The Role of BCG Vaccine in the Clinical Evolution of COVID-19 and in the Efficacy of Anti-SARS-CoV-2 Vaccines

Clinical trial with randomized allocation in two arms (BCG vaccine versus placebo) of volunteers at risk but not yet infected nor vaccinated against SARS-CoV-2. Initially will be evaluated whether BCG has a protective role against severe form of the disease. After participants are vaccinated against COVID-19, it will be evaluated whether BCG favors the vaccine's efficacy. Volunteers will be recruited in three Brazilian states, with at least 250 seronegative in each group. The BCG-trained immunity stimulus will be investigated by assessing cytokines at D0 and D60 in a subsample of 50 participants per group. Until being vaccinated against COVID-19, the participants will be followed for up to 6 months, with visits scheduled every 2 months for interviews and immunoglobulin G (IgG) anti-SARS-CoV-2 antibodies. Those who become symptomatic at any time during the follow-up will be guided and monitored remotely daily until the end of their clinical evolution. After being vaccinated against COVID-19, visits to participants will be adjusted for the time of vaccination (VD), 20 days after the 1st dose (P1) and at least 30 days (P2) after the 2nd dose, with the aim of comparing the efficacy of the anti-SARS-CoV-2 vaccine in the two groups in the short and medium term.

The study's conclusions on the efficacy of BCG in preventing severe COVID-19 will be based on: incidence of SARS-Cov-2 infection (defined as the emergence of IgG over the follow-up period); incidence of illness by COVID-19 (defined as the presence of symptoms among infected participants); intensity and duration of symptoms between cases of COVID-19 and frequency and duration of hospitalizations for COVID-19 in each group. The occurrence, type, frequency and intensity of adverse effects associated with vaccination of adults with BCG will be reported.

The study's conclusions regarding the effect of BCG on efficacy of vaccines against COVID-19 will be based on: frequency of anti-SARS-CoV-2 neutralizing antibodies after the vaccine' 1st and 2nd doses in both groups.

1. OBJECTIVE- To evaluate the role of the BCG vaccine against the severe form of COVID-19 and in improving the efficacy of anti-SARS-CoV-2 vaccines through a two-arm clinical trial comparing BCG versus placebo. Specifically:

   1. Assess whether BCG vaccine is able to stimulate the nonspecific immune response (trained immunity)
   2. Verify whether BCG is able to protect against SARS-CoV-2 infection
   3. Assess whether BCG is able to protect infected with SARS-CoV-2 against illness
   4. Assess whether BCG is able to protect subjects with COVID-19 against its severe form
   5. Evaluate whether BCG can favor the effectiveness of doses of anti-SARS-CoV-2 vaccines
   6. Knowing whether revaccination of adults with BCG causes unexpected adverse reactions
2. METHODOLOGY:

   2.1 STUDY MODEL: Two-arm Clinical Trial - intervention (BCG) versus placebo

   2.2 STUDY POPULATION: Volunteers not yet infected with SARS-CoV-2 or vaccinated against COVID-19, who can be essential service workers with frequent contact with the population: workers in health posts, users of the public health network due to others non-serious illnesses, police officers, employees of pharmacies, supermarkets, stores and others

   Sampling: As the real prevalence of SARS-CoV-2 infection in the eligible population of the different recruitment sites is unknown, a prevalence of 50% was considered (it provides the largest sample size) and estimated that the post-BCG prevalence would decrease at least 10% if its effectiveness to be demonstrated. Thus, it was found a sample of 385 subjects. Considering losses, a minimum of 400 participants will be recruited, with at least 200 in each group.

   2.3. INCLUSION CRITERIA:
   * Never been infected by SARS-Cov-2 - clinical history and absence of anti-SARS-CoV-2 immunoglobulins IgM/IgG antibodies by rapid test (immediate) and serology test (later confirmation)
   * Not having been vaccinated against COVID-19
   * Be at least 18 years old

   2.4. EXCLUSION CRITERIA: • Have been vaccinated with BCG less than 6 months ago

   • Have been vaccinated with BCG more than once in the past
   * Present any classic contraindications for BCG - pregnancy, breastfeeding, acute febrile illness, severe allergy to one of the components, vaccination with other live vaccines in the last month (measles, yellow fever, herpes zoster), immunosuppression for any cause (congenital, illness, use of corticosteroids or immunosuppressants)
   * Have had tuberculosis or contacted someone with tuberculosis
   * Present signs of any current acute illness or have had a severe illness less than 15 days
   * Present a skin lesion at the vaccine application site (right deltoid muscle)
   * History of severe allergic reactions to any product: urticaria, glottis edema, angioedema, respiratory failure, bronchospasm
   * History of serious autoimmune diseases: systemic lupus erythematosus, Crohn's disease, ulcerative colitis, multiple sclerosis, hemolytic anemia, ankylosing spondylitis, pemphigus and others
   * Immunocompromise for any reason: congenital, illness, use of corticosteroids or immunosuppressants
   * Presenting a risk comorbidity for severe COVID-19: poorly controlled hypertension, decompensated heart disease (chronic, congenital or ischemic), pulmonary disease (chronic pneumopathy, poorly controlled asthma, chronic interstitial pneumopathy, others ...), disease advanced chronic kidney disease (use of dialysis), transplantation (of solid organs or bone marrow), uncontrolled diabetes

