Cell Reports Medicine

ArticlePublished August 15, 2022

Multiple BCG vaccinations for prevention of COVID-19 and otherinfectious diseases in Type 1 diabetes
Authors
Denise L. Faustman*1; Amanda Lee2, Emma R. Hostetter2; Anna Aristarkhova2; Nathan C. Ng2 ;Gabriella F. Shpilsky2; Lisa Tran2; Grace Wolfe2; Hiroyuki Takahashi1; Hans F. Dias2; Joan Braley1 ;Hui Zheng1; David A. Schoenfeld1; Willem M. Kühtreiber1

Author’s Affiliations
1
Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02192, USA2
Massachusetts General Hospital, Boston, MA, 02192, USA*Lead Contact: dfaustman@mgh.harvard.edu (D.L.F.)

1

Graphical Abstract

REPEAT BCG VACCINATIONS IN ESTABLISHED TYPE 1 DIABETICS• ≥3 BCG (n=96) or placebo injections (n=48) before COVID-19 pandemic
January 2020

SYMPTOMS• Direct patientreporting• Bi-monthly surveys

BCG VACCINESFOR COVID-19 &INFECTIONPROTECTION

BCG

Placebo

VaccineEfficacy

Posterior
Probability(Vaccine Efficacy)

COVID-19
confirmed by
Point of Careand SARS-CoV-2
Antibodies

1.0%

12.5%

92%

0.99

COVID-19
confirmed by
PCR

0.0%

10.4%

100%

0.99

COVID-19 TESTING• COVID Antibodies• PCR

Cumulative COVID-19 Disease (%)

≥ 3x

BCG
Placebo05Efficacy: 92%
p = 0.009

10

Surveillance Time (months)

15

In Brief
In a randomized, double-blinded, placebo-controlled, Phase 2/3 trial, Faustman et al.
investigate the safety and efficacy of multiple doses of the >100-year old Bacille-CalmetteGuerin (BCG) vaccine against COVID-19. They also determine vaccine efficacy against otherinfectious diseases, which if present would suggest platform protection against new SARS-CoV2 variants.

Highlights• We report on our randomized trial of multi-dose BCG for protection against COVID-19• BCG is safe and has 92% efficacy versus placebo against COVID-19• Findings also suggest platform protection against many infectious diseases• Efficacy takes 1-2 years to manifest, but the protection may last decades

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SUMMARYCONFIDENTIAL

There is a need for safe and effective platform vaccines to protect against COVID-19 and otherinfectious diseases. In this randomized, double-blinded, placebo-controlled Phase 2/3 trial, we
evaluate the safety and efficacy of multi-dose Bacillus Calmette-Guerin (BCG) vaccine forprevention of COVID-19 and other infectious disease in a COVID-19-unvaccinated, at-riskcommunity-based US cohort. The at-risk population are adults with type 1 diabetes. In thisparallel trial design, 144 subjects are enrolled, 96 are randomized to BCG and 48 to placebowith 2:1 randomization, and no drop-outs over the 15-month trial. A cumulative incidence of
12.5% of placebo-treated and 1% of BCG-treated participants meets criteria for confirmed
COVID-19, yielding a vaccine effectiveness of 92%. The BCG group also displays fewer
cumulative infectious disease symptoms and lesser severity, and fewer infectious disease
events per patient, including COVID-19. There are no BCG-related systemic adverse events.
BCG’s broad-based infection protection suggests that it may potentially provide platform
protection against new SARS-CoV-2 variants and other pathogens.
(Clinicaltrials.gov:NCT02081326)

Key words: Bacillus Calmette-Guerin (BCG), COVID-19, infectious diseases, Phase 2/3 trial, hostmicrobe interactions, Hygiene Hypothesis, type 1 diabetes, autoimmune, randomized double
blinded clinical trial, vaccine (Clinicaltrials.gov:NCT02081326)

