Pharmacodynamic of Recombinant Human Interferon Alpha-2b Nasal Drops and Effective Prophylaxis Against SARS-COV-2 Infection

Abstract

The recombinant human interferon alpha-2b (IFN-α2b) nasal drop formulation (Nasalferon) was studied as prophylaxis for SARS-CoV-2. Healthy volunteers between 19 and 80 years of age received 0.5 million international units of IFN in one drop (0.05 mL ) in each nostril, twice a day, for 10 consecutive days. The nondetection of SARS-CoV-2 by real-time polymerase chain reaction was the primary outcome variable. Several IFN-α biomarkers, including intranasal gene expression and innate immune effector activity, were increased in participants who received intranasal IFN-α2b. The study included 2,930 international travelers and 5,728 persons who were their close contacts. The subjects were treated with Nasalferon in January 2021, and 9,162 untreated travelers were included as controls. COVID-19 rate in treated subjects was significantly lower than in untreated subjects (0.05% vs. 4.84%). The proportion of travelers with COVID-19 decreased from 60.9% to 2.2% between December 2020 and February 2021. Furthermore, 1,719 tourism workers also received Nasalferon, and no cases of SARS-CoV-2 infection were detected, whereas 39 COVID-19 cases (10.6%) were reported in 367 untreated subjects. The main adverse events associated with the use of intranasal IFN-α2b were nasal congestion, headache, and rhinorrhea. Our prophylactic health interventions study demonstrates that the daily administration of Nasalferon for 10 days decreases the risk of developing COVID-19 in healthy volunteers.

Introduction

Respiratory viruses are the leading cause of disease in humans. Over 90% of acute respiratory tract viruses, such as the influenza virus, parainfluenza virus, respiratory syncytial virus, rhinovirus, adenovirus, and coronavirus (Weston and Frieman, 2019), produce infections. In December 2019, a new severe acute respiratory syndrome named coronavirus 2 (SARS-CoV-2) emerged in Wuhan, Hubei Province, China, producing the coronavirus 2019 (COVID-19) pandemic (Zhu et al., 2020). SARS-CoV-2 belongs to the same genus of beta-coronavirus as the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS) coronaviruses, but it spreads faster due to its greater ability to replicate, the ease with which it is spread, and its greater pathogenicity (Kandeel et al., 2020).

Acute upper respiratory tract infections affect the nasopharynx, oropharynx, larynx, trachea, ears, and sinuses. Respiratory viruses enter the body through the respiratory tract and are primarily transmitted from person to person by inhaling respiratory droplets or aerosols expelled through coughing and sneezing. The nose and mouth are the main points of entry of the virus, and even the conjunctiva can inoculate infections. The incubation period of respiratory viruses is from 1 to 4 days, and viral replication occurs in the hair cells of the nasal epithelium and nasopharynx (Wang et al., 2020).

Treatment of an acute upper respiratory infection is essentially symptomatic. Generic antiviral drugs are not common, and they have shown reduced effectiveness. Oseltamivir is the most widely known antiviral drug, but it is used only for influenza virus infection (Li et al., 2022). The rest of the medications are usually nonsteroidal anti-inflammatory drugs, which are able to manage fever and sore throat, although none of them can eliminate the virus producing the infection (Azh et al., 2022).

Alpha interferon (IFN-α) is a biological product with immunomodulatory properties and antiviral effects for both DNA and RNA viruses. It is an endogenous protein involved in the immune response of the first line of defense of the body for preventing and eradicating infections caused by any of the known respiratory viruses (Li et al., 2018).

IFN triggers its biological effects when it binds to its specific receptor on the surface of the cell membrane. It thus induces conformational changes in the intracellular chain of the receptor, allowing access to, and activation of, the Janus kinase (Jak) and tyrosine kinase (Tyk) proteins by auto-phosphorylation. Consequently, Jak1 and Tyk2 phosphorylate and activate signal transducers and activators of transcription (STAT) proteins 1 and 2, respectively. STAT1 and STAT2 subsequently bind to a third transcription factor known as the interferon regulatory factor 9 (IRF9 or p48) and form the interferon-stimulated gene factor 3 (ISGF3) (Hu et al., 2021).

ISGF3 is an essential hetero-trimer complex that translocates to the nucleus, leading to the activation and transcription of IFN-induced genes within the interferon-stimulated response element (ISRE), which synthesize proteins with different biological actions (Haque and Williams, 1994). This signaling pathway leads to a state of humoral protection that is able to inhibit viral replication and induce an antiviral response to eliminate the viral particles. These mechanisms of action, within the area of the infected cell, will turn the dominant viral infection into a recessive one and shorten the course of infection (Taniguchi and Takaoka, 2002).

