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International Journal of Hygiene and Environmental Health xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
International Journal of Hygiene and
Environmental Health
journa l homepage: www.elsevier.com/locate/ijheh
In vitro antiviral activity of hypothiocyanite against A/H1N1/2009 pandemic
influenza virus
a,b,∗ a a c a a a
L. Cegolon , C. Salata , E. Piccoli , V. Juarez , G. Palu’ , G. Mastrangelo , A. Calistri
a
Padua University, Department of Molecular Medicine, Padua, Italy
b
Imperial College London, School of Public Health, St. Mary’s Campus, London, UK
c
Alaxia SAS, Lyon, France
a r t i c l e i n f o a b s t r a c t
Article history: Influenza virus spreads via small particle aerosols, droplets and fomites, and since it can survive for a short
Received 13 December 2012
time on surfaces, can be introduced into the nasal mucosa before it loses infectivity. The hypothiocyanite
Received in revised form 28 February 2013 − −
ion (OSCN ), product of the lactoperoxidase/H2O2/SCN system of central airways, is emerging as an
Accepted 3 March 2013
important molecule for innate defense mechanism against bacteria, fungi and viruses. Here we demon-
−
strated that OSCN displays virucidal activity in vitro against the A/H1N1 2009 pandemic influenza virus.
Keywords:
The concentration required to inhibit viral replication by 50% was 2 M when virus were challenged
Influenza prophylaxis −
directly with OSCN before cell inoculation. These values were even lower when inoculated cells were
Influenza treatment −
maintained in contact with enzyme free-OSCN in the culture medium. The last experimental condi-
Infection control −
Disinfection tions better reflect those of tracheobronchial mucosa, where HOSCN/OSCN is retained in the air–liquid
Hypothiocyanite interface and inactivates both the viruses approaching the epithelium from outside and those released
− −
OSCN lactoperoxidase from the inoculated cells after the replication cycle. Importantly no OSCN cytotoxicity was observed
Neuroaminidase in the cellular system employed. The lack of toxicity in humans and the absence of damage on sur- −
In vitro
faces of fomites suggest a potential use of OSCN to avoid mucosal and environmental transmission of
A/H1N1/2009
influenza virus. Since hypothiocyanite is normally present in human airways a low risk of viral resistance
is envisaged. In vivo confirmatory studies are needed to evaluate the appropriate dose, regimen and formulation.
© 2013 Elsevier GmbH. All rights reserved.
Introduction 2007). DUOX employs cytoplasmic nicotinamide adenine dinu-
cleotide phosphate (NADPH) as an electron donor to transfer
It has been recently shown that the epithelium of central two electrons to oxygen, which is reduced to hydrogen peroxide
airways orchestrates an oxidative extracellular microbicidal sys- (H2O2) and then released extracellularly (Grasberger and Refetoff,
−
tem, consisting of the proteins dual oxidase (DUOX) 1 and 2 2006). LPO catalyzes the oxidation of SCN by H2O2 to yield
−
secreted by epithelial cells, lactoperoxidase (LPO) secreted by sub- the antimicrobial oxidizing agent hypothiocyanite ion (OSCN ):
− − −
mucosal glands, and the pseudo-halide thiocyanate anion (SCN ) H2O2 + SCN → H2O + OSCN .
−
secreted by the epithelia (Moskwa et al., 2007; Conner et al., OSCN is an anion in weak equilibrium with the hypothio-
cyanous acid (HOSCN) at neutral pH, where the production of the
former predominates. By contrast, at lower pH HOSCN is the major
product (Tenovuo et al., 1982). The uncharged nature of HOSCN
Abbreviations: ATCC, American tissue culture collection; CO2, carbon diox-
enables prompt penetration into the cell wall (Reiter and Perraudin,
ide; DNA, deoxyribonucleic acid; DTNB, 5 5 dithio-bis-2-nitrobenzoate; DUOX, dual
oxidase; HA, hemagglutinin; HIV, human immunodeficiency virus; HOSCN, hypoth- 1991). Accordingly, the peroxidase system may be considered more
iocyanous acid; FBS, fetal bovine serum; RSV, respiratory syncytial virus; LPO, effective at low pH (Tenovuo et al., 1982). The pKa for the couple
−
lactoperoxidase; MEM, minimal essential medium; MDCK, Madin-Darby Canine
OSCN /HOSCN is reportedly being about 4.85 (Nagy et al., 2009).
kidney; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NA, −
The reactive blend LPO/H2O2/OSCN at the micromolar concen-
neuraminidase; NADPH, nicotinamide adenine dinucleotide phosphate; OD, opti-
−
tration is reported to be bactericidal against several sensitive Gram
cal density; OSCN , hypothiocyanite anion; PBS, phosphate buffered saline; RNA,
ribonucleic acid; rRT-PCR, reverse transcriptase real-time polymerase chain reac- positive and negative bacteria, namely Legionella pneumophila,
−
tion; RSV, respiratory syncytial virus; SCN , thiocyanate; SDS, sequence detection
Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa,
systems software; TCID, tissue culture infectious dose; TNB, 5-thio-2-nitrobenzoic
Listeria monocytogenes, Staphylococcus aureus, Porphyromonas gin-
acid; TPCK, l-1-tosylamide-2-phenylethyl chloromethyl ketone.
