Patients with flu-like symptoms consistent with COVID-19 infection were evaluated at Santa Casa de Misericordia de Santos Hospital emergency ward and screened for the study. Inclusion criteria comprised 1) a positive RT-PCR COVID test; and 2) mild to moderate disease with an oxygen saturation over 93% and no requirement for oxygen. Exclusion criteria included (1) infectious disease: syphilis, hepatitis and HIV; (2) Pregnancy; (3) less than 18 years old; (4) chronic kidney disease; (5) cancer or other immunodeficiencies; and (6) elevated glycated hemoglobin for patients with diabetes. Of 60 patients screened for the study, 39 patients were enrolled. 10 were negative for COVID by PCR and 11 elected not to participate after screening.

Patients were randomized into three cohorts: no Foralumab treatment, nasal Foralumab/Dexa, and nasal Foralumab alone. Treatment was administered in an open label fashion. Patients randomized to Foralumab treatment were visited by a nurse for daily drug administration. 50μg of Foralumab was administered by nose drop to each nostril (total 100μg). Patients receiving dexamethasone received 6 mg of oral dexamethasone on days 1-3. Patients in the control group did not receive foralumab. All arms were allowed to continue background antibiotics. Corticosteroids were not given per protocol but patients were able to access them through self or other healthcare provider outside the hospital.

Foralumab treatment was given for 10 consecutive days. Patients returned to hospital at day 13 for clinical exam and lung CT scan follow up. All patients had a lung CT scan prior to entering the study ( Figure 1 ).

Patients completed a daily clinical outcome questionnaire to assess 15 common COVID-19 related symptoms designed based on FDA guidelines (see below). This scoring system generated a numeric value for each day. Overall wellness was accessed using Baker Wong scale for pain assessment. Patients allocated to the control group did not receive placebo and thus were not visited by the clinical team apart from blood collection on days 5 and 10. For these patients, the daily survey was conducted by phone.

The study was conducted according to the Brazilian regulatory agency for clinical research and good clinical practice guidelines and declaration of Helsinki. Ethical committee approval was granted by Universidade Metropolitana de Santos – UNIMES (CAAE: 38056120.1.0000.5509). The investigators designed the trial, collected the data, and performed the analysis. In addition to providing Foralumab, Tiziana Life Sciences also provided financial assistance to the trial but did not participate in statistical analysis or data interpretation.

Nasopharyngeal swabs were used to screen for COVID-19 by RT-PCR. Clinical laboratory tests were performed at S’agapo Laboratory and included: complete blood counts, IL-6, D-dimer, CRP, COVID-19 Serology, HIV, syphilis, pregnancy, hepatitis, and glycated hemoglobin. White blood counts were measured by flow cytometry and impedance. CRP and glycated hemoglobin were measured by turbidimetry, D-dimer was measured by immunoturbidimetry, and IL-6 was measured by chemiluminescence. IL-18 was measured using ProcartaPlex multiplex assay (Thermo Fisher, EPX450-12171-901).

Lung CT scan was performed at Santa Casa de Misericordia de Santos Hospital using a 16 channel (Toshiba-Alexion) CT scanner. Contrast was not used. Scan coverage was from apex of the lung to the level of bilateral adrenals. Tube voltage was between 100-120Kv. Parenchyma slide thickness was 1mm.

Lung injury consisted of patchy shadowing and ground glass appearance which was graded on a scale of 0 to 4 as follows: 0 = no detectable abnormalities or lung involvement <5%; Stage 1 = mild lung involvement involving approximately 10% of lung area, Stage 2= moderate lung involvement with patchy shadowing and ground glass lesions involving approximately 25% of lung area, Stage 3 = severe confluent ground glass lesions and consolidation involving 25% to 50% of lung area; Stage 4 very severe ground glass lesions and consolidation involving more than half of the lung area.

Baseline lung CT scans obtained on day -2 were compared to scans obtained on day 13. Patients were classified as worsened if they increased by one or more stage, improved if they decreased by one stage and as having marked improvement if they decreased by 2 or more stages. Patients were stable if they did not change stages. Lung CT analysis was performed by three radiologists in a blinded fashion.