The upper respiratory tract mucosa has numerous immune cells expressing IFN-α receptors that have proven their innate ability to suppress viral infections. Intranasal IFN administration leads to the immediate and potent activation of the immune system, which does not depend on serum concentration and produces minimal adverse events (Brice et al., 2019). Scientific papers support the use of IFN-α nasal drops to generate a prophylactic antiviral effect (Lee et al., 2021) and reduce clinical respiratory symptoms in patients with acute respiratory infections, acquired or induced by respiratory viruses (Acosta et al., 2020).

Given the direct effects of type I IFNs-α/β in inhibiting viral replication and the effect of activating immune cell populations to clear virus infections, IFN-α is presented as an ideal antiviral candidate to counteract SARS-CoV-2 infection (Wang and Fish, 2019). The in vitro antiviral activity demonstrated by IFN-α against SARS-CoV-2 infection suggests a possible clinical alternative in the absence of a specific SARS-CoV-2 vaccine or an approved therapeutic drug (Mantlo et al., 2020). An exploratory study, starting in January 2020 with individuals from Wuhan suspected of having COVID-19, provided the first evidence of the therapeutic efficacy of IFN-α2b in COVID-19 (Zhou et al., 2020). Subsequently, Nakhlband et al. (2021), Lu et al. (2022), and Buchynskyi et al. (2023) confirmed the effectiveness of IFN-α in preventing or treating SARS-CoV-2 infection. The key element was a small structural modification of the SARS-CoV-2 genome, making it more susceptible to the antiviral activity of type I IFNs-α/β compared with SARS-CoV-2 and MERS (Lokugamage et al., 2020).

Specifically, IFN-α administered in nasal drops produces an antiviral effect and the reduction of clinical symptoms in patients with induced or acquired respiratory infections (Hayden et al., 1986). An open-label study in the Chinese population during the COVID-19 pandemic reported that IFN-α nasal drops could effectively prevent SARS-CoV-2 infection in susceptible healthy people (Meng et al., 2021).

Human recombinant IFN-α2b produced by the Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba, has demonstrated antiviral efficacy and proven its safety profile for over 37 years (Nodarse-Cuni and Lopez-Saura, 2017). The therapeutic effectiveness of Cuban IFN-α2b in the recovery of COVID-19 patients was initially reported using the parenteral formulation (Pereda et al., 2020). Later, when CIGB introduced the nasal drop formulation, it received an Emergency Use Authorization by the Cuban Regulatory Authority in January 2021.

The group of Cuban biomedical experts for COVID-19 recommended that IFN-α2b nasal drops should be used as a prophylactic intervention for the protection of high-risk individuals exposed to SARS-CoV-2 infection (Garcia-del-Barco et al., 2021).

This report covers the pharmacodynamic (PD) study of the Cuban IFN-α2b nasal drop formulation (Nasalferon) and results from institutional and community health interventions during the COVID-19 pandemic period. A safety profile analysis of the product is also included.

Materials and Methods

Pharmacodynamic study

A PD study coded as RPCEC00000308 in the Cuban Public Registry of Clinical Trials (RPCEC) was carried out in healthy male and female volunteers of over 18 years of age who lived in the neighborhood near the CIGB. All subjects signed the informed consent to participate in the trial (RPCEC, 2020).

The aim of the study was to evaluate several IFN-α biomarkers, including intranasal gene expression and innate immune effector activity with three dose levels of Nasalferon. Nasal drops were available in vials with 2.5 million international units (MIU), 5 MIU, and 10 MIU of IFN-α2b in 2 mL of the solution. The heads of patients were tilted back to administer the Nasalferon, in which one administration, defined as one drop (0.05 mL), was applied in each nostril. The dose depended on the amount of MIU of IFN-α2b in the drop.

The study involved three stages. In the first stage, the patients were included consecutively, and the subjects received 1.0 MIU in one application, once or twice a day for 3 consecutive days. The second stage included another group receiving 1.0 MIU in one application twice a day for 10 consecutive days. The third stage evaluated one application once a day in three random groups of 0.25 MIU, 0.50 MIU, and 1.0 MIU for 3 consecutive days. All subjects received Nasalferon through self-administration, supervised by the nursing staff, at a medical post located in their neighborhood near the CIGB.

For sample collection, an oropharyngeal swab was used in the three stages of the study, before starting the treatment and five times during the course of the study. Blood samples were also collected from individuals in the first and second stages. The biological samples were obtained under optimal biosecurity conditions at the medical post and transferred to specialized laboratories at the CIGB to test for the gene expression of several IFN-α biomarkers and the innate immune effector activity.

