Evaluation of the Effect and Safety of HeberFERON vs Heberon Alpha in Patients Infected with Corona Virus SARS-CoV-2 (Study ESPERANZA/HOPE): Study Protocol for a Randomized Controlled Trial.

Background: As the outbreak of COVID-19 has accelerated, an urgent need for nding strategies to combat the virus is growing. Results from in vitro studies suggest that a combination of IFN type I and Type II may be effective against SARS-CoV. The aim of this study is to investigate the ecacy of treatment with a recombinant IFN alpha 2b and gamma, provided with standard protocol (Kaletra (lopinavir-ritonavir 200/50 mg; 200/100 mg every 12 hour for 30 days; Chloroquine (250 mg) every 12 hours for 10 days) for COVID-19 patients, compared to standard protocol (IFN alpha 2b/Kaletra/Chloroquine) for COVID-19 hospitalized patients, positive diagnosed for SARS-Cov-2. Methods: Hospitalized adult patients with qPCR conrmed SARS-Cov-2 will be enrolled in this open-labeled, single center, prospective, randomized and controlled clinical trial. One hundred and twenty eligible patients with conrmed SARS-CoV-2 positivity by qPCR amplication in oropharyngeal/nasopharyngeal swab samples will be enrolled at “Luis Diaz Soto” Hospital, Havana, Cuba. The primary outcomes are the time to 2019-nCoV RNA negativity in patients and the time until progression to severe COVID-19. Discussion: This will be the rst randomized controlled trial of a potential treatment for SARC-Cov-2 using the combinations of IFNs.


Title {1}
Phase II clinical, randomized, clinical trial of evaluation of the effect and safety of HeberFERON versus Heberon alfa in patients infected with the SARS-CoV-2 coronavirus (Study ESPERANZA/HOPE).

Exclusion Criteria
Exclusion criteria: 1) Patients with decompensated chronic diseases at the time of inclusion (severe arterial hypertension, ischemic heart disease, diabetes mellitus, etc.). 2) Patients with a history of autoimmune diseases. 3) Presence of hyperin ammation syndrome. 4) Serious coagulation disorders. 5) Known hypersensitivity to any of the components of the formulation under study. 6) Pregnancy or lactation. 7) Obvious mental incapacity to issue consent and act accordingly with the study.
Condition patients infected with the SARS-CoV-2 coronavirus.

Intervention
Group A: HeberFERON (Recombinant Interferon Alpha Gamma, 3.5 MIU), subcutaneously, twice a week for three consecutive weeks. Group B (Control): Heberon Alfa R (Recombinant Interferon alfa 2b, 3.0 MIU), subcutaneously, three times per week for three consecutive weeks. All the patients included in the study (as part of the protocol of action foreseen in the country for cases of SARS-CoV-2 in nasopharyngeal exudates) receive basic treatment (in addition to interferon according to study group) with caletra, chloroquine, azithromycin or roce n, depending on the magnitude of respiratory symptoms.  There are three types of Interferons (IFNs): type I (IFN-α and IFN-β), type II (IFN-γ) and type III (IFN-λ). IFNs are proteins created by nature for the rst line defense against pathogens (viruses, bacteria, parasites), a function demonstrated in a group of species at different levels of the evolution chain, con rming their protective role.
Infection with mammalian cell viruses incites the innate immune system to establish a rst line of defense. IFNs play a key role in these events, as they activate the innate immune system and help shape adaptive immunity. IFN-γ is the main modulator in establishing the relationship between these two types of immune responses.
IFNs possess pleiotropic effects that overlap on various cellular functions. IFNs-α and β have a greater antiproliferative and antiviral effect and IFN-γ a superior immunoregulatory activity , .
IFNs-α and -γ exercise their functions through different but related signaling pathways. Studies in animal models have shown that IFNs-α and -γ are essential for antiviral defense and are functionally nonredundant.
Viruses have developed mechanisms to evade the functions of the IFN system by simulating proteins that intervene in the synthesis mechanisms of IFNs, and intracellular signaling to establish the protection mechanisms by these (IFN receptor and other signaling cascade proteins) , .
Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is a positive-sense, enveloped, singlestranded RNA β coronavirus similar to Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) virus. This type of virus is susceptible to the antiviral action of IFNs, but variations have been developed to interfere with this activity.
Viruses have developed mechanisms to evade the functions of the IFN system by simulating proteins that intervene in the synthesis mechanisms of IFNs, and intracellular signaling to establish the protection mechanisms (IFN receptor and other proteins of signaling cascades) ,13 .
SARS-Co, which emerged in 2003 and caused widespread human mortality, encodes at least ve proteins that function as IFN antagonists ,,, , suppressing signaling and production components. These proteins are believed to be responsible for the pathogenesis of SARS-CoV.
The induction of IFNs plays a fundamental role in defending the body against CoV infections. Numerous studies have presented the effectiveness of direct administration of IFN to eliminate these viruses.
IFN-γ has been reported to have an antiviral effect in coronavirus infection. This effect is described mediated by the recruitment of monocytes and T lymphocytes to the area of the infected cells and the production of IFN-γ by them. In the absence of IFN-mediated signaling, infection leads to the death of The higher mortality rate from COVID-19 in elderly patients has been associated with a greater delay between the time of infection and the response of the immune system, caused mainly by the inhibitory effects of the virus on it. Early administration of IFNs could reduce this window of antiviral inactivity and decrease SARS mortality. Combining IFN-γ and IFN-α synergistically could maximize this bene t.