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INTRODUCTION
The BCG vaccine is a >100-year old vaccine originally developed for tuberculosis protection. It is
heralded as the safest vaccine ever developed, with 3-4 billion people already vaccinated andan annual 120 million newborns vaccinated 1. It is highly affordable at about 10-75 cents/dose.
Over the last 17 years, randomized clinical trials and epidemiology studies have shown that the
BCG vaccine protects humans from a multitude of infections, including upper respiratory tract
infections, leprosy, malaria, viral, and bacterial infections 2-16. The first indication of the broad
infectious disease protective abilities of this vaccine came 100 years ago when Albert Calmette,the vaccine’s co-inventor, noted a four-fold decline in child mortality (unrelated to tuberculosis)
in vaccinated children, presumably from broad infectious disease protection 17. These
protective effects appear also when adolescents are re-vaccinated with BCG after the typicalnewborn dose 10. The BCG vaccine may also protect humans from immune diseases such astype 1 diabetes and multiple sclerosis 18-21. The mechanisms behind these wide-ranging
benefits are a topic of active scientific discovery.
With the onset of the SARS-CoV-2 pandemic, some epidemiology studies began touncover an association between neonatal BCG vaccination and reduced morbidity and mortality
from COVID-19, even in older adults decades after the typical neonatal vaccinations, on acountry-by-country basis 22-40. In some other global populations with varying neonatal
exposures, different BCG strains and other populations, benefits were not observed 24,41-47.
Since the US has never vaccinated newborns or adults with BCG, a randomized trial of BCG for
possible protection from SARS-CoV-2 infection offers a clean comparison in a vaccine-naïve
adult US population. This parallel trial was adapted from an ongoing double blindedrandomized trial of BCG for treatment of long-standing adult type 1 diabetes so all cohorts were
fully vaccinated with 3 BCG or placebo vaccinations at the start of the COVID-19 pandemic in
the US, January 1, 2020. Type 1 diabetics represent one of the most vulnerable populations in
the US for morbidity and mortality from COVID-19 48. Type 1 diabetics also have well-known
increased infectious disease risk including COVID-19 49.
As the COVID-19 pandemic worsens, vaccine development assumes center stage. But
antigen-specific vaccines, the focus of most clinical programs, are struggling to keep pace withnew viral variants. The ideal vaccine should be safe, efficacious, affordable, and offer durable
protection against ever-changing viral variants and future infectious disease pandemics. We
conducted a randomized, double-blinded, placebo-controlled platform trial over 15 months(January 1, 2020 to April, 2021) to determine whether the BCG vaccine might offer a platform
vaccine technology for broad-based infectious disease protection, including protection against
COVID-19 in the vulnerable type 1 diabetic population.

RESULTS
We assessed the impact of multi-dose BCG vaccinations for protection from SARS-CoV-2
infections and other infections (Figure 1A). All participants at the start of this parallel study had