Swabs were later placed in an RNA preservation solution (Sigma, USA) at 4°C until purification. Blood was obtained in CPT tubes (Becton Dickinson, USA), and peripheral blood mononuclear cell (PBMC) populations were purified and preserved in the RLT buffer with 1% beta-mercaptoethanol (Qiagen, USA) at −70°C until purification. Additional blood samples were obtained in vacutainers with K3EDTA for IFN-α determinations by enzyme-linked immunosorbent assay and flow cytometry studies.

The study evaluated the quantification of mRNA at the mucosal level using real-time polymerase chain reaction (RT-PCR) to measure the expression levels of IFN-stimulated genes (ISG). Prototypical ISG such as the 2′-5′-oligoadenylate synthetase 1 (OAS1) protein and beta-2 microglobulin (β-2MG) were measured. The STAT1 and STAT3 proteins related to the signaling pathway triggered by the binding between IFN-α and its specific receptor on the cell surface were also assessed. The IFN-α transcript and toll-like receptors (TLRs) TLR3, TLR7, and TLR8 related to both innate and adaptive immune responses to viral infections were also analyzed.

The first and third stages evaluated the expression of all the IFN-α biomarkers and innate immunity effectors 72 h after the start of the treatment. Serum levels of IFN-α were evaluated only in the first stage. The second stage evaluated only the expression of OAS1 at 24, 72, and 120 h after the start of the 10-day treatment, whereas post-treatment evaluations were on days 16 and 23. Biological samples during the treatment were obtained between 30 and 90 min after the administration of IFN-α2b nasal drops.

The qPCR reactions were set up in 20 µL with 300 nM of oligonucleotides, 10 times diluted cDNAs, and LightCycler^®480 SYBR Green-I Master 2x (Roche, Germany), using three replicates per sample. The runs were carried out on a LightCycler^®480II (Roche, Germany) using its own negative control and TATAA Interplate Calibrator (TATAA Biocenter, Switzerland) as the positive control. A standard SYBR Green Probe II program with 45 cycles was used.

The No. of subjects with IFN-α biomarkers and induced innate immunity effectors was calculated. The fold changes of the transcript levels in relation to basal levels of oropharynx and PBMC samples were measured from the crossing points (CP) of amplification curves collected for the RT-PCR. For this, we used the REST 2009 program (Pfaffl et al., 2002) with the CP and efficiency value and estimated the change factor in gene level after normalization with reference genes in both samples. A change factor indicating expression ≥1 was considered relevant.

The processing of biological samples and analytical techniques took place at the biomedical research laboratories of the CIGB. Vázquez-Blomquist et al. (2022) describe the experimental protocol and analytical techniques in an original research article.

Prophylactic health interventions

The prophylactic use of IFN-α2b nasal drops was evaluated and approved by a Research Ethics Committee designed by the Cuban Ministry of Public Health. A community health intervention with Nasalferon was carried out on international travelers and their close cohabiting contacts (CCC) in the cities of Havana and Cardenas (Matanzas Province). Two or three CCC per traveler were placed in the treatment group and one CCC per traveler in the control group. An institutional intervention was also conducted on tourism workers from four hotels located in Varadero, a resort near Cardenas.

Both interventions had a nonrandomized, open-label design to compare groups of treated and untreated individuals. The treated group included subjects between 19 and 80 years of age who received prophylaxis with IFN-α2b nasal drops (Nasalferon, CIGB, Havana, Cuba, 10 MIU/mL, within a vial with 2 mL) in self-administered doses of one drop in each nostril twice a day (2 MIU daily) for 10 consecutive days. Individuals under 19 years of age or over 80 years of age were considered within the untreated control group. Subjects with thimerosal hypersensitivity and those who refused consent were not treated.

The daily absence of clinical and respiratory symptoms and the nondetection of SARS-CoV-2 infection by RT-PCR during the 10-day prophylactic IFN-α2b nasal drop treatment were the primary endpoints of success in both interventions. The database with records of COVID-19 patients diagnosed by the Cuban national health system was monitored up to 30 days after including the last subject.

Safety profile analysis

An active pharmacosurveillance of the Nasalferon safety profile was implemented in subjects enrolled in clinical trials and health interventions. We evaluated the daily safety profile through the identification of adverse events (AE) caused by IFN-α2b nasal drops in the 10-day treatment. The AE classification followed the scales established by the World Health Organization (WHO) for the intensity, severity, and frequency of occurrence in the subjects (WHO, 1992).