∗ givalis, and Actinobacillus actinomycetemcomitans (Moskwa et al.,
Corresponding author at: Padua University, Department of Molecular Medicine,
2007; Conner et al., 2007; Carlsson et al., 1984; Thomas and Aune,
Italy. Tel.: +39 3401005331.
E-mail addresses: [email protected], [email protected] (L. Cegolon). 1978; Ihalin et al., 1998; Reiter et al., 1976).
1438-4639/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.ijheh.2013.03.001
Please cite this article in press as: Cegolon, L., et al., In vitro antiviral activity of hypothiocyanite against A/H1N1/2009 pandemic influenza virus.
Int. J. Hyg. Environ. Health (2013), http://dx.doi.org/10.1016/j.ijheh.2013.03.001
G Model
IJHEH-12668; No. of Pages 6 ARTICLE IN PRESS
2 L. Cegolon et al. / International Journal of Hygiene and Environmental Health xxx (2013) xxx–xxx
Furthermore, fungi as Candida albicans and Candida krusei as Number CRL-2936). Cells were maintained in minimal essential
well as viruses like HIV, herpes simplex 1, echovirus (type 11), and medium (MEM, Gibco Life Technologies) containing 10% heat-
respiratory syncytial virus (RSV), turned out to be sensitive toward inactivated Fetal Bovine Serum (FBS, Invitrogen Corporation) at
◦
LPO system (Lenander-Lumikari, 1992; Mikola et al., 1995; Pourtois 37 C in a humidified atmosphere with 5% carbon dioxide (CO2).
et al., 1990). Although the anti-viral inactivation mechanism of The influenza virus A/California/2009/H1N1 strain was kindly pro-
−
OSCN is yet unknown, it probably modifies viral surface proteins vided by I. Donatelli (Istituto Superiore di Sanità, Rome, Italy). Virus
and prevents the binding or the entry of adenovirus and RSV into was amplified in MDCK cells and the stock was titrated by Tis-
−
the epithelium. Alternatively, OSCN may inhibit the synthesis or sue Culture Infectious Dose 50 (TCID50) assay, following a protocol
assembly of viral nucleic acids and proteins thus preventing the previously described (Klimov et al., 2012). The TCID50 titer was cal-
release of virus from infected cells. culated by using the Excel spreadsheet available for download from
−
Although OSCN has been extensively reported as a product the Yale School of Medicine (www.med.yale.edu/micropath/pdf/
with a broad range of cidal activities, in most cases it was tested as Infectivity%20calculator.xls).
−
a combination of OSCN and the components of the catalytic reac-
tion that ensures a constant production of hypothiocyanite (Björck Reagents
−
et al., 1975; Barrett and Hawkins, 2011). Enzyme free-OSCN is
one of the drug substances of the investigational product ALX- The main chemical reagents employed in this study were: lac-
009 currently developed by Alaxia (Lyon, France), as an inhaled toperoxidase (Alaxia); glucose oxidase (Alaxia); 5 5-dithio-bis-2-
therapy for patients with cystic fibrosis. To our best knowledge nitrobenzoate (DTNB) also called Ellman Reagent (Sigma–Aldrich);
−
enzyme free-OSCN has never been tested against influenza virus sodium thiocyanate (NaSCN, Alaxia SAS) 2 M in physiologi-
before. Influenza A is a respiratory virus that causes annual epi- cal saline solution 0.9% (NaCl, Carlo Erba); d-Glucose (Carlo
demics and occasional pandemics with an unacceptable number Erba); Tris–HCl (Carlo Erba); sodium borohydride (NaBH4,
of deaths and substantial morbidity costs worldwide. Influenza A Sigma–Aldrich); KH2PO4 (Carlo Erba).
viruses belong to the Orthomyxoviridae family. Influenza A viruses
−
are enveloped negative-strand RNA viruses with eight RNA seg- Hypothiocyanite (OSCN ) preparation
ments encoding at least 10 viral proteins (Palese, 2007). The virus
−
particles are enclosed by a lipid envelope, which is derived from For all the experiments, the OSCN solution was freshly pre-
−
the host cellular membrane. Three viral proteins, the surface glyco- pared in sterilized tubes prior to virus addition. OSCN was
proteins hemagglutinin (HA) and neuraminidase (NA) and the M2 produced by LPO catalysis according to Alaxia SAS proprietary tech-
−
ion channel protein, are embedded in the lipid bilayer of the viral nology. pH of freshly enzyme free-OSCN solutions was adjusted to
◦
envelope. HA and NA are essential during viral replication and are 6.8 with 50 mM KH2PO4, and subsequently stored at 4 C, protected
−
the major antigenic determinants (Palese, 2007). Influenza viruses from light until use. Due to the high reactivity of OSCN , prepared
can efficiently transmit between humans via direct or indirect con- solutions were used within 15 min since their production.