Patient reported outcomes were accessed daily using a symptomatology survey based on FDA guidelines (Assessing COVID-19-related symptoms in Outpatient Adult and Adolescent Subjects in Clinical Trials of Drug and Biological Products for COVID-19 Prevention or Treatment) (https://www.fda.gov/media/142143/download). PRO consisted of 15 questions with the following response system: (1) Anosmia (loss of smell): 0=normal, 3=reduced, 5=completely lost). (2) Dysgeusia (loss of taste): 0=normal, 3=reduced, 5=completely lost). (3) Cough: 0=not present, 3 =present some time, 5=present more than half the day. (4) Headache: 0=not present, 3=present some time, 5=present more than half the day); (5) Throat ache: 0=not present, 3=moderate, hurts when swallowing, 5=strong, intense pain when swallowing. (6) Dyspnea: 0=not present, 3= moderate, some lack of air, 5=strong, difficult breathing. (7) Nausea/Vomiting: 0=not present, 3= nausea without vomiting, 5=vomiting. 8) O2 Saturation: 0= > 95, 3 = 94-95%, 5 = 91-93%. (9) Diarrhea was evaluated according to the Bristol Scale (ref) 0=type 0-4, 3=type 5 or 6, 5=type 7. (10) Rhinorrhea: 0=not present, 3=nose with mucus, 5=runny nose (liquid)); (11) Abdominal pain: 0=not present, 3=moderate, 5=intense). (12) Myalgia: 0=not present, 3=moderate, 5=intense (full body). (13) Fever: 0=not present, 3 = 37-38C, 5= >38.0C. (14) Conjunctivitis: 0=absent, 5=present. (15) Appetite: 0=normal, 3=reduced, 5=completely lost. General well-being (how you are feeling today) was assessed using the Baker Wong scale for pain assessment (0-10). The maximum possible score was 85.

We also accessed COVID-19 symptoms reported at day -2 as compared to symptomatology at day 13. For this, we stratified patient reported symptoms according to Domains as follows: Domain 1 (weakness, fatigue, inappetence, body ache, backpain); Domain 2 (fever, chills, sweating); Domain 3 (nausea, diarrhea, epigastric pain); Domain 4 (ageusia); Domain 5 (anosmia); Domain 6 (runny nose, odynophagia, sneezing); Domain 7 (headache, anxiety, eye pain, dizziness); Domain 8 (cough, dyspnea, chest pain). Patient was scored for one symptom in each domain.

The demographic characteristics of the three treatment groups were summarized using means/standard deviations for continuous outcomes and number/percentages for dichotomous outcomes. The patient reported outcomes were measured daily for 10 days, and the change with time in each treatment group was estimated using a linear mixed model with a random intercept and slope. The fixed effects in the model were two indicators for the Foralumab groups, day, and two day by treatment group interaction terms. The estimated slope in each treatment group, and the pairwise group comparison of the slopes are reported. For the presence of symptoms at each domain, the number/percentage for each domain was calculated prior to starting the treatment and at day 10, and the percentage was compared between the treatment groups using a chi-squared test. The total number of domains at each time point was compared across the treatment groups using a Kruskal-Wallis test. For biomarkers, we used linear mixed effects regression model with a random intercept to compare the three groups in terms of change from baseline (day -2) at day 5 and day 10. The primary analysis was the three-group comparison, and this was completed using appropriate contrasts from the linear mixed model. Comparisson between the treatment groups for lung injury (Lung CT scan) was calculated using a chi-squared test. A p value of < 0.05 was considered statistically significant. All analyses were performed with R software and graph prism was used for graph representation.

The baseline demographics are shown in Table 1 . Thirty-nine patients participated in the study, 64.1% females (n=25) and 35.9% males (n=14). The mean age of subjects was 41.1 ± 14.2. with control subjects 33.5, Foralumab/Dexa subjects 41, and Foralumab subjects 44.5. The majority of patients were white. Twenty-three (41%) patients had one or more comorbidities with the most common being obesity. Obesity (BMI>30 Kg/m²) occurred in 37.8% of patients and was observed in all groups (31). Grade 2 obesity was observed in one control subject and one subject in the Foralumab group. Eleven patients (28.2%) reported the use of corticosteroids (prednisolone or dexamethasone) prior to entry in the study. Patients that did not use corticosteroids prior to the study were randomized to the control or the Foralumab group. Patients that used corticosteroids prior to the study were randomized to the control or Foralumab/Dexa group.

Drug use during the study: Twenty-three patients (58.9%) received azithromycin prescribed their physicians. Two patients took the anti-helminth drug ivermectin and 5 patients took the drug acetylcysteine. At the 13day follow-up visit, 5 patients in control group reported taking up to 5 days of off label steroids during the study.