Heberon alpha R®
Heberon Alfa R® (IFN alfa-2b) is a drug produced in Cuba by the Center for Genetic Engineering and Biotechnology (CIGB), which has remained a product with proven antiviral e cacy and an adequate safety pro le for 34 years.
The evidences are different for preventive use and the treatment at early stage of the disease, so Heberon Alpha R is one of the interventions included in the Cuban protocol for the management of COVID-19 and about 76,900 doses have been supplied to the National Health System by the CIGB since March 2020. The current Cuban recommendation for patients is to use recombinant interferon alfa 2b (Heberon alfa 2b) 3 million of units intramuscular routs thrice a week at early stage.
Today there are several clinical studies with IFN alpha-2b in COVID-19 registered at clinicaltrials.gov and guideline issued by Expert Committees of WHO, Singapore, South Korea and US Institutions recommend the clinical use of IFN alpha for the treatment and prevention of COVID-19. However, the use of IFN in advance stages of the disease has been inconsistent.

HeberFERON®
HeberFERON® (IFN-α2b + IFN-γ) is a drug produced in Cuba by CIGB, which was approved in Cuba by CECMED in 2016 for the treatment of basal cell carcinomas (BCC), and since then it has been routinely administered to more than 3,000 patients with advanced, high-risk or multiple BBC in the country.
HeberFERON® has been extensively studied through physicochemical studies, biological activity in vitro and in vivo, in studies of pharmacokinetics, pharmacodynamics in humans , , animalsand toxicology , .
The proteins, enzymes and metabolites mediating the antiviral effect (2-5 OAS, β-2 microglobulin, Neopterin) of IFNs are synergistically stimulated by HeberFERON 28,29 . The main intracellular signaling factor common to both IFNs, STAT-1 7 has also been synergistically stimulated and has been shown to be a target of SARS-CoV antagonism on the IFN system 12 .
These evidences support the use of this therapeutic candidate for the control of COVID-19 in the early stages of the disease and in patients positive for the virus.
We have decided to compare these two formulations of IFNs to de ne the antiviral of IFN formulations activity against SARS-CoV-2 in a controlled randommized clinical trial.

Objectives {7}
Taking into account that the pharmacodynamics of HeberFERON® is superior to that of the IFNs separately, based on the stimulation of the genes of antiviral activity involved in the defense of the organism against COVID-19, between 2-5 times more, and that both IFN alpha and IFN gamma demonstrate antiviral activity against coronavirus infection synergistically, it is expected that patients treated with HeberFERON® achieve a reduction in the time until the SARS-CoV-2 RNA negative (absence of the virus according to the technique of qPCR in real time) with 20% superiority over the group treated with Heberon alpha R®.