−
tact and by respiratory droplets. Control of influenza is particularly OSCN concentration was measured by using a sulfhydryl com-
important at the beginning of pandemic events and relies upon a pound [5-thio-2-nitrobenzoic acid (TNB)] which adsorbs at 412 nm.
number of measures such as (Couch, 2000; Heymann, 2008): TNB was produced by a NaBH4-mediated reduction of DTNB. In par-
ticular, 40 mg of DTNB [5,5 -dithiobis(2-nitrobenzoic acid)] were
• added to 20 mg of sodium borohydride (NaBH ) and brought to
surveillance (notification, isolation of infectious cases, protection 4
100 mL with Tris–HCl buffer 0.5 M pH 7, to obtain the reactive solu-
of contacts);
• tion. The absorbance (optical density, OD) of the samples (0.25 mL
preventive hygiene rules (hand-washing, health education on
reactive in 2.65 mL water and 0.1 mL sample) was measured and
transmission via unprotected coughs/sneezes, from hand to
compared to the reference (0.25 mL reactive in 2.75 mL water). mucous membranes);
−
• OSCN concentration was calculated by employing the following
annual vaccination of healthy adults and specific vulnerable populations; formula:
•
anti-viral treatment as a supplement to vaccination when imme- − ( OD) × 3 × 1000
OSCN (mM) =
diate maximum protection is recommended. Anti-viral drugs
13, 600 × 2 × 0.1
should be considered in individuals at high risk of complications −
where OD = (reference OD) − (OSCN solution OD); 3 = total vol-
due to influenza, hospitalized and during community outbreaks.
ume; 0.1 = sample volume; 13,600 = molar extinction coefficient of
DTNB; 2 = correction factor for the stoichiometric reaction (2 moles
At present, two classes of antiviral drugs are approved for −
TNB reacts with 1 mole OSCN to produce 1 mole DTNB).
influenza therapy: M2 ion channel blockers (adamantanes) and
NA inhibitors. The development of resistance is a major obstacle −
Evaluation of OSCN virucidal effect
to the usefulness of both classes of anti-influenza agents. There-
fore new strategies to prevent and control influenza with different 5
MDCK cells were plated in triplicate at a density of 1 × 10 cells
mode of action and/or less resistance-induction potential are highly
per well, on a 24-well plate, 24 h before viral inoculation. Different
welcome and recommended. −
concentrations of enzyme-free-OSCN (0, 3, 6, 12, 25, 50, 75 and
The aim of this study was therefore to evaluate, for the first time,
− 100 M) were combined with an equal volume of 30 TCID50 viral
the in vitro virucidal effect of enzyme free-OSCN against pandemic
suspension, freshly prepared from the stock in MEM, vortexed and
influenza virus strain A/California/2009/H1N1 as a viral prototype. ◦
incubated at 37 C for 1 h. After two washings with PBS, cells were
◦
incubated for 1 h at 37 C with 2 g/mL TPCK-treated trypsin (Wor-
Materials and methods thington Biochemical Corporation) plus 200 l viral suspensions
−
treated or untreated (control) with OSCN .
Cell culture and viruses Cells were then kept in MEM with 10% FBS, in the presence of the
−
appropriate enzyme-free-OSCN concentration or left untreated.
The Madin-Darby Canine Kidney (MDCK) epithelial cells were Cell supernatants were collected at fixed times post-viral inoc-
purchased from the American tissue culture collection (ATCC ulation, and the viral yield was quantified by a specific reverse
Please cite this article in press as: Cegolon, L., et al., In vitro antiviral activity of hypothiocyanite against A/H1N1/2009 pandemic influenza virus.
Int. J. Hyg. Environ. Health (2013), http://dx.doi.org/10.1016/j.ijheh.2013.03.001
G Model
IJHEH-12668; No. of Pages 6 ARTICLE IN PRESS
L. Cegolon et al. / International Journal of Hygiene and Environmental Health xxx (2013) xxx–xxx 3
transcriptase Real-Time-PCR (rRT-PCR). In specific experiments, A
120 0 µM 3 µM 6 µM 12 µM
the rRT-PCR was also performed on viral RNA extracted from 25 µM 50 µM 75 µM 100 µM
infected cells. The number of viral genomes obtained from the cel-
100
lular supernatants or from the cellular extracts were expressed as
percentage (%) of the respective values observed in the case of cells 80
−
inoculated with the viral suspension not treated with OSCN (set 60
as 100%).
For rRT-PCR, viral RNA was extracted from the 200 l cellular 40
viral yield (%)
supernatant by employing the QIAamp Viral RNA Mini Kit (Qiagen,
Milan Italy) or by RNeasy Mini Kit for cellular pellets (Qiagen, Milan 20
Italy), following the manufacturers’ instruction.
