and out-come of an influenza virus infection is influenced by viral and host factors. Host risk factors, like obesity or pregnancy, became evident during the recent swine flue pandemics [4]. Furthermore, genetic factors in humans associated with higher susceptibility to influenza infections and severe disease outcome have been suspected for the 1918 pandemics, as well as the H5N1 human infections [5-9]. Multiple serine protease activities in the lung are also implicated in mediating influenza infection [10]. Therefore, blocking damage of influenza virus activated proteases in lung epithelia by the use of serine protease inhibitors may provide an alternative approach for treatment of influenza infection. Furthermore, host serine proteases are essential for the influenza virus life cycle because the viral haemagglutinin (HA) is synthesized as a precursor that requires proteolytic maturation.


We have developed the serine protease inhibitor (serpin) antithrombin III (ATIII) as a potential broad-spectrum viral inhibitor [11-15]. This protein belongs to a group of serpins which are part of the early physiologic response to viral infection that includes mannose binding lectins, soluble CD14, defensins, antimicrobial peptides, neutrophils, monocyte/ macrophage and natural killer cells [16]. We feel that this molecule is well suited since it has an unusual long 72 h half-life in lungs that is longer than that of current small molecule inhibitors. Together, this longer half-life and the induction of an intrinsic anti-viral host-cell defense typical for ATIII [11, 13, 14] might prevent the occurrence of drug resistant influenza strains.

II. MATERIAL AND METHODS Ethical statements This study was conducted in accordance with the approval of the Institutional Animal Care and Use Committee of Utah State University. The work was done in the AAALACaccredited Laboratory Animal Research Center of Utah State University. Initial accreditation was granted on Feb 10, 1986 and has been maintained to the present time (last renewal: March 31, 2010). The Animal Welfare Assurance Number is A3801-01 and was last reviewed by the National institutes of Health on April 7, 2010 in accordance to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (2002 Edition) and expires on February 28, 2014. Int Trends Immun. Author manuscript; available in PMC 2014 May 28.

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Source of ATIII


For our studies we used cGMP produced ATIII which was more than 95% pure as tested by C4 HPLC or by coomassie blue staining of sodium dodecyl sulfate-polyacrylamide gels. Serum derived ATIII (Talecris, Durham, NC) was used for the in vitro studies. For the in vivo efficacy studies we used human recombinant ATIII produced by GTC Biotherapeutics (Framingham, MA) in transgenic goats. In vitro human influenza virus inhibition assays


We measured viral inhibition by ATIII with the following methods: 1) inhibition of virusinduced cytopathic effect (CPE), as determined by visual (microscopic) examination of the cells, 2) increases in the uptake of the vital dye neutral red (NR) into cells (NR Assay), and 3) virus yield reduction (Virus Yield Assay) as determined by endpoint dilution titration in MDCK cells. All tests have been previously described [17]. CPE inhibition data were expressed as the 50% effective concentration (EC50); toxicity was measured by determining the 50% cytotoxic (cell-viability) concentration (IC50) in stationary cell monolayers; and the selectivity index (SI) was determined as IC50/EC50. Eight one-half log10 concentrations of the test compounds were evaluated in triplicate wells containing influenza virus-infected MDCK cells. Standard placebo-treated virus controls, toxicity controls, ribavirin and normal-medium controls were included in all assays. In vivo efficacy studies for ATIII using mouse-adapted H1N1 We tested the in vivo efficacy of ATIII using an influenza A/NWS/33-m (= mouse adapted) (H1N1) virus infection in mice, and compared it with ribavirin. ATIII III (400 μg, 200 μg, 100 μg/mouse) in 50 μl was administered intranasally once a day for 5 days starting 2 hours prior to virus challenge. 75 mg/kg (300 μM/kg) ribavirin was tested as a control with twice daily intraperitoneal (i.p.) administrations. Influenza A/NWS/33-m (H1N1) virus was administered intranasally at approximately 5 × 102 pfu (~3 × mLD50). Statistical analysis


Survival curves for in vivo evaluations were initially compared by the Mantel-Cox log-rank test, and statistical significance was found. Subsequently, pairwise comparisons of survival curves were made using the Gehan-Breslow-Wilcoxon test with Bonferroni corrected threshold of significance for the number of treatment groups evaluated. Calculations were made using the Prism 5.0 software program (GraphPad Software, San Diego, CA).

