Can non-invasive transcranial Direct Current Auricular Vagus Nerve Stimulation (tDC-aVNS)
inhibit Cellular and Systemic Inflammation in COVID-19, reducing risk of longer
hospitalization, chronic morbidity and mortality?

A clinical research protocol to determine if non-invasive trans-cranial Direct Current
auricular Vagus Nerve Stimulation (tDC-aVNS) decreases pro-inflammatory Cytokines and
Chemokines in COVID-19: a Proof of Concept Trial of adults with moderate to severe
symptomology, at greatest danger of SARS-Cov2 Cytokine Storm.

Objectives:

To observe how many patients with moderate-severe forms of Covid-19 will need to be
admitted to the ICU with hyper-inflammation (Cytokine Storm) after application of Subtle
Pulse Electro-biological Recovery Apparatus (SPERA) in addition to the Standard of Care
(SOC), comparing tDC-aVNS from Prescription group and a Control group who receive only
SOC.

SOC observations for all patients will include: a CT scan prior to admission or in the
first days after admission. Blood samples collected for hemoleucogram with FBC,
coagulation parameters, D-dimers, fibrinogen, CRP, procalcitonin, LDH, ferritin, ALT,
AST, urea, creatinine, glucose, sodium, potassium, GGT, bilirubin. The clinical status
(symptoms and clinical signs) will be checked daily and the vital signs: BP, pulse rate,
temperature, oxygen saturation will also be measured regularly. An ECG will be routinely
performed and cardiologist's advice will be sought on anticoagulation treatment.
Additional investigations will be performed upon request as needed (such as inflammatory
cytokines, etc). Further objective measures will include: time to clinical recovery,
clinical status, hospital length of stay, number of days with supplemental oxygen and/or
non-invasive ventilation, mortality rate.

Introduction:

The primary challenge in hospital wards treating patients with SARS-Cov-2 is to identify
safe and effective therapies to prevent or resolve cellular and systemic inflammation
caused by this novel virus without compromising protective innate autoimmune responses.
Those with severe infection are at high risk of acute complications, intractable new
co-morbidities and lengthy hospitalization. Physicians struggle to resolve pulmonary
inflammation before occurrence of Acute Respiratory Distress Syndrome (ARDS). Patients
can progress rapidly to the devastating hyper-inflammatory response and tissue damage
known as Cytokine Storm (CS) with coagulation, multi-organ failure and greatest danger of
mortality. Avoiding this conclusion and restoring homeostasis presents a double-edged
sword if pharmacology for inhibiting inflammation also compromises the innate immune
system. Therefore, the investigators will conduct an open label, interventional, proof of
concept clinical trial to assess the safety and effectiveness of non-invasive, subliminal
amperage, auricular Vagus Nerve stimulation (aVNS), of the adult human anti-inflammatory
pathways in hospitalized patients with moderate-severe Covid-19.

SPERA delivers digital subliminal pulse stimulation transcranially (tDCS) to auricular
branches of the Central Nervous System (CNS), it is claimed, by modulating afferent and
efferent fibres of the Vagus Nerve (VN). Murine and human studies have demonstrated the
efficacy and safety of vagus nerve stimulation (VNS) in the resolution of inflammatory
insults including sepsis. Surgically implanted electrodes have hitherto been employed to
overcome impedance of skin and bone to deliver therapeutic pulse frequencies to VN. SPERA
however, employs a non-invasive auricular access to the vagal afferent - efferent
feedback loop, which moderates and mediates homeostasis and inflammation from brain
throughout the body.

Methods and materials:

To identify patients with comparably moderate-severe expression of COVID-19 we will adopt
criteria set out in Table 4 of the Carricchio predictive model (Caricchio R, et al.
Preliminary predictive criteria for COVID-19 cytokine storm. Ann. Rheum Dis 2020; 0:
1-8.) Those patients who meet this criteria remain three times longer in hospital and
suffer six times higher loss of life. Our early ability to predict which patients will
most probably develop CS could help target inflammatory cytokine-inhibition strategies
(including non-invasive aVNS), shorten hospitalization, reduce mortality and improve
clinical outcomes.