RNA from all samples were amplified by rRT-PCR assay 0
0 1020304050
and run in an ABI Prism 7900HT Fast Real-Time PCR System
hours post inoculation
(Applied Biosystems) using the Superscript III Platinum One-
B 120
step rRT-PCR kit (Invitrogen). The selected target sequence
100
mapped at the level of the M1 encoding region. Real time PCR 100
primers and probe were selected using Primer-Express Soft-
80
ware (Applied Biosystem). The forward and reverse primer
sequences were 5 -AgATgAgTCTTCTAACCgAggTCg-3 and 5 - 60 50.26
CTgCAAAgACACTTTCCAgTCTCTg-3 , respectively. An MGB probe
40 29.14
5 -TCAggCCCCCTCAA- 3 contains a fluorescent reporter dye viral yield (%)
(6-carboxyfluorescein, FAM) at the 5 -end and a non fluorescent 20
3.8
quencher at the 3 -end. The 25 l reaction volume contained 5 l 0.49 0.97 0.68 0.63
0
of extracted RNA, 1X Superscript III Platinum One-step qRT-PCR 0 102030405060708090100
reaction mix, 0.5 l of internal reference dye (ROX) as a passive Concentration (µM)
reference, 0.2 M of probe and 0.4 M of primers for the matrix
− ◦
Fig. 1. Virucidal effect of OSCN . (A) Influenza virus was incubated for 1 h at 37 C
gene RNA. The following thermal profile was used: a single cycle
−
◦ ◦ with increasing OSCN concentrations; MDCK cells were then inoculated and incu-
of reverse transcription for 30 min at 45 C, 2 min at 95 C for
bated for additional 48 h. Viral yield was evaluated by quantification of released
reverse transcriptase inactivation and DNA polymerase activation
viral particles. Percentage (%) of viral growth (Y-axis) over time post inoculation
◦ −
followed by 40 amplification cycles of 15 s at 95 C and 1 min at (X-axis) at different enzyme free OSCN concentrations. (B) IC50 evaluation at 48 h
◦
±
60 C each (annealing-extension step). Triplicate negative and pos- post inoculation. Data presented as mean standard deviation (SD) of the results of
the three independent experiments.
itive controls were included in each experiment. Each fluorescent
reporter signal was measured against the ROX signal to normalize
for non-PCR-related fluorescence fluctuations between samples. to the manufacturer’s instructions. OD was determined by measur-
The data were collected at the annealing step of each cycle and the ing absorbance at 550 nm. All experiments were conducted at least
threshold cycle for each sample was calculated by determining the in triplicate.
point at which the fluorescence exceeded the threshold limit.
The level of influenza virus produced by infected cells was mea- Results and discussion
sured by absolute quantitative rRT-PCR. Absolute quantification
relates the fluorescent PCR signal to input copy number using an Results external calibration curve.
−
The calibration curves used in our absolute quantification In order to evaluate the potential virucidal effect of OSCN
is based on known concentrations of DNA standard molecules against influenza virus, we selected the pandemic influenza virus
(recombinant plasmid DNA). Indeed, the PCR fragment ampli- strain A/California/2009/H1N1, as viral prototype. After assessing,
® ® −
fied from the viral genome was ligated into the pCR 2.1-Topo by MTT assay, that no significant OSCN cytotoxicity was observed
kit (Invitrogen) according to the manufacturer’s instructions. The in MDCK cells (data not shown), a panel of different concentra-
−
resulting plasmid, pTA-M-FLU, was accurately quantified by a fluo- tions of OSCN was combined with viral suspensions and incubated
◦
rimetric Picogreen method and a serial dilution was used as a for 1 h at 37 C. As described in materials and methods section
5
reference for influenza virus quantification. A six-point standard 10 MDCK cells were inoculated and kept in MEM with 10% FBS.
6
curve was fitted by serial dilution (from 5 × 10 to 50 copies) of At twenty-four and 48 h post-inoculation cell supernatants were
the pTA-M-FLU plasmid. This standard curve showed a linear trend collected and the viral yield was quantified by rRT-PCR. The molec-
throughout the mentioned points. The unknown viral RNA copy ular method was selected due to its higher sensitivity. The number
number was automatically calculated by interpolation of values of genomes obtained was expressed as percentage (%) of those
from the above standard curve, employing the Sequence Detection obtained from MDCK infected with viral suspensions not treated
− −
Systems Software (SDS, Applied Biosystem). with OSCN (0 M OSCN ), the latter being set as 100%. As clearly
−
shown in Fig. 1A, OSCN reduced the viral yield in a dose depend-
−
Evaluation of OSCN cytotoxic effect ent manner. In particular, starting from a concentration of 12 M,
−
incubation of influenza virus with enzyme free-OSCN had a strong
−
The possible cytotoxicity of OSCN against MDCK was assessed effect on viral ability to infect the targeted cells. In particular, at
−
by MTT assay (Cell Proliferation Kit I, Roche Diagnostics GmbH, 48 h post-inoculation the OSCN concentration capable of reduc-
Mannheim, Germany), following the manufacturers’ instruc- ing the viral infectivity by 50% (IC50) was roughly 2 M (Fig. 1B).
−
tions. Briefly, cells were plated in quadruplicate at a density of Overall, these results indicated that OSCN directly inactivated the
× 3
5 10 cells per well, on a 96-well plate. The cytotoxic activity of pandemic influenza virus, with a potent virucidal effect.
− −
OSCN was determined on exponentially growing cells in com- As expected, when the appropriate enzyme free-OSCN concen-
plete medium at 2, and 24 h post-incubation with the appropriate tration was added following viral inoculation (immediately after
−
hypothiocyanite concentration. The MTT dye was added according and 24 h after the inoculation with enzyme free-OSCN -treated
Please cite this article in press as: Cegolon, L., et al., In vitro antiviral activity of hypothiocyanite against A/H1N1/2009 pandemic influenza virus.