III. Results In vitro human Influenza virus inhibition by ATIII We found that ATIII was most active for Flu A (H1N1) viruses with yielded EC50 values ranging from 30 to 150 nM (Tab. I) and was most active for influenza A H1N1 isolates California/07/2009 and New Caledonia/20/99. We did not detect cell toxicity in our tests for ATIII. Our experiments yielded routinely SI values of greater than 10. We found that inhibition was HA dependent with decreasing efficacy in the order of H1N1 > H3N2 > H5N1 >> Flu B. Ribavirin yielded an EC50 between 12.3 μM (NR assay) and 5.3 μM

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(Visual Assay) against influenza A/California/07/2009 (H1N1) virus. Thus, ATIII was 96fold (NR Assay) or 177-fold (Visual Assay) more active than ribavirin for this strain. ATIII elicited inhibition for influenza A/Perth/16/2009 (H3N1) that was 390-fold (NR Assay) and 238-fold (Visual Assay) stronger than ribavirin. There was a 390-fold (NR Assay) or 150fold (Visual Assay) greater ATIII treatment effect than ribavirin against Influenza A/ Duck/MN/1525/81 (H5N1). In vitro mouse-adapted influenza virus inhibition by ATIII We also measured ATIII inhibition using mouse-adapted influenza virus strains to select the most suited strain for our in vivo testing. Most strains were strongly inhibited by ATIII with 16-176-fold higher efficacies than that found for ribavirin in the control experiments (Tab. II). In vivo efficacy test using mouse-adapted influenza virus


We then tested the effects of treatment with ATIII or ribavirin using an influenza A/NWS/33-m (H1N1) virus infection in mice. ATIII III (400 μg (6.9 nmol), 200 μg (3.45 nmol), 100 μg (1.72 nmol)/mouse) in 50 μl was administered intranasally once a day for 5 days starting 2 hours prior to virus challenge. Seventy-five mg/kg (300 μM/kg) ribavirin was tested as a control with twice daily intraperitoneal (i.p.) administrations. Influenza A/NWS/33-m (H1N1) virus was administered intranasal at approximately 5 × 102 pfu (~3 x mLD50). Using 10 mice per group, we observed a 20% reduction in mortality in the high-dose group (344 nM/kg or 20 mg/kg) when mice were evaluated over a 21-day period compared to a 100 % reduction in the ribavirin control group (Fig. 1A). Treatment with ATIII delayed the mean time to death, but the results were not statistically significant compared to placebo. We found that the two surviving mice recovered faster in body weight compared to the ribavirin group (Fig. 1B). In vitro we found that ATIII was 157-fold more active than ribavirin for this isolate, the use of 1000-fold lower amounts in vivo might explain the lack of inhibition. We feel that a more direct application to the lung tissue is necessary to see a more potent effect on inhibition in vivo.


IV. DISCUSSION ATIII showed superior in vitro efficacy compared to ribavirin, which was not confirmed in vivo. Our in vivo data suggest that we might need to change the intranasal administration to inhalation administration to increase the possible drug load in the lung tissue. Additionally, it might be necessary to change the formulation to increase mucin penetration. Nevertheless, we feel that ATIII is uniquely positioned to add to current treatment options. We have not tested at which step ATIII blocks viral replication. This was beyond the scope of this publication. It is possible that ATIII inhibits several steps in the virus’ lifecycle. Serpins are found to block entry and intracellular replication of HIV [18]. Additionally, the drug might neutralize lung proteases known to increase influenza virus replication, decrease lung tissue damage and lymphocyte infiltration as described earlier for another serine protease inhibitor [10]. ATIII might also have further advantages: ATIII limits bacterial outgrowth and lung