AE intensity could be mild (when not requiring treatment), moderate (when requiring a specific therapy), and severe (when the modification of the original therapeutic schedule was necessary). Any AE resulting in death was classified as serious according to the consequence scale. Furthermore, any AE related to the hospital admission or prolongation of the patient’s stay, persistent significant disability, or life-threatening conditions was considered serious. The terminology established by WHO for naming and coding the clinical information regarding medicinal treatments (WHO Adverse Reaction Terminology, WHO-ART) was used (WHO, 2009). For adverse events frequency, we used the criteria of the Council of International Organizations of Medical Sciences (CIOMS) that grouped the AE by frequency based on the No. of affected subjects (CIOMS, 1995).

Statistical analyses

Descriptive statistics to estimate the measures of central tendency and dispersion (mean and extreme values) were used. The statistical tests depended on the type of distribution of the data. We analyzed the association between qualitative variables through contingency tables and the Fisher’s exact test, or chi-square was calculated for proportion comparison. Moreover, to compare independent samples, we used the t-test or the nonparametric Mann–Whitney test (U test). A nonparametric Wilcoxon test was used to evaluate the difference between two correlated samples. Through the odds ratio, we measured the association between treatments and outcomes.

Results

Pharmacodynamic study

Ninety-one healthy volunteers were included from July 2020 to October 2020. There were 54 women (59.3%) and 37 men, averaging 53 years of age, which ranged from 19 to 85 years old. No statistically significant differences were observed in the mean ages between the genders (women were 53.1 years old and men were 53.8 years old; P = 0.650).

The first period included 24 subjects, where the first 12 patients received Nasalferon twice a day and the following 12 received it once a day for 3 consecutive days. Forty individuals were treated twice a day for 10 consecutive days in the second period, and 27 subjects placed in three randomized groups of nine individuals received one application during 3 consecutive days in the third period.

Table 1 shows the No. of subjects expressing the IFN-α biomarkers and innate immunity effectors after the IFN-α2b nasal drop treatment at all three dose levels studied. PD activation or immune activity was confirmed in all subjects, although transcript expression in the entire set of samples tested was not obtained. Each IFN-α biomarker or innate immune effector analyzed was found in more than 50% of the subjects with an activated expression of transcripts in both oropharynx and PBMC. No significant differences between genders were found in gene expression.

Table 1.

Patients with Pharmacodynamics Biomarkers and Innate Immunity Effectors Expression After IFN-α2b Nasal Drops Treatment at All Three Dose Levels Studied

Dose (MIU)	OAS1		β-2MG		TLR3		TLR7		TLR8		IFN-α	STAT1	STAT3
Dose (MIU)	OM	PB	OM	PB	OM	PB	OM	PB	OM	PB	OM	PB	PB
0.25 q.d	4/9	NE	NE	NE	2/9	NE	2/9	NE	3/9	NE	2/9	NE	NE
0.25 q.d	44.4%				22.2%		22.2%		33.3%		22.2%
0.50 q.d	5/9	NE	NE	NE	5/9	NE	5/9	NE	4/9	NE	5/9	NE	NE
0.50 q.d	55.6%				55.6%		55.6%		44.4%		55.6%
1.0 q.d	18/21	NE	NE	NE	16/21	NE	16/21	NE	15/21	NE	16/21	NE	NE
1.0 q.d	85.7%				76.2%		76.2%		71.4%		76.2%
1.0 b.i.d	47/50	24/40	6/11	6/12	2/11	7/8	2/4	6/8	3/5	6/8	NE	7/8	8/8
1.0 b.i.d	94.0%	60.0%	54.5%	50.0%	18.2%	87.5%	50.0%	75.0%	60.0%	75.0%		87.5%	100.0%
Total	74/89	24/40	6/11	6/12	25/50	7/8	25/43	6/8	25/44	6/8	23/39	7/8	8/8
Total	83.1%	60.0%	54.5%	50.0%	50.0%	87.5%	58.2%	75.0%	56.8%	75.0%	59.0%	87.5%	100.0%

b.i.d, twice a day; β-2MG, beta-2 microglobulin; IFN-α, interferon alpha; MIU, million international unit; NE, not evaluated; OAS1, 2′-5′-oligoadenylate synthetase 1 protein; OM, oropharyngeal mucosa; PB, peripheral blood; q.d, once a day; STAT, signal transducers and activators of transcription; TLR, toll-like receptor.

In subjects showing activation of all or at least one of the transcripts, we found a dose-dependent global and specific gene expression of IFN-α2b (Fig. 1). The lowest proportions of subjects with PD activity and immune activation were observed in those using 0.25 MIU and 0.50 MIU doses. Both doses showed a 50% reduction of subjects presenting gene expression of all transcripts.