Int. J. Hyg. Environ. Health (2013), http://dx.doi.org/10.1016/j.ijheh.2013.03.001
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A 0 µM 3 µM 6 µM 12 µM 120 25 µM 50 µM 75 µM 100 µM
100
80
60
40 viral yield viral yield (%)
20
− ◦
Fig. 3. Effect of OSCN on viral entry. Influenza virus was incubated for 1 h at 37 C
−
0 with 6 M and 50 M OSCN concentrations; MDCK cells were then inoculated
0 10 20 30 40 50 and harvested at 5 h post-inoculation. Viral entry was evaluated by quantification
hours post inoculation
of viral genomes in the inoculated cells. Percentage (%) of viral entry (Y-axis) after
−
treatment with enzyme free-OSCN concentrations of 6 M and 50 M respectively
B 120
(X-axis). Data presented as mean ± standard deviation (SD) of the results of the three
independent experiments. 100 100
80 −
free-OSCN in the culture medium post viral inoculation (Fig. 2A).
60 These last experimental conditions better reflect those in the
−
tracheobronchial mucosa, where HOSCN/OSCN is retained in the
40
viral yield viral yield (%) air–liquid interface and inactivates both the viruses approaching
the epithelium from outside and those released from the infected
20
7.59 cells after the replication cycle.
2.19 0.02 0.02 0.02 −
0.1 0.02
0 OSCN has been extensively reported as a product with a broad
−
0 10 20 30 40 50 60 70 80 90 100 range of cidal activities. Published studies used a mixture of OSCN
Conce ntration (µM)
and components of its catalytic reaction (Pruitt and Reiter, 1985;
−
− Shin et al., 2005), whereas enzyme free-OSCN preparations were
Fig. 2. OSCN effect on influenza virus particles released from MDCK-infected cells. −
◦ − employed in the present work. Enzyme free-OSCN might have less
(A) Influenza virus was incubated for 1 h at 37 C with increasing OSCN concen-
−
trations; MDCK cells were then inoculated and appropriate OSCN concentrations risk of adverse/allergic reactions in humans.
added for additional 48 h. Viral yield was evaluated by quantification of released HOSCN concentrations in biological matrices are generally
viral particles. Percentage (%) of viral growth (Y-axis) over time post inoculation
unknown, except for saliva where it was reported to be 10 M on
−
(X-axis) at different enzyme free OSCN concentrations. (B) IC50 evaluation at 48 h
− average (Thomas et al., 1980), or in the range 20–60 M (Tenovuo
post inoculation (in persistent OSCN presence). Percentage (%) of viral growth (Y-
− et al., 1982), or equal to 31, 25, 30 M in resting, stimulated whole
axis) by enzyme free OSCN concentrations (X-axis) at 48 h post-inoculation. Data
presented as mean ± standard deviation (SD) of the results of the three independent saliva and stimulated parotid saliva, respectively (Jalil, 1994).
experiments.
In upper airways (nose) and eyes, indirect evidence sug-
gests that mucosal secretions lack the lactoperoxidase-based
defense mechanism (Tenovuo et al., 1986; Marcozzi et al., 2003;
virus), the effect of the compound was significantly enhanced
Mastrangelo et al., 2005, 2009). This probably contributes to the
(Fig. 2A). In this case, the IC50 was even lower than 2 M and at
reported strong survival of influenza virus in the nasal mucus
10 M over 99.9% of viral growth was culled (Fig. 2B). However,
(Schaffer et al., 1976). Moreover, viral shedding has been detected
when untreated viral suspensions were employed for the inocu-
in nasal secretions (Couch, 1995). Viruses are shed in high num-
lation, no antiviral effect was observed after addition of enzyme
− bers, and shedding may occur before symptom onset and continue
free-OSCN 1.5 h post viral inoculation (data not shown). This lat-
− for several days or weeks after symptoms have ceased (Barker
est result further supports the hypothesis of an OSCN direct action
et al., 2001). Nasal secretion droplets containing infectious virus
on the viral particles without significant effects on cells viability.
particles are generated via coughing, sneezing, and talking and are
In order to further investigate the mechanism of enzyme free-
− easily transmitted over considerable distances (Aitken and Jefferies,
OSCN virucidal effect, the amount of intracellular viral genomes
− 2001). Influenza A virus can be frequently detected on fomites too
was measured by rRT-PCR 5 h post-inoculation with OSCN -treated
− (Boone and Gerba, 2005).
viral suspension. At OSCN concentrations of 6 M and 50 M viral
It is still debated which of the three transmission modes
genomes were respectively reduced to 65.8% and 35.96% as com-
(airborne, droplets, fomites) is responsible for most influenza infec-
pared to the control (Fig. 3). This data are in line with the hypothesis
tions (Tellier, 2007; Brankston et al., 2007; Lemieux et al., 2007). If
of a virucidal effect of the compound due to a direct damage of the
touching is important then hand washing and fomites disinfection
viral particles and thus to a reduced infectivity of influenza virus.
offer a major defense. If droplets are important, simple barriers,
In conclusion, under the experimental conditions assayed,
− such as a surgical mask, will stop transmission; if bioaerosols are
enzyme free-OSCN demonstrated a significant virucidal effect and
important, specialized respirators are needed, and indoor disinfec-
this effect is clearly dose-dependent.