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injury in Streptococcus pneumoniae pneumonia when administered via inhalation suggesting a novel role as a cationic antimicrobial protein [19]. This might be useful during influenza virus infections with Streptococcus pneumoniae coinfections. These coinfections are typically associated with greater loss of lung tissue remodeling/wound repair, higher inflammation, more Ca2+ signaling and apoptosis than influenza infections alone, and are suspected to be the reason for the severity of the 1918 influenza virus and 2009 H1N1 pandemic [20]. There is a lack of activation of repair processes after influenza virus infection that might be restored by ATIII since there are disease scenarios where ATIII restored lung's self-repair mechanism. This was true for example after smoke inhalation-induced lung injury [21]. A vast knowledge base of coagulation related treatment in the lung exists [22-25] which lowers the risk of the drug's development. Additionally, ATIII is very stable against degradation: it has a 72 h half-life in blood much longer than most small molecule inhibitors.


In summary, our data indicate that ATIII might block HA activation by a host protease(s) necessary to gain its fusion ability to host cell membrane and thereby block the infection process. It is also conceivable that the use of ATIII may exert additional indirect beneficial effects by suppressing proteases that are released from infiltrating immune cells directly or by desensitizing of inflammatory cell receptors. This anti-inflammatory activity may suppress a hyper-inflammatory response in severely influenza virus infected individuals, which has been described to be detrimental in humans and in animals.

V. CONCLUSIONS


Our data describe the inhibition of influenza virus H1N1 through serpin ATIII. We also found inhibition of other influenza strains with decreasing efficacies H1N1>H3N2>H5N1>>Flu B. ATIII was routinely more effective than ribavirin for all strains tested except Flu B. The in vitro efficacy did not transfer to feasible in vivo inhibition. Lack of in vivo efficacy calls for the use of new formulation techniques to target lung tissue and allow sufficient penetration. This was also true for another anti-viral indication of ATIII where only high in vivo efficiencies (up to 50.000-fold of current treatments) were generated after applying of a tissue and cell targeting ATIII-formulation [13].

Acknowledgments This study was funded by contracts HHSN272201100019I and HHSN272201000039I from the Virology and Respiratory Diseases Branches, Division of Microbiology and Infectious Diseases, National Institute for Allergy and Infectious Diseases, National Institutes of Health, U.S.A.

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Figure. 1. Effects of treatment with ATIII and ribavirin on survival and body weight during an influenza A/NWS/33 (H1N1) virus infection in mice

ATIII (shown as μg/mouse in a group of 10) was given intranasally once a day for 5 days starting 2 hours prior to virus challenge. Ribavirin (mg/kg/day) was administered i. p. twice a day for 5 days starting 4 hours after virus exposure. The placebo control and ribavirin groups received intranasal saline administrations similar to the ATIII treatment group. *** P1720

>23

Visual

30

>1720

>55

>1720

>55

Virus Yield Flu A (H1N1)

New Caledonia/20/99

EC90

30

Neutral Red

55

>1720

>31

Visual

64

>1720

>27

>1720

>12.5

>1720

>13

Virus Yield

140

Flu A (H1N1)

NWS/33

Neutral Red

133

Visual

143

>1720

>12

Flu A (H1N1)

Solomon Islands/03/2006

Neutral Red

122

>1720

>14

Visual

150

>1720

>11

>1720

>1.3


Flu A (H1N1)

PR/8/34

Neutral Red

1340

Visual

>1720

>1720

0

Flu A (H3N2)

California/7/04

Neutral Red

100

>1720

>17

Visual

64

>1720

>27

>1720

>2.2

Virus Yield Flu A (H3N2)

Flu A (H3N2)

Flu A (H5N1)

Flu A (H5N1)