Trial Design {8}
The investigation was designed as an open-labeled, single center, prospective, randomized and controlled clinical trial. One hundred and twenty eligible patients with con rmed SARS-CoV-2 positivity by qPCR ampli cation in oropharyngeal swab samples will be enrolled at "Luis Diaz Soto" Hospital, Havana, Cuba, based on a power of 80%, and a level of con dence set at 95%, while also considering a dropout rate of 5%. Patients will be block randomized individually to one of two treatment arms by means of random computer-generated lists, with an allocation ratio of 1:1, with block sizes of six patients.
Blood, nasopharyngeal swab, sputum, stool, and urine samples will be collected for laboratory examinations, which also includes hematological analysis, 6. Pregnancy or lactation, 7. Obvious mental inability to issue consent and act accordingly on study.
Who will take informed consent? {26a} 1. Patients will be asked for written consent to participate after having been duly informed about the characteristics of the trial, objectives, bene ts and possible risks. They will have the necessary time to decide (minimum 24 hours). Likewise, they will be informed of the right to participate or not and to withdraw their consent at any time, without exposing themselves to limitations for their medical care or other retaliation.
2. The investigator should obtain the patient's written consent only after making sure they understood all the information offered. The procedure will be provided by means of a standard writing; in language easily understood by the patient (it should not be technical, but practical). Neither the investigator nor the trial staff can in uence the patient's decision to participate or continue in the trial.
All patients who provide informed consent will be randomly allocated into one of two study groups.
Additional consent provisions for collection and use of participant data and biological specimens {26b} The consent for collection and use of participant data and biological specimens is included in the informed consent

Intervention group
The intervention group will receive the standard of care as well as, Kaletra and chloroquine as described 33 , and HeberFERON. HeberFERON will be administered two times per week at 3.5 MIU for 3 weeks.

Control group
The control group will receive standard of care as well as Heberon Alpha R, Kaletra, and chroloquine. Heberon Alpha R will be administered three times per week at 3.0 MIU for 3 weeks, with Kaletra and chloroquine as described in the CUBAN NATIONAL ACTION PROTOCOL FOR COVID-19 Version 1.4 .33 .
Criteria for discontinuing or modifying allocated interventions {11b} In the event of serious adverse events (with proven causality) the administration of the product will be interrupted and the required measures will be taken depending on the event and the possible expedited report of the same will be considered. The occurrence of the event will be noti ed to the monitor within a period not exceeding 24 hours.
The person responsible for the investigation, the monitors and the team of investigators, will carry out the corresponding investigations in order to determine the component causing the undesired effect.
After recovery from the adverse event, the patient may restart treatment according to the treatment scheme has been received. If severe toxicity occurs again, treatment is permanently discontinued.

Strategies to improve adherence to interventions {11c}
Prior to the preparation of this protocol, a criteria uni cation workshop was held with the participation of clinical specialists and opinion leaders involved in the project, where the experimental evidence in animals and humans, as well as the elements of rationality, were presented and discussed. The analysis, discussion and mastery of the protocol will favor adherence and compliance with GCP by all researchers.
The trial monitors will make quality monitoring visits at all stages of its execution, ensuring strict compliance with the provisions of the protocol.
In the case report form (CRF) there is a Dipirone or acetaminophen will be indicated to relieve fever, headache, and pain if necessary, depending on the doses listed below, taken from the AHCPR "Guidelines for the Management of Acute Pain" [ii] .  We hope to enroll a sample size of one hundred and twenty patients for this study, based on a power of 80%, and a level of con dence set at 95%, while also considering a dropout rate of 5%. One hundred and twenty eligible patients with con rmed SARS-CoV-2 positivity by qPCR ampli cation in oropharyngeal swab samples will be enrolled at Luis Diaz Soto Hospital, Havana, Cuba. Patients will be block randomized individually to one of two treatment arms by means of random computer-generated lists, with an allocation ratio of 1:1, with block sizes of six patients.

Concealment mechanism {16b}
There will be a centralized randomization at CIGB. The randomization procedure will be carried out by the Supply Group of the Direction of Clinical Investigations at CIGB. Patients will be block randomized individually to one of two treatment arms by means of random computer-generated lists, with an allocation ratio of 1:1, with block sizes of six patients.

Implementation {16c}
The information of the treatment group (A or B) will only be known after the patient is included. At the time of inclusion (after the selection criteria have been veri ed and written informed consent has been obtained), the clinical investigator will give the information to the CIGB monitors (who will visit the hospital daily and remain at their posts command). After collecting the general patient information to include (initials, date and time), will assign the inclusion number and the corresponding treatment (consecutive, according to a random list in their possession). For the purposes of this study, a data entry system will be generated at OpenClinica, which is a free software platform for protocol con guration and CRF design, which allows the electronic capture, storage and management of data.

Assignment of interventions: Blinding
The data entry will be carried out in duplicate (independently by two operators) for the subsequent process of automatic comparison and correction of the bases, necessary for statistical analysis with accurate information from the trial. For debugging errors, the data that does not match the one registered in the original CRF will be corroborated to avoid confusion. The comparison will be repeated until no differences are found between the databases. This process will guarantee the cleanliness of the data included in the databases that will later be used in the statistical analysis. This activity will be recorded, so that it can be traced to national and foreign inspections and / or audits.