FIG. 1.

IFN-α2b nasal drops upregulate the expression of antiviral PD biomarkers and innate immunity effectors. Quantification of mRNA for the human genes expression levels was measured in oropharynx swabs and blood samples using the RT-PCR technique. The proportion of patients with activation of all or at least one of the transcripts at any of the five sampling times after starting treatment is shown. Dose-dependent gene expression of IFN-α2b was found. Statistically significant differences by chi-square test for comparison of proportions were observed. The lowest proportions of subjects with PD activity and immune activation after 0.25 MIU and 0.50 MIU doses were obtained. Both doses showed a 50% reduction of subjects with gene expression for all transcripts. Number inside the bar is the quantity of subjects expressing transcript. N = total of subjects. P < 0.05: statistical significance level. b.i.d, twice a day; IFN-α2b, interferon alpha-2b; MIU, million international units; mRNA, messenger ribonucleic acid; PD, pharmacodynamics; q.d, once a day; RT-PCR, real-time polymerase chain reaction.

Statistically significant differences were found between 0.25 MIU and 1.0 MIU and between 0.50 MIU and 1.0 MIU applied twice daily. However, between each dose and its immediately higher level, there were no significant differences. Results revealed that reducing the dose of Nasalferon from 1.0 MIU to 0.5 MIU applied once daily does not eliminate the gene expression of antiviral activity or the innate immune response activation. Reducing the application frequency from twice to once a day did not change the effect of the product.

The proportion of subjects expressing OAS1 showed no statistical difference with sampling time (Fig. 2). In 23/40 (57.5%) subjects, there was a sustained expression from 24 h to 120 h. Its expression was detected in 24/40 (62.5%) subjects tested on the 13th day of the last administration of Nasalferon, including 11 (27.5%) with a sustained expression as of the first day of the treatment.

FIG. 2.

OAS1 expression remains raised during treatment with IFN-α2b nasal drops. Quantification of mRNA for the expression levels of human genes encoding OAS1 was measured in oropharynx swabs and blood samples using the RT-PCR technique. The proportion of patients with OAS1 expression at each sampling time after starting treatment is shown. A sustained expression of the PD antiviral biomarker was detected. No statistically significant differences by chi-square test for comparison of proportions were observed. Number inside the bar is the quantity of subjects expressing OAS1. N = total of subjects. P < 0.05: statistical significance level. mRNA, messenger ribonucleic acid; OAS1, 2′-5′-oligoadenylate synthetase 1; PD, pharmacodynamics; RT-PCR, real-time polymerase chain reaction.

IFN-α biomarkers and innate immune effectors showed a sustained expression. The expression on the third day of the treatment showed at least a 1-fold increase in all the transcripts (Fig. 3). The average expression of OAS1 in the oropharyngeal mucosa increased up to 3-fold and 6-fold after 24 h and 72 h, respectively. Two subjects had an 8-fold increase at 24 h, and eight had more than a 10-fold increase at 72 h. The maximum peak of expression occurred at 72 h in a subject with a 65-fold increase from the baseline value. The expressions in PBMC compared with oropharynx were 4-fold to 6-fold lower.

FIG. 3.

Fold changes of transcript levels on the third day after Nasalferon treatment started with respect to basal levels in oropharynx and PBMC. Samples were collected in the first hour after the last IFN-α2b nasal drop application. A change factor in gene expression levels was reported after normalization with reference genes GAPDH (Glyceraldehyde-3-phospate dehydrogenase), YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta), and HMBS (Hydroxymethylbilane synthase). A change factor indicating expression ≥1 was considered relevant. Higher activation in oropharynx was statistically different than in PBMC. β-2MG, beta-2 microglobulin; IFN-α, interferon alpha; IFN-α2b, interferon alpha-2b; OAS1, 2′-5′-oligoadenylate synthetase 1; PBMC, peripheral blood mononuclear cells; PD, pharmacodynamics; STAT, signal transducers and activators of transcription; TLR, toll-like receptor.

STAT1 and STAT3 were only quantified in PBMC and showed increased expressions in 87.5% of the samples. In nasopharyngeal swab cells, it was impossible to study these transcripts due to the inaccurate expression levels of the genes and their poor yields of total RNA.

More than 50% of the subjects evaluated showed increases in TLRs without a homogeneous pattern in the expression of transcripts in the oropharynx. Greater increases were detected in PBMC samples. The levels of the β-2MG transcripts increased in 50% of the subjects but not as much as with OAS1 or STATs.