43 BCG vaccinations or 3 placebo vaccinations over a 2 year time period preceeding the
pandemic.
The Trial, Which had No Drop-Outs, Found that Multi-Dose BCG Was Safe
All 144 participants were US citizens not previously vaccinated with BCG and had long-standing
type 1 diabetes, a co-morbid condition for worse COVID-19 disease and symptoms (Figure 1B)49. None of the participants was lost to follow-up or dropped out during the current 15 monthparallel study. A total of 150 participants were enrolled for the first study on type 1 diabetes; atthe start of this parallel clinical trial (January 2020) evaluating the effects of BCG multi-dosing
on overall infections and COVID-19 specific disease, there were 96 BCG-treated and 48 placebotreated. These recipients were all vaccinated with BCG or placebo in the two years prior.
Unlike antigen specific vaccines no BCG-related systemic adverse events occurred in any of theparticipants during the vaccination time period. BCG vaccines do cause local skin reactions thatusually appear at 2-4 weeks. No excess local reactions were reported as adverse events. During
this time period of this trial, other COVID-19 vaccines were not yet available and therefore had
no influence on this study.
The BCG Group Had Fewer Cases of Confirmed COVID-19 than the Placebo Group, with
Vaccine Efficacy at 92%
The cumulative incidence of confirmed COVID-19 in the BCG cohort was 1 out of 96 (1%),
whereas it was 6 out of 48 (12.5%) in the placebo group at the end of the 15-month COVID-19
trial. The criteria for a confirmed case of COVID-19 were developed early in the pandemic prior
to widespread availability of PCR. Therefore, the criteria required (see Methods and Figure S1)
were reporting at least 1 FDA-defined symptom of COVID-19 plus positive findings on ≥5 out of
10 assays (9 SARS-CoV-2 IgG antibody binding assays and a tenth assay that included PCR
testing for viral RNA). Because of the limited PCR availability, several subjects were diagnosedon basis of symptoms plus multiple positive serologies only. The efficacy of BCG (vs placebo) for
preventing a confirmed case of COVID-19 was 92% (Figure 2). Monte Carlo statistics resulted ina Posterior Probability (with Vaccine Efficacy >30%) of 0.99. Since the current standard ofdiagnosis for COVID-19 is based on PCR, we also calculated BCG efficacy based on at least one
FDA-defined symptom of COVID-19 plus a positive PCR alone. We found no symptomatic
subjects that were PCR-positive in the BCG group (0%), whereas 5 symptomatic and PCRpositive subjects were in the placebo group (10.4%). These results indicate an efficacy of 100%
at 0.99 Posterior Probability if only PCR results are considered. This can also be readily
visualized in the PCR cumulative graph (Figure 3B), which shows the first detected COVID-19
case based on PCR at 7 months into the trial, whereas serology testing for SARS-CoV-2
antibodies revealed the first positive cases as early as two months (Figure 2B).
There were no deaths from COVID-19 in either the BCG group or the placebo group.
Heatmap Comparison Shows Higher SARS-CoV-2 Antibody-level Presence and Intensity in thePlacebo Group Over the BCG Group
Heatmap comparisons of SARS-CoV-2 antibody-level presence and intensity (Z-scores versus
pre-COVID baseline) show a significant increase in infections and the intensity of the antibody
response in symptomatic subjects in the placebo over the BCG group (Figure 3A). These data5

are in symptomatic subjects. For completeness we also present the COVID-19 antibodies in alltrial subjects, with and without symptoms. The heatmap of all trial participants again shows