The investigators will prioritize those most likely to develop COVID Cytokine Storm
(COVID-CS) and requiring early therapeutic intervention to resolve acute immune system
dysfunction. SARS-Cov2 associated CS is considered to be a unique form of a
hyper-inflammatory response. Criteria classifying other forms of CS, Macrophage
Activation Syndrome (MAS), Haemophagocytic Lymphohistiocytosis (HLH) and the HScore, does
not sufficiently identify and predict COVID-CS. Therefore, patient selection for our
clinical trial of SPERA will be made using the Caricchio predictive model identifying
those at high risk of progressing to CS. Patients, fulfilling this triage criteria,
present with symptoms comprising three clusters of laboratory analysis:

- Inflammation;

- Cell death and tissue damage;

- Prerenal electrolyte imbalance. These clusters accurately predict viral infection
severity most likely to develop into CS in COVID-19, thereby raising the urgency of
early intervention to inhibit pro-inflammatory damage and restore autoimmune
dysfunction.

The investigators will consider patients with a positive RT-PCR test for COVID-19, having
moderately-severe symptoms, expressed by extensive fatigue, confusion, abdominal and
respiratory symptoms (according to the Caricchio severity criteria clusters 4-6, which
demonstrate a sensitivity of 0.85 and a specificity of 0.8) and evidence of COVID-19
pneumonia on the chest CT scan among those hospitalized at "Victor Babes" Infectious
Diseases and Pulmonology Clinical Hospital, Timisoara.

The patients agreeing to enrol in the study by signing the Informed Consent Form will
receive a tDC-aVNS anti-inflammatory prescription by applying the SPERA device 4+4 hours
each day, between 1 and 7 days, in addition to the standard of care. The control group
will consist of hospitalized patients with COVID-19, matched by age, sex and severity of
disease. In the study group we will differentiate at what time the SPERA aVNS
anti-inflammatory prescription was applied following onset of symptoms (first, second
week of infection).

A total of 30 male and female adults with moderate-severe Covid-19 symptomology will be
enrolled to receive the SPERA anti-inflammatory digital prescription by trans-cranial
Direct Current - auricular Vagus Nerve Stimulation in addition to the standard of care.
All subjects will undergo an initial clinical evaluation including vital signs
measurement and a blood draw between 07:30 and 9:30am, followed by stimulation at
10am-13.45pm, with tDC-aVNS Prescription. The SOC Control group will receive no SPERA
digital stimulation, while patients in the tDC-aVNS prescription group will receive a
second session of stimulation between 14:15pm -18.00pm. Additional blood samples will be
drawn again 24 hours after the first stimulation session. Samples will be measured for
inflammatory markers and pro-inflammatory cytokines (biomarker IL-6) using ELISA.
Statistical comparative analyses within and between groups will be calculated.

Intervention:

All patients will receive the same clinical standard of care. The active treatment group
will additionally receive SPERA aVNS, a digital anti-inflammatory prescription group. The
SPERA device will produce a subliminal low amperage, low-voltage electrical square wave
pulse with zero-charge component (at pre-programmed frequencies-Hz, variable
wavelengths-milliseconds and pulse trains/pauses). Upon auricular Cymba Conchae (CC)
montage application of the device, it will generate subtle pulses at a maximum output of
10V and subliminal 10-12 microAmp current, delivering a pre-programmed digital
inflammation-inhibiting prescription (10 volunteers), or sham stimulation (10
volunteers), the Control SOC group will receive no aVNS stimulation (10 volunteers).
Anod/Cathode ionized carbon or metallic auricular ear-clip electrodes applied by the
research team will enable delivery of auricular digital vagus nerve stimulation. In the
tDC-aVNS prescription group the investigation team will place the device electrodes on
the ears at the CC designated point of stimulation. In the inflammation-inhibiting
tDC-aVNS prescription group there will be no noticeable tingling or sensation of
electrical pulse during the stimulation sessions. (SPERA) devices will be applied to
right and left ears of the tDC-aVNS prescription group in the CC montage throughout the
4+4 hour aVNS stimulation sessions.