   2.5. RECRUITMENT OF PARTICIPANTS The volunteers will be recruited in three different Brazilian states - Minas Gerais state (Juiz de Fora city), Amazonas state (Manaus city) and Pará state (Belém city). Their approach will be based on the viability and logistics of each recruitment site, considering the local dynamics of anti-COVID-19 vaccination by age. Those who meet the inclusion criteria and accept to participate in the study, must sign the Informed Consent Form (ICF). By lot, the participant will be allocated to Group 1 or 2, to receive placebo (BCG solvent) or BCG vaccine.

   2.6. INCLUSION AND FOLLOW-UP OF PARTICIPANTS 2.6.a -- Evaluation of BCG against the severe form of COVID-19:

   The subjects will be contacted at D0 (day of inclusion and intervention), D60, D120 and D180, as well as on any day of symptom onset in order to assess the presence and evolution of a SARS-CoV-2 infection. A standardized questionnaire will be developed and tablets will be programmed with the REDCap software to conduct interviews at each visit to investigate demographic data, past and current illnesses, vaccination history, risk activities and symptoms suggestive of COVID-19. Participants will be instructed to inform the team as soon as present any suspicious symptoms. In each scheduled visit, the participants will also undergo a venous blood collection to:

   - On D0 - Search of IgM/IgG antibodies by rapid test and IgG by serology (enzyme immunoassay-ELISA) in all subjects and search of pro-inflammatory cytokines Tumor Necrosis Factor α (TNF-α), interferon γ (IFN-γ), interleukin (IL)1β, IL-4, IL-6 and IL-10 in 50 participants from each group.

   - On D60 - Search of anti-SARS-CoV-2 IgG by ELISA in all participants and paired cytokine search (participants who did it on D0).
   * On D120 and D180 - Search of anti-SARS-CoV-2 IgG by ELISA.
   * If Symptom - Daily follow-up to guide diagnostic confirmation of SARS-CoV-2 infection in the local health network and to monitor clinical evolution. Furthermore, the patient will fill a table of daily symptoms, which will be collected at the next scheduled visit. In case of hospitalization, data of clinical evolution will be obtained from medical records.

   2.6.b -- Evaluation of BCG´s capacity to improve the vaccine anti-SARS-CoV-2 efficacy:

   From the moment the participants are vaccinated against covid-19, the visits will be adjusted to the vaccination schedule, as follows:

   - VD - at the time of 1st dose of the vaccine

   - P1 - 20 days after the 1st dose

   - P2 - at least 30 days after the 2nd dose On all these occasions, neutralizing anti-SARS-CoV-2 antibodies will be tested in all participants.

   2.7. INTERVENTION The inclusion in the BCG or Placebo Group will be done by lot, whose knowledge will be restricted to the main researcher and the professional who will perform the intervention. The participants and the technician who will carry out the laboratory tests will not know it.

   - BCG Group - BCG vaccine (live attenuated Mycobacterium bovis (Bacillus Calmette-Guérin) from the Serum Institute of India Ltd. Intradermal dose of 0.1 ml applied at the distal insertion of the right deltoid.

   Expected adverse effects of BCG can be tenderness, swelling and a reddened papule at the site which will become a 1 cm-diameter liquid ulcer that will close in 2 or 3 months, almost always leaving a scar of the same size. Other possible side effects are: headache, malaise, fever, ulcer greater than 1 cm, delayed healing (up to 6 months), enlarged lymph nodes. A rare serious complication is local or axillary node abscess due to error in administration with subcutaneous deposit of the vaccine. Thus, BCG will be managed by a rigorously trained professional. There is also reference to the spread of the vaccine bacillus causing organ damage in immunocompromised individuals (an exclusion criterion). Subjects with a severe reaction will receive adequate follow-up and treatment.

   -Placebo Group - Application of 0.1 ml intradermal of BCG vaccine solvent.

   2.8. INFECTION DETECTION Current SARS-Cov-2 infection will be identified by rapid test to detect IgM/IgG antibodies (only on D0 as an exclusion criterion) and by serology to detect IgG antibodies on every visit. Through positivity for IgG throughout the follow-up, the investigators will identify the participants who became infected between visits throughout the follow-up.

   2.9. CLINICAL EVOLUTION - The clinical status of symptomatic infected people will be defined as:
   * MILD STATUS (Influenza Syndrome-SG) - Fever accompanied by cough or sore throat or loss of smell/taste and at least one of the following symptoms: headache, myalgia or arthralgia. Less common symptoms may be: body pain, nasal congestion, diarrhea, abdominal pain, rash, discolored fingers and toes, conjunctivitis.
   * MODERATE STATUS - Tiredness or slight difficulty in breathing on small efforts and/or increase in the intensity of the underlying symptoms after 4 days of illness and/or increase in the number of underlying symptoms after 4 days of illness and/or persistence of symptoms for more than one week.
   * SERIOUS STATUS (Severe Acute Respiratory Syndrome) - SG associated with deficit in the respiratory system (difficulty breathing, cyanosis, oxygen saturation \<95%, tachypnea, partial pressures of oxygen in alveolar gas/fraction of inspired oxygen (PaO2/FiO2) \<300 and/or pulmonary infiltration \>50% in 24- 48h), deficit in the cardiovascular system (hypotension, weak pulse) or change in mental status.
   * CRITICAL STATUS - Respiratory failure, septic shock and / or multiple organ dysfunction, loss of speech or movement.