Flu B


Flu B

Flu B

Victoria/3/75

Perth/16/2009

Vietnam/1203/2004H

Duck/MN/15/25/81

Florida/4/2006

Shanghai/361/02

Sichuan/379/99

775

Neutral Red

>1720

>1720

0

Visual

1029

>1720

>1.3

Neutral Red

21

>1720

>81

Visual

55

>1720

>31

Neutral Red

345

>1720

>5

Visual

345

>1720

>5

Neutral Red

52

>1720

>33

Visual

90

>1720

>19

Neutral Red

620

>1720

>3

Visual

>1720

>1720

0

Neutral Red

>1720

>1720

0

Visual

517

>1720

>3

Neutral Red

>1720

>1720

0

Visual

>1380

>1720

>1.2


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TABLE II


In vitro efficacy of ATIII in mouse-adapted influenza virus strains measured as EC50 by neutral red and visual assay compared to ribavirin. SI is given for ATIII. Concentrations are in nM. Virus

Strain

Assay

EC50

SI

Fold Ribavirin activity

Flu A (H1N1)

New Caledonia/20/99-m

Neutral Red

100

>16

176

Visual

114

>13

75

133

>13

157

Flu A (H1N1)

NWS/33-m

Neutral Red Visual

143

>12

149

Flu A (H1N1)

Solomon Islands/03/2006-m

Neutral Red

122

>14

157

Visual

150

>12

123

>1.3

16

Flu A (H1N1)

PR/8/34-m

Neutral Red

1340

Visual

>1720

0

7

87

Visual

>1720

0

Anti-HSV activity of serpin antithrombin III.

Species-Specific Serological Detection for Schistosomiasis by Serine Protease Inhibitor (SERPIN) in Multiplex Assay.

Serpin-serine protease binding kinetics: alpha 2-antiplasmin as a model inhibitor.

A bumblebee (Bombus ignitus) venom serine protease inhibitor that acts as a microbial serine protease inhibitor.

Antithrombin Chimeras Provides Insight into the Serpin Specificity of Coagulation Proteases.

Exploration of binding and inhibition mechanism of a small molecule inhibitor of influenza virus H1N1 hemagglutinin by molecular dynamics simulation.

Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

Serine protease SP105 activates prophenoloxidase in Asian corn borer melanization, and is regulated by serpin-3.

The inhibition of human plasma kallikrein by antithrombin III.

Inhibition of purified plasma kallikrein by antithrombin III and heparin.

Safety of virus inactivated antithrombin III concentrate antithrombin III immuno (AT III).

In vivo behaviour of some antithrombin III-protease complexes.

Return of pandemic H1N1 influenza virus.

X protein of hepatitis B virus resembles a serine protease inhibitor.

A vaccinia serine protease inhibitor which prevents virus-induced cell fusion.

Degradation of plasma proteins by the trypsin-like enzyme of Porphyromonas gingivalis and inhibition of protease activity by a serine protease inhibitor of human plasma.

Inhibition of the presentation of dengue virus antigen by macrophages to B cells by serine-protease inhibitors.

Thrombin, collagen and A23187 stimulated endogenous platelet arachidonate metabolism: differential inhibition by PGE1, local anesthetics and a serine-protease inhibitor.

Selective Inhibition of Prostasin in Human Enterocytes by the Integral Membrane Kunitz-Type Serine Protease Inhibitor HAI-2.

Inhibition of cathepsin A activity by the potato protease inhibitor.

In silico modification of oseltamivir as neuraminidase inhibitor of influenza A virus subtype H1N1.

3,4-Dichloroisocoumarin, a serine protease inhibitor, inactivates glycogen phosphorylase b.

Respiratory syncytial virus (RSV) entry is inhibited by serine protease inhibitor AEBSF when present during an early stage of infection.

Increased antibody responses to human papillomavirus type 16 L1 protein expressed by recombinant vaccinia virus lacking serine protease inhibitor genes.