Research Design:

Hospitalised volunteers with moderate-severe COVID-19 will be recruited at the designated
research facility for two sessions during two consecutive days. The Standard Operating
Procedure (SOP) for SPERA COVID-19 Clinical Trials, with informed consent, will be
rigorously applied by SPERA Trained Workers (STW). Patients from each group will undergo
an initial blood draw DAY1 between 07:30 and 9:30am, followed by stimulation with
tDC-aVNS Anti-inflammation digital Prescription (active treatment group); or no SPERA,
just SOC (control group). Stimulation for tDC-aVNS Prescription group will last for four
hours in the morning and four hours in the afternoon DAY1 from 10.00-13.45 and
14.15-18.00. The subjects will be examined again at the designated research facility the
following morning. On DAY2, research staff will ascertain any changes to subject's
medical history or adverse events, electrocardiographic findings, and vital signs and
perform a physical examination. A second blood draw will be taken on DAY2, 24 hours after
the first stimulation session. The primary endpoint of this study will be the time to
recovery defined as the time to hospital discharge with or without home oxygen
requirement. Secondary outcomes include the number of patients requiring admission to the
ICU, hospital length of stay, number of days with supplemental oxygen and/or non-invasive
ventilation, mortality rate and detection of percent change in inflammatory markers and
pro-inflammatory cytokines (IL-6).

Hypothesized Results:

Signs of systemic inflammation will be prominent in the COVID-CS cohort, indicating
significantly higher levels of ferritin, C-Reactive Protein (CRP), LDH and fibrinogen.
CRP, an acute-phase reactant protein of hepatic origin, increases following interleukin-6
(IL-6) secretion by macrophages and T cells. Harmful inflammation will be confirmed by
the elevation of IL-6 in most COVID-19 patients, but significantly higher (Caricchio et
al. mean: 35 vs 96pg/mL) in COVID-CS. This clinical study will observe whether a
significant percent decrease occurs following SPERA digital prescription in levels of
inflammation; cell death and tissue damage; prerenal electrolyte imbalance; with the
pro-inflammatory cytokine IL-6 as a biomarker. This clinical trial of tDC-aVNS will be
conducted in hospitalized volunteers with moderate-severe COVID-19 infection. In
mammalian studies of sepsis and VNS, levels of pro-inflammatory cytokines were inhibited,
including IL-1b, IL-6, tumor necrosis factor alpha (TNFa) levels, and chemokine,
interleukin IL-8, macrophage inflammatory protein [MIP]-1a, and monocyte chemo-attractant
protein [MCP]-1. In SOC only group, if consistent with previous studies there might be a
significant percent increase in pro-inflammatory biomarkers at 24 hours (p<0.05). At 24
hours, in the tCD-aVNS Prescription group, according to previous studies, might be
observed to have a greater percent decrease in pro-inflammatory biomarker concentration
(p<0.05) compared to SOC group. The tCD-aVNS prescription group, based on previous
published VNS research, might have a greater percent decrease in inflammatory markers and
pro-inflammatory cytokines at 24 hours (p<0.05) in comparison to SOC group.

Conclusions:

If SPERA non-invasive VNS down-regulates (inhibits) inflammatory cytokine release (IL-6,
IL-1b, TNFa etc, analyzing IL-6 as primary biomarker) as hypothesized, it may be that
non-invasive auricular tDCS of the VN could safely resolve and prevent CS risk in
COVID-19. It is supposed that VNS restores dysfunctional afferent - efferent
communication, which may be a cause of unregulated hyper-inflammatory insults. tDC-aVNS
would be an inexpensive, novel and potentially ubiquitous treatment for cytotoxic
cellular and systemic inflammation. This could be an adjunct or alternative to
cytokine-neutralizing and anti-inflammation pharmacology, without unwelcome autoimmune
system side-effects. Further clinical trials could extend this tool in prophylaxis, ICU,
and chronic recovery stages of SARS-Cov2. If proven safe and effective, clinical benefits
would be reduced morbidity and mortality; less time in hospital; quicker and more
complete viral clearance. Moreover, it is possible that that on-going SPERA VNS
prescription research, applied to COVID-19 co-morbidities, will identify beneficial
electro-chemical pathways related to stress, pain and memory, for example through
activation of specific neurotransmitters and neuropeptides (dopamine, endorphine,
serotonin). This digital health science is young but may become highly beneficial in the
absence of universal vaccines and treatments for the scale and inflammatory novelties of
SARS-Cov2, its variants and mutations.