the presence of COVID-19 infections almost exclusively in the placebo subjects (Figure S2).
Cumulative graphs for the anti-COVID-19 antibody tests to different regions of the virus
are shown in Figure 2B. Number at risk data are shown in Figure S4. The identity of the SARSCoV-2 protein region eliciting an antibody response, mostly the Spike protein, is denoted by the
Roman numerals listed in Figure S1. Six protein regions eliciting an antibody response werefrom viral Spike Protein 1, two were located in Spike Protein 2, and one viral region was specificfor antibodies against viral RNA polymerase. Almost all COVID-19 viral regions included in thesetests show significantly increased accumulation of antibody reactivity in the Placebo group ascompared to the BCG group, except for COVID-19 viral epitope II.
The BCG Group (vs Placebo Group) Exhibited Fewer Infections per Patient and Fewer
Infectious Disease Symptoms and Severity assessed by the Total and Average Infectious
Disease Index
We analyzed infectious disease events collected as “adverse events” by the Medical Dictionary
for Regulatory Activities (MedDRA) classification coding (Figure S3B). Prior clinical trial datasuggests BCG vaccination exhibits “off-target” effects, i.e., viral and bacterial infectious disease
prevention (and, in this study, prevention of SARS-CoV-2 viral infections) beyond its intended
use in tuberculosis protection 22-25,27,37-39,50}. Using the time period of the current COVID-19
trial, we analyzed infectious disease symptoms including COVID-19 and other infections. The
cumulative infectious diseases reported by the subjects and represented as cumulative
infections per patient were significantly fewer in the BCG vs placebo group (Poisson modelcomparing adverse events rates p=0.004; Figures 4A and S3B). These findings are likely torepresent the minimum protection. Some clinical trial data for other off-target effects of the
BCG vaccine suggest the time period for BCG clinical effectiveness is often in the two- to threeyear range after vaccinations begin 19,20. To look at this time period of BCG onset of vaccine
effectiveness but in the setting of infection protection, the clinical trial infectious disease
adverse events were broken into the pre-COVID trial period during which the three BCG
vaccines were administered versus the subsequent 15-month time period of this current clinical
trial (Figure 4B). While comparison of infectious adverse events in BCG versus placebo groups
during the current trial had a significant Poisson distribution (p=0.004), there was no significant
difference in all infections during the pre-COVID trial period (Poisson p=0.46). These datasuggest that, just like other reported off-target events of the BCG vaccine, it might similarly
take about 2 years after the first vaccine for maximal effectiveness for the platform infectious
disease protection.
Our intent was to evaluate the severity of COVID-19 symptoms with BCG treatment ascompared to placebo in confirmed COVID-19 patients, but this was not possible because only
one subject in the BCG group fit our criteria for confirmed COVID-19. We therefore analyzed
the symptoms in symptomatic BCG- and placebo-treated participants--regardless of whether
they were confirmed COVID-positive--to understand the impact of BCG on overall infectious
disease severity.
Using data from symptom surveys based on FDA guidelines 51 that were completed
every two months, we calculated a Total and Average Infectious Disease Index (Figure S3A).
6

