In their efforts to provide care for patients with novel coronavirus (SARS-CoV-2) disease
(COVID-19) infection, many healthcare workers around the globe exposed to SARS-CoV-2 got
infected and died over the past two months. Quarantined nurses and physicians have become the
norm in regions with COVID-19 patients, putting at risk the overall functionality of the
regional healthcare system. Other than strict contact-precautions, no proven vaccination or
targeted therapy is available to prevent COVID-19 in healthcare workers. Inhaled nitric oxide
gas (NO) has shown in a small clinical study to have antiviral activity against a Coronavirus
during the 2003 SARS outbreak. We have designed this study to assess whether intermittent
inhaled NO in healthcare workers might prevent their infection with SARS-CoV-2.

Background: After almost two months of fight against COVID-19 infection, on February 24, more
than 3,000 physicians and nurses were reported as contracting COVID-19 disease in Wuhan
(China). Fatalities among those healthcare workers were reported to be related to SARS-CoV-2
infection. Implementation of strict contact protections for all healthcare personnel is
essential to decrease and contain the risks of exposure. However, despite best efforts,
dozens of thousands of healthcare providers have been quarantined for at least 14 consecutive
days in Wuhan alone. Similarly data have been reported in Italy, several healthcare providers
have been quarantined, developed pneumonia and died. Most recent information from Italy
reported that 12% of healthcare workers are infected.

The shortage of hospital personnel, especially in the critical care and anesthesiology
domains, led many hospitals to postpone indefinitely scheduled surgical procedures, including
cardiac surgery or oncological procedures. Only urgent and emergent cases are performed in
patients without symptoms (i.e., absence of fever, cough or dyspnea), no signs (i.e.,
negative chest CT for consolidations, normal complete blood count) and a negative test on
SARS-CoV-2 reverse transcriptase (rt)-PCR. If time does not allow for thorough screening
(i.e., after traumatic injury), such patients are considered to be infected and medical staff
in the OR are fully protected with third degree protections (i.e., N95 masks, goggles,
protective garments and a gown and double gloving).

Rationale. In 2004 in a collaborative study between the virology laboratory at the University
of Leuven (Belgium), the Clinical Physiology Laboratory of Uppsala University (Sweden) and
the General Airforce Hospital of China (Beijing, China), nitric oxide (NO) donors (e.g.
S-nitroso-N-acetylpenicillamine) greatly increased the survival rate of infected eukaryotic
cells with the coronavirus responsible for SARS (SARS-CoV-1), suggesting direct antiviral
effects of NO. These authors suggest that oxidation is the antiviral mechanism of nitric
oxide. A later work by Akerstrom and colleagues showed that NO or its derivatives reduce
palmitoylation SARS-CoV spike (S) protein affecting its fusion with angiotensin converting
enzyme 2. Furthermore, NO or its derivatives reduce viral RNA synthesis in the infected
cells. Future in-vitro studies should confirm that NO donors are equally effective against
SARS-CoV-2, as the current virus shares 88% of its genome with the SARS-CoV [3]. However, at
present it is reasonable to assess that a high dose of inhaled NO might be anti-viral against
SARS-CoV-2 in the lung. The virus is transmitted by human-to-human contact and occurs
primarily via respiratory droplets from coughs and sneezes within a range of about 1.5
meters. The incubation period ranges from 1 to 14 days with an estimated median incubation
period of 5 to 6 days according to the World Health Organization [1]. COVID-19 disease is
mainly a respiratory system disease, but in the most severe forms can progress to impair also
other organ function (i.e., kidneys, liver, heart). Nitric oxide gas inhalation has been
successfully and safely used for decades (since 1990) in thousands of newborns and adults to
decrease pulmonary artery pressure and improve systemic oxygenation.

Recently at the Massachusetts General Hospital, a high dose of inhaled NO (160 ppm) for 30 -
60 minutes was delivered twice a day to an adolescent with cystic fibrosis and pulmonary
infection due to multi-resistant Burkholderia cepacia. There were no adverse events to this
patient, blood methemoglobin remained below 5% and lung function and overall well-being
improved.

Clinical Gap. Thousands of healthcare workers have been infected with SARS-CoV-2 and
contracted COVID-19 despite their best efforts to prevent contamination. No proven vaccine is
available to protect healthcare workers against SARS-CoV-2.

Hypothesis. Due to genetic similarities with the Coronavirus responsible for SARS, it is
expected that inhaled NO gas retains potent antiviral activity against the SARS-CoV-2
responsible for COVID-19.

Aim. To assess whether intermittent delivery of inhaled NO gas in air at a high dose may
protect healthcare workers from SARS-CoV-2 infection.

Observational group: daily symptoms and body temperature monitoring. SARS-CoV-2 RT-PCR test
will be performed if fever or COVID-19 symptoms.

Treatment group: the subjects will breathe NO at 160 parts per million (ppm) for two cycles
of 15 minutes each at the beginning of each shift and before leaving the hospital. Daily
symptoms and body temperature monitoring. SARS-CoV-2 RT-PCR test will be performed if fever
or COVID-19 symptoms. Safety: Oxygenation and methemoglobin levels will be monitored via a
non-invasive CO-oximeter. If methemoglobin levels rise above 5% at any point of the gas
delivery, inhaled NO will be stopped. NO2 gas will be monitored and maintained below 5 ppm.

Blinding. The treatment is not masked.