tion spraying might be beneficial. However, face masks can make
breathing difficult, are frequently improperly donned (Cummings
Discussion et al., 2007) and might not provide reliable protection (Weber et al.,
1993). Airborne transmission is considered the predominant trans-
Overall, the results of this study support a dose-dependent mission route for pandemic and epidemic influenza viruses that
−
virucidal activity of enzyme-free OSCN against influenza virus, have circulated among humans so far (Herfst, 2012). If, as it appears,
without any cytotoxic effect. The concentration required to inhibit airborne transmission plays a major role with influenza (Tellier,
−
viral yield by 50% was 2 M when viruses were challenged directly 2006; Weber and Stilianakis, 2008), sprayed OSCN could be used
−
with OSCN before cell inoculation. These values were even for influenza prophylaxis of the indoor air, mucosae (airways, eyes)
lower when MDCK cells were maintained in contact with enzyme and fomites, since it is devoid of biologic toxicity and does not
Please cite this article in press as: Cegolon, L., et al., In vitro antiviral activity of hypothiocyanite against A/H1N1/2009 pandemic influenza virus.
Int. J. Hyg. Environ. Health (2013), http://dx.doi.org/10.1016/j.ijheh.2013.03.001
G Model
IJHEH-12668; No. of Pages 6 ARTICLE IN PRESS
L. Cegolon et al. / International Journal of Hygiene and Environmental Health xxx (2013) xxx–xxx 5
harm surfaces. NA inhibitors are recommended for the prevention lactoferrin. This relationship does not represent a true conflict of
of influenza in patients who have not been immunized or who may interest because Alaxia did not provide any financial support to the
not develop immunity from the vaccine (Zachary et al., 2012). How- study. V.J.P. provided scientific and technical advice for the pro-
−
ever, these antiviral drugs are few and can generate resistance and duction of OSCN only. L.C., C.S., E.P., G.M., G.P., A.C. did not accept
adverse effects. Moreover, NA inhibitors are not seemingly able to honoraria or other payments from Alaxia or other pharmaceutical
prevent asymptomatic infections, and decrease but do not interrupt industries. No other conflicts of interest have to be declared.
nasal shedding of seasonal influenza viruses, the latter persisting in
the upper airways after up to five days of treatment (Jefferson et al.,
−
2006). In this context, OSCN could constitute another valuable Acknowledgments
measure contributing to chemoprophylaxis of influenza.
There could be problems in using the anti-viral and anti- This research was supported by grants from the Veneto Region
−
bacterial OSCN in anatomical districts (such as nose) where (2012) and the University of Padua (ex-60% funds). Alaxia provided
permanent and transient colonization by many microorganisms the reagents and the proprietary technology for the production
−
including viruses is normally present. Nonetheless, hypothiocyan- of OSCN . We thank I. Donatelli for providing the influenza virus
ite is going to have an effect on commensals and parasites of A/California/2009/H1N1 strain.
−
the airways epithelium. Furthermore, as OSCN is herewith pro-
posed to be used in short term prophylaxis, the airways microflora
References
would quickly restore after the end the treatment. Indeed, no major
changes occurred in salivary microflora of 20 patients using tooth-
− Aitken, C., Jefferies, D.J., 2001. Nosocomial spread of viral disease. Clin. Microbiol.
paste and mouth-rinse containing OSCN (generated by the LPO
Rev. 14, 528–546.
system) for 4 weeks (Kirstilä et al., 1996). While it is unpredictable Barker, J., Stevens, D., Bloomfield, S.F., 2001. Spread and prevention of some common
−
viral infections in community facilities and domestic homes. J. Appl. Microbiol.
whether OSCN could impair the long term innate defense against
91, 7–21.
infectious diseases in humans, it has been reported that in vivo
Barrett, T.J., Hawkins, C.L., 2011. Hypothiocyanous acid benign or deadly? Chem. Res.
inhibition of airway LPO in sheep led to a significant decrease in Toxicol. 25, 263–273.
Björck, L., Rosén, C.G., Marshall, V., Reiter, B., 1975. Antibacterial activity of the lac-
bacterial clearance from the airways (Gerson et al., 2000). Consid-
− toperoxidase system in milk against pseudomonads and other gram-negative
ering OSCN mechanism of action against a vast range of viruses
bacteria. Appl. Microbiol. 30, 199–204.
and micro-organisms, this highly reactive compound does not seem Boone, S.A., Gerba, C.P., 2005. The occurrence of influenza A virus on household and
to target a specific protein, but rather to have a more general day care center fomites. J. Infect. 51, 103–109.
Brankston, G., Gitterman, L., Hirji, Z., Lemieux, C., Gardam, M., 2007. Transmission
cidal effect. Therefore it is very unlikely that specific mutations
− of influenza A in human beings. Lancet Infect. Dis. 7, 257–265.
would allow influenza virus to evade the effect of OSCN , and thus
Carlsson, J., Edlund, M.B.K., Hanstrom, L., 1984. Bactericidal and cytotoxic effects of
develop drug resistance. Furthermore, as hypothiocyanite is nor- hypothiocyanite-hydrogen peroxide mixtures. Infect. Immun. 44, 581–586.
Conner, G.E., Wijkstrom-Frei, C., Randell, S.H., Fernandez, V.E., Salathe, M., 2007. The
mally present in the human body and especially in human airways
lactoperoxidase system links anion transport to host defense in cystic fibrosis.
a low risk of resistance against other pathogens is envisaged. Lastly,
FEBS Lett. 581, 271–278.