Indexes for each individual patient were calculated separately and then averaged across all
subjects in the BCG and placebo cohorts (Figure 4C). Comparing only symptomatic patients, theTotal Covid Index in the BCG cohort (48±11, n=31) was significantly reduced vs placebo(152±70, n=20; p=0.04). The Average Infectious Disease Index also showed a significant
decrease (BCG 13±2, n=31, versus placebo 23±7, n=20; p=0.04). This indicates that BCG
vaccination reduced the severity and duration of all infectious disease symptoms as comparedto placebo. Symptomatic patients in the BCG group also reported significantly fewer number ofdays of missed work as compared to the placebo group (Figure 4D; BCG 0.77 ± 0.28; placebo
2.26 ± 0.84, p=0.02). All individual average symptom scores (12 out of 12) were more severe in
the placebo group vs the BCG group (Figure 4E Left panel). For 11 out of 12 symptoms there
also was a higher percentage of patients in the placebo group versus BCG group (Figure 4E
Right panel).
BCG recipients had comparable or milder infectious disease symptoms compared to theirhousehold members, whereas most placebo recipients had more severe symptoms comparedto their household members
We on an exploratory basis studied infection disease severity of trial participants compared to
non-diabetic adults living in the same household. We collected infectious disease symptom
information for trial participants and for cohabitating adult partners in 20 households (13 BCG
and 7 placebo) and determined the differences in Total Infectious Index for each household(Figure 5A, S3A). The data shows BCG recipients had comparable or milder symptoms ascompared to their household members, whereas most placebo recipients had more severe
disease as compared to their household members. The symptoms scores between participants
and household members were compared between the BCG-treated group and the placebotreated group using a Two-Sample Wilcoxson test (p=0.049, 2-tailed). The stacked horizontal
bars in Figure 5B show the distributions of the infectious disease symptom scores in eachgroup. BCG recipients overall had milder symptoms as compared to placebo recipients andeven as compared to non-diabetic household member controls. Number at risk data are shownin Figure S4.
This data supports previously published data that type 1 diabetics not only have more
severe COVID-19 but also have more severe and more infectious events disease events 49.
Lastly, the distribution of symptoms in Figure 5B shows once again that for BCG-treated trial
participants individual symptoms were minimized with vaccinations compared to placebo.
DISCUSSION
This randomized, double-blinded, placebo-controlled trial shows that multi-dose BCG
vaccination is safe and prevents COVID-19 with an efficacy of 92% to 100% (depending on casedefinition) relative to placebo. This trial was conducted in the US in previously unvaccinated atrisk type 1 diabetic subjects. BCG-vaccinated adults also had reduced incidence and severity of
all infections. This randomized double blinded study now is a rigorous manner of formal testingand substantiates a large body of both epidemiological, observational and clinical trial evidence
that BCG vaccines might in some settings provide a broad platform for infectious diseaseprotection. This data is from a study conducted in the US, as a multi-dose BCG vaccine therapy
7and administered two years prior to the pandemic to allow sufficient time for full effectiveness
2-16.
This clinical trial has several strengths. First, this trial is, to our knowledge, the firstpeer-reviewed randomized double-blinded trial of multi-dose BCG for COVID-19 protection andinfection protection in a US population. A prior trial of single-dose BCG, with immediate COVID19 disease monitoring, showed neither protection from COVID-19 infections nor protection
from disease severity 7. This trial was likely too short in duration to find an effect based on theevidence from other off-target effects (see below) indicating years to show clinical efficacy.
Second, our trial uses a very potent strain of BCG, Tokyo-172. BCG strain differences for otheroff-target indications are important and this strain of BCG exhibits some of the highest in vitro
potency and is highly immunogenic 52-57. Indeed it is now appreciated that Japan as a country
with mandatory BCG vaccines and one of the oldest populations in the world has remarkable
resistance to COVID-19 58. Third, the diabetic study population is high-risk for infections,suggesting that the vaccine is efficacious in populations susceptible to infections. Fourth, thestudy uses rigorous molecular methods to define a case of COVID-19 based on both PCR and/or
SARS-CoV-2-directed immunoglobulin production to very specific regions of the virus to confirm
current or past infection. Fifth, there was no patient attrition. Sixth, the subjects are from the
US. This is important because all subjects, prior to enrollment, were confirmed by diagnostics
and by history to be unexposed to tuberculosis and lacking prior BCG vaccinations. The US has
never had a country policy of neonatal BCG vaccinations. Two properties of the vaccine areespecially noteworthy. BCG’s 100-year strong safety record and its international use as aneonatal vaccine should help to overcome vaccine hesitancy. Seventh, this trial was conductedin subjects who all had negative serology for prior COVID-19 infections and also had not yet
received the approved Moderna and Pfizer vaccines that could confound the effect of BCGs
protection alone. Finally, the BCG vaccine is affordable, which is especially important in thedeveloping world where it offers almost immediate availability.
In conclusion, the BCG vaccine effectively protects against COVID-19 and provides broad
infectious disease protection as now tested with a formal double blinded and randomized
clinical trial. Also the BCG vaccine is safe, effective, affordable, and potentially protective
against every changing viral variants of the COVID-19 pandemic, based on its broad-based
protection against other infections.
Limitations of the Study
The most important limitation of our trial is that off-target effects take time tosystemically manifest, but, once they do appear, they may offer broad-based infectious diseaseprotection over the long-term. BCG does not work as fast as the antigen-specific COVID-19
vaccines and also likely requires multiple treatments over time to be effective, at least in BCGnaïve adults. Antigen-specific vaccines generally take weeks to show an effect althoughprotection is limited by a specific strain and then downstream by short durability. In the case of
the BCG vaccine, the off-target platform protection from infections has a slower onset (months
to years) but offers perhaps life-long durability. The other off-target effects of BCG are in its
reset of autoimmune diseases, benefits that take 2 years to show an effect 18-21. Nonetheless,the benefits may last for decades thereafter as well, at least 8 years in randomized clinical8