Baseline serum levels of IFN-α2b in PBMC increased in 6 out of 12 subjects on the third consecutive day with the Nasalferon treatment. No statistically significant differences were found using Wilcoxon matched-pairs signed-rank test.

Prophylactic health interventions

Two health interventions were made in January 2021 that included 29,082 subjects, with 14,804 women (50.9%) and 14,278 men. Their average age was 43 years old, ranging from 19 to 85 years. Of these subjects, 10,377 (35.7%) were treated with Nasalferon. Statistically significant differences in gender were found in the treated subject (5,113 females for 49.3% and 5,264 males, P = 4 × 10⁻⁵).

Out of 2,930 travelers arriving on international flights, who would be living with their family or friends, we treated 5,728 CCC in the community intervention from January 1 to January 31, 2021. The travelers and CCC received the drug; the nursing staff at the medical post trained them for the self-administration of the product, and they completed the treatment at home. At the same time, 9,162 untreated travelers and 9,162 CCC were included as controls. The average age of the subjects was 44 years old.

Table 2 shows statistically significant differences in COVID-19 infection rates between the treated and the untreated subjects in both regions. The inclusion in Havana started on January 8 after detecting 138 cases in 2,507 international travelers (COVID-19 infection rate = 5.5%), and at the end of the month, there were 367 cases in 11,150 subjects (COVID-19 infection rate = 3.3%). The inclusion in Cardenas started on January 17 after detecting 63 cases in 390 international travelers (COVID-19 infection rate = 16.2%), and the rate was reduced to 79 cases in 942 subjects as of January 31 (COVID-19 infection rate = 8.4%). Our data suggest that the treatment with intranasal IFN-α2b reduced the risk of developing COVID-19 in healthy volunteers. Specifically, after applying Nasalferon in only 24.2% of the international travelers in Havana and Cardenas in January, there was a reduction in the incidence of COVID-19 of 46.8% in this population at the end of the month.

Table 2.

COVID-19 Rate in Community Health Intervention in Travelers and Close Cohabiting Contacts

	Treatment		No treatment
	Travelers	CCC	Travelers	CCC	P value^b
Rate^a (%)
Havana	2 / 2,825 (0.07)	0 / 5,411 (0.00)	365 / 8,325 (4.38)	557 / 8,325 (6.69)	10⁻⁵
P value	0,118^c		10^−5d
Cardenas	1 / 105 (0.95)	1 / 317 (0.32)	78 / 837 (9.32)	26 / 837 (3.11)	10⁻⁵
P value	0,436^c		10^−5d
Total	3 / 2930 (0.10)	1 / 5,728 (0.02)	443 / 9,162 (4.84)	583 / 9,162 (6.36)

COVID-19 rate = COVID-19 cases number / No. of subjects.

Fisher’s exact test for treated versus untreated subjects.

Fisher’s exact test.

Chi-square test.

CCC, close cohabiting contacts.

The four CCC subjects (three men and one woman, P = 0.625) who developed SARS-CoV-2 in the treated group showed a mild COVID-19 infection with a satisfactory recovery. The untreated group had 1,026 COVID-19 cases diagnosed, 521 women (50.8%) and 505 men, with no statistically significant differences between genders (P = 0.518).

Four hotels having 2,100 tourism workers and with a detected active spread of COVID-19 were selected for an institutional intervention carried out from January 17 to February 9, 2021. The inclusion consisted of 1,719 healthy tourism workers (81.9%) who received the self-administered treatment of Nasalferon supervised by the nursing staff at the medical post of the hotel. The control group consisted of 381 untreated healthy workers. All subjects were 39 years old on average.

The first case of COVID-19 in this population of tourism workers was detected on January 8, 2021, and the number of new cases increased during the following nine days before the intervention. The use of IFN-α2b nasal drops eliminated the spread of the virus in an average of 5 days (range of 1–8 days) in the four hotels, and the last subject with SARS-CoV-2 infection was detected on January 25. No cases of SARS-CoV-2 infection were reported in the Nasalferon group, and there were 39 (10.2%) cases of COVID-19 in the untreated group. Our data reveal a rapid and effective prophylactic effect of intranasal IFN-α2b in confined populations. Specifically, Nasalferon prevented the infection of SARS-CoV-2 between individuals working in closed facilities. Whether or not the treated and untreated subjects were separated within the hotel institution, the use of the product interrupted viral transmission and prevented new COVID-19 cases during the following 15 days (62.5%), up to the end of the intervention.