IgM and IgG anti-COVID antibodies were measured on day -2 and day 10 of the study. Nine subjects (23.1%) had anti-COVID IgM antibodies on day -2 which increased to 27 subjects (69.2%) at day 10. Six subjects (15.4%) had anti-COVID IgG antibodies on day -2 which increased to 32 subjects (82%) at day 10. No differences in the development of IgM or IgG antibodies were observed between the groups.

Patients completed a daily clinical outcome questionary to assess 15 common COVID-19 related symptoms. There were no differences in patient reported outcome among groups ( Table 2 ).

We compared patient symptomatology at day -2 vs. day 13 according to the 8 Domains (eg., fever, gastrointestinal symptoms, and respiratory symptoms) described in methods above. At day -2, patients had been experiencing symptoms for an average of 6 days in an average of 5 Domains. As shown in Table 3 , most patients improved during the course of the study with no major differences between the treatment groups. At the end of the study 23 of 39 patients (58.9%) were asymptomatic; 8 of 16 (50%) in the control group, 6 of 11 (54.5%) in the Foramulab/Dexa group and 9 of 12 (75%) in the Foralumab group. Among the 16 patients that remained symptomatic at the end of the study, anosmia (Domain 5) and cough (Domain 8) were the most common symptoms. There were anecdotal reports of rapid recovery from anosmia and ageusia in both Foralumab treated groups. Of note, our study was not designed to determine long-term effects of Foralumab on COVID-19 symptomatology.

Eleven patients (28%) experienced an adverse event including headache (n=4) burning in the nostril (n=1), retrosternal pain (n=2), pustular lesions and itching in cervical area (n=1), dysuria (n=1), tachycardia associated with anxiety (n=1) and insomnia (n=1) ( Table 4 ). No serious adverse events were observed, and all patients completed the study. The incidence and severity of adverse events was determined according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0.

Lymphocyte, monocyte and neutrophil counts were obtained on days -2, 5 and 10 ( Table 5 ). No reduction in white blood cells was observed with treatment. Lymphocytes and monocytes were increased in the Foralumab/Dexa cohort on day 5; p=0.002 and p=0.038, respectively but not on the last day on dosing. Lymphocytes were not elevated significantly above Baseline in the foralumab alone group or the control group. No differences in neutrophil counts were observed. In three group comparisons, a difference was observed in the control vs. the Foralumab/Dexa group and the Foralumab vs. Foralumab/Dexa for lymphocytes at day 5; p= 0.008 and p=0.027, respectively.

We quantified serum levels of IL-6, CRP and D-dimer on days -2, 5 and 10. IL-6 and CRP levels may be linked to worse outcome in COVID-19 (32, 33) and anti-IL-6 therapy is being investigated as immunotherapy in COVID (34–36). As shown in Figure 2 Foralumab resulted in a 69% reduction in IL-6 levels at day 10 (p=0.031) and 85% reduction in CRP at day 10 (p=0.032).

As shown in Table 6 , three group comparisons showed a difference between Control vs Foralumab at a day 5 (p=0.01) and at day 10 (p=0.031) and in CRP on day 10 (p=0.032). No significant differences were observed in D-dimer serum levels among groups. We also measured serum IL-18. Paired analysis showed that IL-18 in the Foralumab group was 46.1 (± 15.5) pg/ml before treatment and 37.6 (± 12.6) pg/ml after treatment (p=0.054). There were no significant changes in serum IL-18 following treatment in the Foralumab/DEXA (p=0.16) or Control groups (p=0.43).

Computerized tomography (CT) of the lung was obtained prior to treatment at day -2 and at study completion on day 13 and analyzed as described in the methods. Lung CT scan was not obtained in 2 control subjects and 1 Foralumab/Dexa subject. Each patient was classified as worse, stable, improved or markedly improved. As shown in Table 7 , 1/10 of the Foralumab/Dexa subjects worsened. 10/14 control subjects, 2/10 Foralumab/Dexa subjects and 2/12 Foralumab subjects remained stable. Regarding improvement, because 6 patients in the control group and 2 in the Foralumab/Dexa group had no lung involvement on day -2, they were not able to improve. Thus, improvement occurred in 3/8 control, 1/8 Foralumab/Dexa and 5/12 in the Foralumab group. Marked improvement was observed in 1/8 control, 6/8 in the Foralumab/Dexa and 5/12 in the Foralumab group. Thus, marked improvement was predominantly observed in subjects receiving Foralumab/Dexa or Foralumab alone. Control vs. Foralumab/Dexa, p=0.01 and Control vs. Forlumab/Dexa+Foralumab, p=0.04 (chi-square analysis) ( Figure 3 ).