trials. Many of the epidemiologic studies show off-target protection after neonatal or adultvaccination, including one study showing >60 years of protection from a neonatal BCG vaccine
19,59. This “long immunity,” is both long to start and perhaps long-lasting. The slow onset oftherapeutic effects and perhaps lifelong durability could be due to the gradual migration of the
BCG organism from the vaccine injection site to the bone marrow, where it may infect resident
stem cells, a process that is hastened by the IV administration of BCG 19,60,61. Longitudinalmechanistic data from our lab shows that BCG over time alters key metabolic and immune
signaling pathways via methylation of genes over a 2-3 year time period 61-63. Others haveshown that more acute changes are brought about by chemical modifications (methylation,acetylation, etc) of the histones in the promoters and enhancers of the genes. 65,66. The slow
reset of key immune genes by BCG-driven methylation and de-methylation of select host genes
and in almost all lymphoid cell types such as both T cells and monocytes, shows this “longimmunity” is multi-lineage. This could be due to possible stem cell driven re-modeling of thehuman immune system by this very synergistic organism. This randomized double blindedclinical trial documents in humans the broad infectious benefits of host microbe interactions.
Other potential limitations for this BCG study, as well as other studies that test COVID19 vaccines in specialized patient populations, such as type 1 diabetic subjects, health care
workers or the elderly, is that the translation of vaccine efficacy to the general public, incountries with diverse populations and different neonatal vaccination histories, may not always
be applicable.
ACKNOWLEDGEMENTS
We thank all the participants who volunteered for this study with their dedication to continue
to participate through the COVID-19 pandemic. We specifically thank Dr. Deneen Vojta for her
insight and advocacy. We thank UnitedHealth Group for their insight to support this clinical
trial. We also thank Dr. Miriam Davis for her editing of the manuscript with the highest of
standards.
AUTHOR CONTRIBUTIONS
All authors met the criteria for authorship set by the International Committee of Medical
Journal Editors. DF was the principal investigator and WK was the co-principal investigator ofthis randomized clinical trial. EH, NN, and GW were clinical trial coordinators and interactedwith participants, gathered participant data and worked on formatting Figures with processed
data. JB role was a registered nurse tracking vaccine side effects and seeing subjects. AL, AA,
GS, LT, HT, HD performed various assays related to serum obtained from patients. HZ and DS
performed the statistical analysis with the data being provided by WK. All authors had access tothe blinded data. The data interpretation and data verification was performed at many levels
but most by DF, DS, HZ, and WK. Authors DS, HZ and WK were unblinded subjects thatprocessed the data; as outlined the original randomized trial is still active and ongoing. Blinded
audits of data intergrety and FDA compliance were performed by an outside monitoring
9