−
as OSCN did not prove to be cytotoxic, at least in our experimental Couch, R.B., 2000. Prevention and treatment of influenza. N. Engl. J. Med. 343 (24),
1778–1787, December 14.
setting, we do not expect it to be toxic for the airways epithe-
Couch, R.B., 1995. Medical Microbiology. University of Texas Medical Branch,
lium, which is also known to be protected by a mucus layer and Galveston1–22.
characterized by a fast cellular turn-over. Cummings, K.J., Cox-Ganser, J., Riggs, M.A., Edwards, N., Kreiss, K., 2007 May. Respi-
rator donning in post-hurricane new orleans. Emerg. Infect. Dis. 13 (5), 700–707.
However, in view of the above uncertainties a precautionary
− Gerson, C., Sabater, J., Scuri, M., et al., 2000. The lactoperoxidase system functions in
approach may be however appropriate, restricting usage of OSCN
bacterial clearance of airways. Am. J. Respir. Cell Mol. Biol. 22, 665–671.
in the most urgent medical situations; a leading indication for its Grasberger, H., Refetoff, S., 2006. Identification of the maturation factor for dual
oxidase: evolution of an eukaryotic operon equivalent. J. Biol. Chem. 281,
use could be wide short-time prophylaxis during major influenza
18269–18272.
pandemics, or on specific targeted patients in milder annual epi-
Herfst, S., 2012. Airborne transmission of influenza A/H5N1 virus between ferrets.
demics. Science 336, 1534–1541.
In conclusion, under the experimental conditions assayed, Heymann, D.L., 2008. Control of Communicable Diseases Manual. American Public
− Health Association, Washington, DC.
enzyme free-HOSCN/OSCN demonstrated a significant dose-
Ihalin, R., Loimaranta, V., Lenander-Lumikari, M., Tenovuo, J., 1998. The effects of
dependent virucidal effect in vitro. Although the present study
different (pseudo) halide substrates on peroxidase-mediated killing of Acti-
−
found that influenza virus was susceptible to HOSCN/OSCN nobacillus actinomycetemcomitans. J. Periodontal Res. 33, 421–427.
Jalil, R.A., 1994. Concentrations of thiocyanate and hypothiocyanite in the saliva of
in vitro, the viral inhibition should be, however, confirmed in vivo
young adults. J. Nihon Univ. Sch. Dent. 36 (December (4)), 254–260.
evaluating at the same time the appropriate dose, regimen and for-
Jefferson, T., Demicheli, V., Rivetti, D., Jones, M., Di Pietrantonj, C., Rivetti, A., 2006.
mulation. Successful drug administration depends in fact upon a Antivirals for influenza in healthy adults: systematic review. The Lancet 367,
303–313.
formulation compatible for aerosol formulation.
Kirstilä, V., Lenander-Lumikari, M., Söderling, E., Tenovuo, J., 1996 Dec. Effects of oral
hygiene products containing lactoperoxidase, lysozyme, and lactoferrin on the
Authorships composition of whole saliva and on subjective oral symptoms in patients with
xerostomia. Acta Odontol. Scand. 54 (6), 391–397.
Klimov, A., Balish, A., Veguilla, V., Sun, H., Schiffer, J., Lu, X., Katz, J.M., Hancock,
G.M. conceived the study and drafted the paper; L.C. was respon-
K., 2012. Influenza virus titration, antigenic characterization, and serological
sible for the integrity of the work as a whole, and drafted the paper; methods for antibody detection. Methods Mol. Biol. 865, 25–51.
Lemieux, C., Brankston, G., Gitterman, L., Hirji, Z., Gardam, M., 2007. Questioning
E.P. carried out the laboratory work and contributed to data analy-
aerosol transmission of influenza. Emerg. Infect. Dis. 13, 173–175.
sis; A.C. and C.S. designed the study, analyzed/interpreted the data
Lenander-Lumikari, M., 1992. Inhibition of Candida albicans by the
and contributed to the drafting of the paper; G.P. supervised the peroxidase/SCN/H2O2 system. Oral Microbiol. Immunol. 7, 315–320.
laboratory work and managed the research team; V.J.P. provided Marcozzi, G., Liberati, V., Madia, F., Pizzinga, A., de Feo, G., 2003. Effect of hormone
replacement therapy on lacrimal fluid peroxidase activity in woman. Maturitas
technical advice and contributed to the drafting of the paper.
45 (3), 225–229.
Mastrangelo, G., Zanibellato, R., Fadda, E., Lange, J.H., Scoizzato, L., Rylander, R., 2009.
Conflicts of interest Exposure to hydrogen peroxide and eye and nose symptoms among workers in
a beverage processing plant. Ann. Occup. Hyg. 53 (March (2)), 161–165.
Mastrangelo, G., Zanibellato, R., Fedeli, U., Fadda, E., Lange, J.H., 2005. Exposure
V.J.P. is employed by Alaxia, a pharmaceutical company that
to hydrogen peroxide at TLV level does not induce lung function changes: a
−
develops ALX-009, a fixed drug combination of OSCN and longitudinal study. Int. J. Environ. Health Res. 15 (August (4)), 313–317.