Con dentiality {27}
The protocol's medical specialists, the promoter, the monitors and auditors appointed by the promoter will guarantee that the personal data of the subjects included in the protocol are treated in accordance with the provisions established in Law 15/1999 on data protection personal nature and the regulations that develop it. Likewise, the anonymity of the subjects included and the protection of their identity will be maintained; no personal data of the subjects of the protocol will be transferred, except in those circumstances allowed by law.
Monitors and auditors appointed by the promoter may access clinical information and documentation on the subjects included, in order to verify the accuracy and reliability of the data, but must not collect the personal identi cation data of the subjects. Access to this data should also be provided to the inspectors of the competent health authorities.
The results of the clinical trial, as well as all the work and reports carried out and all the industrial property rights derived from it, are the exclusive property of the promoter. The latter is committed to disseminating them, once the protocol is nished and whether they are negative or positive, in public access media.
The publication of the results, by the medical specialists of the hospital institutions, in scienti c magazines or books and the oral presentations or posters at scienti c events, workshops or meetings, must be carried out in agreement with the promoting center.
Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} The promoter center will be in charge of transmitting, operationally, the procedure for the extraction, identi cation and conservation of the biological samples that will be transferred to the CIGB according to The transfer of samples from the healthcare unit will be the responsibility of the promoter center, which will guarantee the transportation, specialized personnel and resources necessary to carry out these operations with the maximum quality and compliance with GCP, as established by the procedures. in force (4.40.120.01 and 4.40.123.07) and their biosafety protocols.

Statistical methods
Statistical methods for primary and secondary outcomes {20a} We will compare the study endpoints among the two arms using time-to-event methods with the Cox proportional-hazards model. The different categorical variables will be analyzed using the one-way analysis of variance. To describe the e cacy and safety of the IFN regimens, Kaplan-Meier estimates and a multivariate Cox proportional hazards model will be used to compare severity of patients and adverse events among the two arms during the study period at week four. A p-value of <0.05 will be deemed to confer statistical signi cance.

Interim analyses {21b}
'Not applicable' Methods for additional analyses (e.g. subgroup analyses) {20b} 'Not applicable' Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c} 'Not applicable' Plans to give access to the full protocol, participant level-data and statistical code {31c} 'Not applicable' Oversight and monitoring Composition of the coordinating centre and trial steering committee {5d} Steering Committee The Steering Committee, led by the principal investigator, will be responsible for overseeing the conduction of the trial, providing training for new sites, ensuring compliance with the study procedures, addressing challenges that occur at all sites, reviewing serious adverse events and formulating the statistical analysis plan.
For clinical trials at CIGB, the function of coordinating centre is playing by the Direction of Clinical Research that is the responsible for design, endpoint adjudication, data management, statistical analysis and nal report for the clinical trial.
Monitors The trial monitors will carry out quality monitoring visits at all stages of its execution, ensuring strict compliance with the provisions of the protocol.
The ow of primary protocol information to and from health institution will be guaranteed by the CIGB.
The research product (HeberFERON), CRF and other models will be delivered / collected by the CIGB, directly by the study monitors. The monitors, in the quality monitoring visits, will collect the CRFs of those patients who concluded their participation in the trial. The Supply Group at CIGB is responsible for the adequate supply of medicines and medical supplies, as well as the collection of the product under investigation (dispensed and not dispensed).
The investigator should have a report of the number of patients included, the detected adverse events, the study departures and the causes of these, as well as any other relevant information during the course of the trial, for when they are requested during the quality monitoring visits.

Adverse event reporting and harms {22}
In this study, the occurrence of serious adverse events is not expected, due to short time of exposure and low doses of treatment. However, if they occur, treatment will be suspended and the required measures will be taken depending on the type of event.
Special attention should be paid to known adverse events for HeberFERON. The "Product Manual" (attached to the protocol) describes the behavior against the most signi cant adverse reactions (reported in other entities). The investigator responsible for the study at the participating institution will be responsible for the diagnostic evaluation and follow-up of patients with adverse events.
In the event of serious adverse events (with proven causality) the administration of the product will be interrupted and if this has not been concluded, the required measures will be taken depending on the event and the possible expedited report of the same will be considered.
The occurrence of the event will be noti ed to the monitor within a period not exceeding 24 hours. The person responsible for the investigation, the monitors and the team of investigators, will carry out the corresponding investigations in order to determine the component causing the undesired effect. After recovery from the adverse event, the patient may restart treatment according to the treatment scheme they were receiving, if the inclusion in their group had not been stopped due to exceeding the maximum level of toxicity. If severe toxicity occurs again, treatment is permanently discontinued Noti cation of adverse events It will consist of two stages: the immediate noti cation of all serious adverse events and the reporting of serious and unexpected adverse events in relation to causation, which will be independent of each other.
Immediate noti cation will be made to the national regulatory agency in Cuba, Center for State Control of the Quality of Medicines (CECMED), within the rst 72 hours after the Promoter becomes aware of the serious (serious) and unexpected adverse event and will be mandatory.
The model of the report of serious (serious) and unexpected adverse events in relation to causation and the attached documentation regarding the adverse event presented must be submitted to CECMED, as soon as possible and never after seven calendar days if the adverse event is fatal or compromises the subject's life, if not, the reporting time will be 15 days.