company (Advanced Clinical Inc) as well as blinded compliance audits once per week by EH, DF,
GW, NN. All primary data will be shared at this time but is still blinded data since the primary
outcome trial is still ongoing until approximately August 2023. All unblinded will be shared after
the primary trial is completed with patient identifiers removed for HIPPA compliance.
DECLARATION OF INTERESTS
None of the authors have any ownership rights to the study drug, the Tokyo 174 strain of BCG.
None of the authors consult or receive research support from Japan BCG Laboratory, themanufacturer and owner of the study drug. Funding for this trial was from non-profit sources
and not provided by the drug owner. We thank UnitedHealth Group for their insight to support
this clinical trial.
Inclusion and diversity statement
All subjects for eligility were chosed based on non-bias selection for sex or select ethnic
diversity.
FIGURE LEGENDS
Figure 1. Flow Diagram and Characteristics of ParticipantsThis flow diagram represents all enrolled participants from January 1, 2020 to April, 2021 forthis double-blinded randomized clinical trial testing repeat Tokyo-172 BCG vaccination vsplacebo for COVID-19 protection. All 144 subjects were followed for 15 months with a 2:1
randomization and no drop-outs. Data collection for this trial ended on 4/20/21, the date whensubjects started to receive provisionally approved COVID-19-specific vaccines. The geographicallocation of the participants within the US are shown in Figure S5.
Figure 2. BCG Vaccine Efficacy and Diagnostic Confirmation of COVID-19(A) Shown is the cumulative incidence of confirmed COVID-19 as a primary endpoint. During
the 15-month surveillance time, one BCG recipient out of 96 (1.0%) met our criteria formolecularly confirmed COVID-19. In contrast, 6 out of 48 placebo recipients met the criteria(12.5%). Fisher’s Exact testing showed a significant difference (two-tailed p=0.006). Calculated
vaccine efficacy was 92% and the posterior probability (vaccine efficacy >30%) was 0.99. Thiswas calculated using the Monte Carlo method. Vaccine efficacy was defined as (p1–p2)/p1x100
where p1 is the percentage COVID-positive subjects in the placebo group and p2 is thepercentage COVID-positive subjects in the BCG group. Our criteria for confirmed COVID-19 (seetext) required a combination of COVID-19 symptom(s) and ≥5 of 10 positive antibody assays
including PCR, if available. Since many current clinical trials only define confirmed COVID-19 bysymptom(s) and positive PCR testing, the PCR-only group was studied separately for vaccine
efficacy (Figure 2A and Figure 3B).
(B) Cumulative findings from each of the 10 diagnostic tests used to confirm COVID-19 (alongwith positive symptoms). These tests included the presence of COVID-19-specific antibodies to10various SARS-CoV-2 virus epitopes through protein display (I-VIII), antibodies to the receptor
binding domain with an ELISA test (IX) and Point of Care testing (X) that included PCR. Ourcriteria for having confirmed COVID-19 required at least 5 of 10 detection methods to bepositive, along with symptom(s). For the antibody assays, a patient was considered positive
when the test resulted in a Z-score of ≥ 3. In the cumulative graphs, the x-axis data shows thetime-period of the 15-month trial. The Y-axis shows the cumulative percentage of positive
subjects. Except for the point of care graph (X), all other graphs represent the percentage of
BCG and placebo patients with a Z-score of ≥3 for the anti-SARS-CoV-2 antibody binding to agiven protein region of the virus, i.e., the average antibody level during the COVID trial period
was at least 3 standard deviations greater than the average level in the period preceding the
COVID trial (baseline). The percentiles at the top-right of each graph represent the calculated
vaccine efficacy if this test alone were used to diagnose COVID-19 disease. Respective efficacy
and Fisher’s Exact p values for each COVID-19 antibody test were as follows: (I) 100%,p=0.0007; (II) 80%, p=0.089; (III) 91.7%, p=0.009; (IV) 93.8%, p=0.001; (V) 90%, p=0.024; (VI)87.5%, p=0.004; (VII) 94.4%, p=0.0003; (VIII) 83.3%, p=0.009, (IX) 90%, p=0.009; (X) 91.7%,
p=0.029. For all graphs, BCG n=96, Placebo n=48. Figure S1 shows the viral protein regions foreach anti-COVID antibody tested. Number at risk data for each cumulative graph are shown in
Figure S4.
Figure 3. Reduced COVID-19 Disease Markers after BCG Vaccination
A. The heatmaps show Z-scores for antibodies against SARS-CoV-2 in BCG- and placebo-treated
symptomatic subjects (BCG n=26, Placebo n=21). Since some of the Z-scores in the left map are
faint, the right map shows which Z-scores are ≥ 3. The Roman numerals at the bottom of eachlane denote antibodies against SARS-CoV-2 protein regions as listed in Figure S1. The maximum
Z-score in the left heat map was 126.
B. Cumulative incidence of confirmed COVID-19 defined by symptomatic subjects testing
positive solely by PCR. The BCG group (n=96) had no symptomatic subjects who were PCR
positive (0%), whereas the placebo group (n=48) had 5 symptomatic and PCR positive subjects(10.4%). This difference was significant (Fisher’s Exact p=0.0036) and translated to a vaccine