Please cite this article in press as: Cegolon, L., et al., In vitro antiviral activity of hypothiocyanite against A/H1N1/2009 pandemic influenza virus.
Int. J. Hyg. Environ. Health (2013), http://dx.doi.org/10.1016/j.ijheh.2013.03.001
G Model
IJHEH-12668; No. of Pages 6 ARTICLE IN PRESS
6 L. Cegolon et al. / International Journal of Hygiene and Environmental Health xxx (2013) xxx–xxx
Mikola, H., Waris, M., Tenovuo, J., 1995. Inhibition of herpes simplex virus type Shin, K., Wakabayashi, H., Yamuchi, K., Teraguchi, S., Tamura, Y., Kurokawa, M.,
1, respiratory syncytial virus and echovirus type 11 by peroxidase-generated Shiraki, M., 2005. Effect of orally administered bovine lactoferrine and Lac-
hypothiocyanite. Antiviral Res. 26, 161–171. toperoxidase on influenza virus infection in mice. J. Med. Microbiol. 54,
Moskwa, P., Lorentzen, D., Excoffon, K.J., Zabner, J., McCray Jr., P.B., Nauseef, W.M., 7174–7723.
Dupuy, C., Banfi, B., 2007. A novel host defense system of airways is defective in Tellier, R., 2006. Review of aerosol transmission of influenza A virus. Emerg. Infect.
cystic fibrosis. Am. J. Respir. Crit. Care Med. 175, 174–183. Dis. 12, 1657–1662.
Nagy, P., Jameson, G.N.L., Winterbourn, C.C., 2009. Kinetics and mechanisms of the Tellier, R., 2007. Transmission of influenza A in human beings. Lancet Infect. Dis. 7,
reaction of hypothiocyanous acid with 5-thio-2-nitrobenzoic acid and reduced 759–760.
glutathione. Chem. Res. Toxicol. 22, 1833–1840. Tenovuo, J., Lehtonen, O.P., Aaltonen, A.S., Vilja, P., Tuohimaa, P., 1986. Antimicrobial
Palese, P., 2007. Orthomixoviridae, fifth ed. Lippincott Williams & Wilkins, Philadel- factors in whole saliva of human infants. Infect. Immun. 51 (January (1)), 49–53.
phia, PA. Tenovuo, J., Pruitt, K.M., Thomas, E.L., 1982. Peroxidase antimicrobial system of
Pourtois, M., Binet, C., Van Tieghem, N., Courtois, P., Vandenabbeele, A., Thiry, L., human saliva: hypothiocyanite levels in resting and stimulated saliva. J. Dent.
1990. Inhibition of HIV infectivity by lactoperoxidase-produced hypothiocyan- Res. 61, 982–985.
ite. J. Biol. Buccale 18, 251–253. Thomas, E.L., Aune, T.M., 1978. Lactoperoxidase, peroxide, thiocyanate antimicro-
Pruitt, K., Reiter, B., 1985. Biochemistry of peroxidase system: antimicrobial effects. bial system: correlation of sulfhydryl oxidation with antimicrobial action. Infect.
In: Pruitt, K.M., Tenovuo, J.O. (Eds.), The Lactoperoxidase System. Marcel Dekker, Immun. 20, 456–463.
Inc., New York, pp. 143–178. Thomas, E.L., Bates, K.P., Jefferson, M.M., 1980. Hypothiocyanite ion: detection of the
Reiter, B., Marshall, V.M.E., Bjorck, L., Rosen, C-G., 1976. Nonspecific bactericidal anti-microbial agent in human saliva. J. Dent. Res. 59, 1466–1472.
activity of the lactoperoxidase-thiocyanatehydrogen peroxide system of milk Weber, A., Willeke, K., Marchioni, R., et al., 1993. Aerosol penetration and leakage
against Escherichia coli and some gram-negative pathogens. Infect. Immun. 13, characteristics of masks used in the health care industry. Am. J. Infect. Control.
800–807. 21, 167–173.
Reiter, B., Perraudin, J-P., 1991. Lactoperoxidase: biological functions. In: Everse, J., Weber, T.P., Stilianakis, N.I., 2008. Inactivation of influenza A viruses in the environ-
Everse, K.E., Grisham, M.B. (Eds.), Peroxidases in Chemistry and Biology, Vol. I. ment and modes of transmission: a critical review. J. Infect. 57, 361–373.
CRC Press, Boca Raton, FL, pp. 144–180. Zachary, K.C., Hirsch, M.S., Thorner, A.R., 2012. Prevention of seasonal influenza
Schaffer, F.L., Soergel, M.E., Straure, D.C., 1976. Survival of airborne influenza virus: in adults. Available at: http://www.uptodate.com/contents/prevention-of-
effects of propagating host, relative humidity, and composition of spray fluids. seasonal-influenza-in adults?source=search result&search=prevention+
Archives of Virology 51, 263–273. influenza&selectedTitle=1∼150 (accessed May 2012).
Please cite this article in press as: Cegolon, L., et al., In vitro antiviral activity of hypothiocyanite against A/H1N1/2009 pandemic influenza virus.
Int. J. Hyg. Environ. Health (2013), http://dx.doi.org/10.1016/j.ijheh.2013.03.001