In Table 3 there is an overall analysis of both interventions. There were statistically significant prophylactic effects in SARS-CoV-2 infection within the high-risk population treated with IFN-α2b nasal drops compared with the nontreated group. Nasalferon prevented 5.7 SARS-Cov-2 infections in every 100 subjects treated, which means that 1 out of 18 individuals is protected.

Table 3.

Prophylactic Effects with IFN-α2b Nasal Drops (Nasalferon) in SARS-CoV-2 Infection High-Risk Population

	Treatment	No treatment
COVID-19 rate^a (%)	4 / 10,377 (0.04)	1,065 / 18,705 (5.69)
P value^b	10⁻⁵

COVID-19 rate = COVID-19 cases number / No. of subjects.

Fisher’s exact test for treated versus untreated subjects.

Safety profile analysis

The study of the Nasalferon safety profile included 6,391 subjects. The adverse events were of 50 types, and there were 1,002 reports in 654 (10.2%) individuals. The main and most frequent AE (≥1.0%) according to the CIOMS scale were headache (4.3%), rhinorrhea (1.5%), and fatigue (1.3%). Moreover, 13 AE (26.0%) ranked as uncommon (≥0.1% to <1.0%), and 34 AE (68.0%) were considered rare (≥0.01% to <0.1%). There were 930 (92.8%) mild AE and no reports of severe AE. All AE associated with the use of intranasal IFN-α2b were mostly local, short-lived, and reversible, not requiring any concomitant medication and not having any negative repercussion on the adherence to the treatment.

Discussion

The sustained increase in the overall incidence of COVID-19 emphasized the urgent need to identify a drug with antiviral activity against SARS-CoV-2. The clinical trial conducted by Zhou et al. provided valuable preliminary evidence of the benefits of the IFN-α2b treatment and set the grounds for the use of this drug as part of public health strategies to contain the transmission of SARS-CoV-2 and accelerate the elimination of the virus in COVID-19 patients (Zhou et al., 2020). The respiratory mucosa, as the main entry point of SARS-CoV-2, is of greater interest in the nasal administration of IFN-α, either for the prevention or the treatment of the disease, as was suggested during the COVID-19 pandemic (Higgins et al., 2020).

Pharmacodynamic studies are able to evaluate the capability of IFN formulations to activate their own intracellular signaling cascade pathway and produce antiviral action. OAS-directed ribonuclease L is responsible for viral genome degradation and one of the main effector pathways of the IFN-mediated antiviral response (Sadler and Williams, 2008). Levels of OAS are commonly used in PD studies because individuals undergoing IFN-α treatment should display high levels of OAS activity. Hence, the expression of this protein is a classical surrogate marker of the antiviral activity of IFN, which usually corresponds to the success of the treatment (Schoggins, 2019). Because of this, we used OAS1 as the primary transcript evaluated.

TLR3, TLR7, and TLR8 are essential components of the innate and adaptive immune response for the recognition and neutralization of viral nucleic acids. The functions of the TLRs lead to the activation of IRF-3 and IRF-9, which promote a type I IFN induction (Uematsu and Akira, 2007). Hence, the immunomodulatory potential of IFN-α2b nasal drops evaluated through TLRs gene expression was considered to be the correct routine in our work.

Zedan et al. (2020) and Hatton et al. (2021) reported the necessary IFN-α signaling pathway for the suppression of viral replication in the olfactory epithelium. Our finding of the gene expression of eight transcripts, including STAT1 and OAS1, is considered sufficient evidence to affirm that the IFN-α signaling system is actively involved in the antiviral responses originating within the olfactory mucosa after Nasalferon administration. The sustained expression of OAS1 in the oropharynx from 24 to 120 h after starting treatment indicated that Nasalferon leads to a relevant sustained PD activity in the critical entry area of the viral respiratory infection. The lack of significance between IFN-α serum concentration at the baseline and on the third day after starting the Nasalferon treatment confirms the local and significant antiviral activity. This finding allows us to affirm that nasal drops remain in the nasopharyngeal cavity without any relevant absorption into the systemic bloodstream.

Our study in healthy volunteers demonstrated the excellent PD potential of Nasalferon to induce the temporary reinforcement of the immune system in high-risk populations and vulnerable groups in recent, ongoing, or immediate direct exposure to respiratory viruses. Gene expression occurred irrespective of the gender in a population with a predominance of women. We consider that the fact that the biological effect is not affected by unequal gender distributions is a significant advantage for the drug.

Nasalferon represents an important noninjectable and locally applied alternative, which is highly useful in health strategies against extremely communicable respiratory viruses. Drugs applied as nasal drops are of simple and friendly ambulatory use in the primary health system, through the self-application of the drug by the patients without interrupting their daily activities. Specifically, Nasalferon optimizes medical and therapeutic management because one vial contains the No. of IFN-α2b doses required for the complete treatment.