   2.10. EVALUATION OF THE HUMORAL RESPONSE PROFILE At D0, as an inclusion criterion, the rapid test for detection of anti-SARS-CoV-2 IgG/IgM antibodies (TR COVID-19 IgM/IgG "BioManguinhos-Fiocruz") with 100% specificity for IgM and IgG and sensitivity of 85.7% (4-7 days) and 90% (\> 8 days) for IgM and 92.8% (4-7 days) and 90% (\>8 days) for IgG. Also on D0 and on all visiting days and in case of symptoms, IgG will be detected by a commercial enzyme immunoassay (recombinant ELISA - "Euroimmun Brasil Medicina Diagnóstica") with the S1 subdomain of the Spike protein as the capture antigen, in order to detect infections occurred between visits (seroconversion).

   To assess the efficacy of anti-SARS-CoV-2 vaccines, anti-SARS-CoV-2 neutralizing antibodies will be investigated using the SARS-CoV-2 Surrogate Virus Neutralization Test-sVNT ("Genscript") kit, which provides qualitative and semi-quatitative (titer in UI/ml) results. This investigation will be done 20 days after the 1st dose and at least 1 month after the 2nd dose of the vaccine.

   2.11. CYTOKINE ANALYSIS - It will be measure the proinflammatory cytokines IL-1β, IL-4, IL-6, IL-10, TNF-α (R\&D Systems) and IFN-γ ("Sanquin") at D0 and D60 to assess the effectiveness of BCG in stimulating the non-specific immune response.

   2.12. ETHICAL ISSUES - The project was approved by the National Commission for Ethics in Research with Human Beings-CONEP. Volunteers who wished to participate signed the Informed Consent Form. An Independent Data and Safety Monitoring Committee (IDSMC) will monitor the study to ensure it is ethically non-violent and data credible, and may recommend modifications or suspension of the study if necessary. Through the Research Electronic Data Capture-REDCap software, a data manager will provide information about the study progress any time it be requested. The results of the exams will be provided to the participants. Intense reaction to BCG will receive due care from the research team and will be notified to CONEP and IDSMC within 24 hours. Participants will be encouraged to receive the COVID-19 vaccine when it becomes available to them.

   2.14. STATISTICAL ANALYSIS OF RESULTS - Through the Statistical Package for the Social Sciences (IBM SPSS v.22) and Stata (v.14.0) programs, the outcomes will be analyzed comparatively between the groups, being defined as failures. Analyzes will be performed for the intention-to-treat population (total recruits) and per protocol population (with full follow-up) using the Kaplan-Meier method. Proportions will be calculated with 95% confidence intervals and compared by Chi2 and Fisher's exact test. Survival curves will be compared by log rank test. Logistic regression will identify the set of independent variables that can explain the binary dependent variables, such as serological reactivity.

Eligibility Criteria:
INCLUSION CRITERIA:

* Never been infected by SARS-Cov-2 - clinical history and absence of anti-SARS-CoV-2 IgM/IgG antibodies by the rapid test (immediate) and by serology test (later confirmation)
* Not having been vaccinated against COVID-19
* Be at least 18 years old

EXCLUSION CRITERIA:

* Have been vaccinated with BCG less than 6 months ago
* Have been vaccinated with BCG more than once in the past
* Present any classic contraindications for BCG - pregnancy, breastfeeding, acute febrile illness, severe allergy to one of the components, vaccination with other live vaccines in the last month (measles, yellow fever, herpes zoster), immunosuppression for any cause (congenital, illness, use of corticosteroids or immunosuppressants)
* Have had tuberculosis or contacted someone with tuberculosis
* Present signs of any current acute illness or have had a severe illness less than 15 days
* Present a skin lesion at the vaccine application site (right deltoid muscle)
* History of severe allergic reactions to any product: urticaria, glottis edema, angioedema, respiratory failure, bronchospasm
* History of serious autoimmune diseases: systemic lupus erythematosus, Crohn's disease, ulcerative colitis, multiple sclerosis, hemolytic anemia, ankylosing spondylitis, pemphigus and others
* Immunocompromise for any reason (congenital, illness, use of corticosteroids or immunosuppressants)
* Presenting a risk comorbidity for severe COVID-19, such as: poorly controlled hypertension, decompensated heart disease (chronic, congenital or ischemic), pulmonary disease (chronic pneumopathy, poorly controlled asthma, chronic interstitial pneumopathy, others ...), disease advanced chronic kidney disease (use of dialysis), transplantation (of solid organs or bone marrow), uncontrolled diabetes