Expedited report of adverse events
To do the same, you must follow the instructions of procedure 4.40.039.01 in force at the CIGB Directorate of Clinical Investigations. Any serious unexpected adverse event that arises must be evaluated with a view to the expedited report.
For any serious unexpected adverse event, the causal relationship with the drug should be established to assess whether it is classi ed as an adverse reaction. According to the de ned terms, an adverse reaction is any harmful and involuntary event that has a reasonable possibility of a causal relationship between the medicinal product and the adverse event, meaning as reasonable causal relationship the existence of evidence, facts and arguments that suggest a relationship of causality.
In the event that the investigator classi ed the adverse event as reasonably related to the medication as serious and unexpected, said specialist will proceed to the expedited report of said reaction. Its realization will include: The immediate report by the specialist to the monitor as representative of the promoter. This must within the rst 24 hours of occurrence. The information may be communicated by phone, fax, email or in person.
The immediate report by the promoter to the CECMED. This must occur in the following periods: The monitor's expedited report to CECMED will be carried out using the corresponding model. This model will be lled DOUBLE. Quality monitoring and control will be carried out by the monitors, quality control managers, consultants and specialists responsible for the test. These visits will also serve to discuss any aspect of the protocol that the researcher suggests.
Execution check visits will be made daily from the beginning of the study. The rst Quality Control visit will be made immediately after the rst patient is included. Some limitations to our study design should be noted. The blinding was not feasible, it will maintained for laboratory SARS-CoV-2 RNA detection by PCR, that is one of the endpoint of the study.
Trial status.
The HOPE trial has already been approved by the Institutional Review Boards at "Luis Diaz Soto" Hospital and Regulatory Authority for Medicines, CECMED. Enrollment for this study began in April 11, 2020, and has enrolled one hundred patients as of May 26, 2020. The recruitment will be completed will be completed in the second week of June 2020.

Abbreviations
[Review for completeness prior to nalizing protocol -all abbreviations mentioned within the protocol must be included; some are provided below, use/modify as Declarations accountable for all aspects of the work. FHB, critical revision of the manuscript for important methodologically content, approval of the nal version to be published. HNC, drafting of the manuscript. YDR, analytical plan, approval of the nal version to be published and agreement to be accountable for all aspects of the work. CMS, analytical plan. ICL, critical revision of the manuscript for important methodologically content. IEM, clinical research coordinator at the hospital, analytical plan. VMG, approval of the nal version of the protocol. GNG, revision of the manuscript for important intellectual content.

Funding {4}
This work was supported by Center for Genetic Engineering and Biotechnology and Ministry of Health of Cuba.

Availability of data and material {29}
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate {24}
The HOPE study is approved by the Ethics Committee of "Luis Diaz Soto" Hospital in Havana, Cuba and by CECMED The study will be conducted in accordance with the ethical principles of the Declaration of Helsinki and the International Council for Harmonization-Good Clinical Practice guidelines. Informed consent will be obtained from patients. Site investigators will explain the objectives of the trial and its potential risks and bene ts to patients during the process of obtaining consent. No compensation is provided for enrollment in the trial. Patient personal data are de-identi ed.

Consent for publication {32},
The consent for publication is included in informed consent that sings the patient -I agree that the blood or tissue samples obtained for the study may be used in the future for new analyzes related to the disease or study drugs not provided for in the current protocol (genetic analyzes are excluded, as long as they are not part of it of the study objectives): Competing interests {28}.
Authors IBR, FHB, HNC, YDR, CMS, ICL, VMG and GGN, are employees of the Center for Genetic Engineering and Biotechnology, Havana network where Heberon Alpha R and HeberFERON are produced.
The other authors have no competing interests.