The national Cuban protocol for COVID-19 care includes the use of IFN-α2b as one of the antiviral health measures. International travelers arriving in Cuba through the airport and tourism workers are the two most exposed populations to the direct infection by SARS-CoV-2. This supports the evaluation of the antiviral effects of Nasalferon in both groups (Cañete et al., 2021). Taken as a whole, the open and confined groups of subjects evaluated, resulting in a positive outcome, indicates that IFN-α2b nasal drops are a viable option that may benefit any population with viral respiratory diseases. Our data demonstrated that it is possible to obtain relevant prophylactic effects in a population, even when not treating all persons. These findings emphasize that the increase in the gene expression of the biomarkers is clinically materialized through the expected immunomodulatory and antiviral biological effects of this drug.

The fact that there is an identical pattern of results in two regions with different clinical research groups supports the accuracy of the methodology used and reveals the occurrence of a nonrandom prophylactic effect. The difference in the clinical outcomes between treated and untreated subjects highlights the decisive role of Nasalferon within the array of health and hygiene measures implemented in Cuba to minimize the risk of infection by SARS-CoV-2. The prophylactic effect was not related to gender, in spite of having more men than women in the treated group. The fact that gender does not affect the clinical outcome results in an advantage for the drug.

Nasalferon has proven to be a very safe medication with a treatment schedule associated with a low probability of AE that is not dose dependent. Results during the COVID-19 pandemic highlight the fact that IFN-α2b nasal drops can be used reliably, safely, and effectively for prophylaxis or to attenuate the amount and intensity of symptoms and reduce the duration of the disease. The mild and short course of the respiratory symptoms observed in a few subjects with COVID-19 after the prophylactic application of Nasalferon not only supported the clinically relevant impact of using this approach but also introduced the potentially beneficial use of the intranasal route for therapeutic purposes. Our clinical results with Nasalferon agree with prophylactic evidence obtained by the prospective open-label trial of recombinant human IFN-α1b nasal drops to prevent COVID-19 in healthy Chinese medical staff from an epidemic area in Shiyan City, Hubei Province (Meng et al., 2021).

The limitation of the PD results of this study is that it only analyzed the expression of OAS1. Other prototypical ISGs such as IFN-induced proteins with tetratricopeptide repeats (IFITs), the C-X-C motif chemokine ligand 10 (CXCL10), and the Mx protein would have to be evaluated. Another limitation is the nonuniform number of patients analyzed in the data available on the biomarker and immune system effectors.

In spite of these limitations, we demonstrated the activation of the IFN-α mechanism of action in the oropharynx after the administration of our IFN-α2b nasal drop formulation. We also demonstrated the upregulation of the classic ISG OAS1 and other molecular and cellular elements related to the induction of innate and adaptive immune responses in the mucosa and peripheral bloodstream. The immunomodulatory and antiviral prophylactic effects of Nasalferon in high-risk SARS-CoV-2 infection populations were proven by two health interventions.

In summary, our study highlighted that the treatment with IFN-α2b may be effective for health strategies under the conditions of uncontrolled SARS-CoV-2 transmission. The effectiveness and safety profile of Nasalferon for other acute viral respiratory diseases must be confirmed in further clinical trials.

Footnotes

Acknowledgment

The authors are grateful to Jose Enrique Brito Leon and Carolina Rodriguez Rius from CIGB in Havana, Cuba, for supplying the drug for the PD study and health interventions. The authors also thank Adriana La Torre Cinza and Miriam Ribas Hermelo for the review, grammatical corrections, and edits made on the article.

Authors’ Contributions

H.N.C.: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Writing—original draft (lead); Methodology (equal). O.B.: Data curation (equal); Methodology (equal); Project administration (supporting). R.C.: Investigation (equal); Resources (equal). D.V.B: Formal analysis (equal); Investigation (equal). D.Q.: Formal analysis (equal); Investigation (equal). A.A.B.: Resources (lead); Reviewing and editing (equal). G.G.N.: Conceptualization (equal); Methodology (equal). A.A.: Supervision; Project administration (supporting). I.M.: Conceptualization (equal); Project administration (lead).

Author Disclosure Statement

H.N.C., D.V.B., D.Q., A.A., and G.G.N. are employees in the CIGB, Havana network, where Nasalferon is produced. The rest of the authors have no competing interests at all, such as relevant affiliations or financial involvement with any organization or entity with a financial interest in, or financial conflict with, the subject matter or materials discussed in the article. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Funding Information

No funding was received for this article.

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