efficacy of 100% with 0.99 posterior probability. There was low availibility of positive PCR tests
at Point of Care locations during the first 7 months of the trial. This, together with the need toperform the PCR test in a narrow window of about two weeks to be positive, is the reason why
we designed the 9 non-PCR methods shown in Figures 2A,B and 3A.
Figure 4. Cumulative Infections and Infection Severity
A.Cumulative total infectious diseases during the 15 months of surveillance. This cumulative
figure shows all infections per patient, including all COVID-19 events, within the BCG group(blue) compared to the placebo group (red). Included are the infections for which multiple
subject events were documented in both BCG and placebo groups. Comparison by means of a
Poisson model yields a significant difference with p=0.004 (BCG n=24 out of 96 T1D total,
Placebo n=20 out of 48 T1D total).
B. Cumulative infections for two different time periods: pre-COVID-19 pandemic (the 2.5 year
period prior to this trial, i.e., the Pretrial) and during the COVID-19 pandemic (the Current Trial
period of 15 months). The Pretrial time period was when all clinical trial subjects received their11≥ 3 BCG vaccines or placebo vaccines; the Current Trial was when the subjects were monitored
during the 15-month observation during the COVID-19 pandemic (total patients BCG n=96, Pbon=48). The lack of a statistical difference in the number of infections between BCG andplacebo groupsin the Pretrial period suggests that a longer length of time than 2.5 years isnecessary to realize BCG’s maximal infectious disease protection. It appears that, during theentire period of 15 months, prior BCG vaccinations were protecting from COVID-19 and otherinfections.
** p < 0.01
C. Infectious Disease Index for symptomatic patients. Symptomatic patients in the BCG-treatedgroup had significantly reduced Total Infectious Disease Symptom Index (placebo 152 ± 70
(n=20) vs BCG 48 ± 11 (n= 31), p = 0.04, single tail and unpaired) as well as Average Infectious
Disease Symptom Index (Placebo 23 ± 7 (n=20) and BCG 13 ± 2 (n=31), p=0.04, single tail andunpaired). We first calculated total and average symptom scores per patient and thencalculated average and s.e.m. of each of these for BCG and placebo cohorts separately (*Student’s T-test p < 0.05, 1-tailed, unpaired).
D. Patients in the BCG cohort reported significantly fewer days of missed work during infections
as compared to the placebo group (* p=0.02). Missed work was reported by 7 out of 32 BCG
patients and by 8 out of 17 Placebo patients
E. The number of patients that reported at least one symptom were 20 out of 48 in the Placebogroup and 33 out of 96 in the BCG group. The placebo group had more severe average
symptoms as compared to the BCG group for 12 out of 12 symptoms (Left panel). The numberof patients in BCG and placebo groups that reported each symptom was then expressed (Rightpanel). For 11 out of 12 symptoms there was a higher percentage of symptomatic patients inthe placebo group as compared to the BCG group. Statistical analysis by Student’s T-test (1tailed, unpaired, * p < 0.05).
Figure 5. Infection Symptoms of Trial Participants vs. Adult Household Members.
A. Infection symptoms in BCG and placebo groups compared to non-diabetic adult partners
living in the same households. We collected surveys of symptoms of infectious diseases from alltrial participants and household members of 13 BCG families and 7 placebo families. The BCGtreated trial participants had comparable or lower Total Infectious Symptoms Indexes ascompared to their partners living in the same household, whereas most placebo-treated trial
participants had more severe symptoms as compared to their partners. Statistical analysis of
the differences by Two-sample Wilcoxon testing was significant (2-tailed; p=0.049; BCG n=13
and Placebo n=7). Left panel: XY-plot of Symptom Index of Trial Participants versus their
Household Members. Right panel: the same data is also shown as a XY-plot of Household
Members versus the difference of Trial Partipants and their Household Members. This plot
makes it easier to visualize which groups had milder or more severe disease by introducing
positive and negative differences.
B. Distribution of individual infectious disease symptoms in BCG-, placebo-treated participants
and infected household members across the four scoring possibilities (0 no symptoms; 1 mild; 2
moderate; 3 severe symptoms). Number at risk data are shown in Figure S4.

12

STAR★Methods
RESOURCE AVAILABILITY
Lead Contact
Further information and requests for resources and reagents should be directed to and will be
fulfilled by the lead contact, Denise Faustman (dfaustman@mgh.harvard.edu)
Materials availability
This study did not generate new unique reagents.
Data and Code availability
Any additional information required to reanalyze the data reported in this paper is available
from the lead contact upon request.
Additional Resources
This trial is listed on clinicaltrials.gov (https://clinicaltrials.gov/ct2/show/NCT02081326)
Key resources table
REAGENT or RESOURCE
Antibodies

SOURCEIDENTIFIER

Japan BCG Laboratory,
Tokyo, Japan

N/A

Bacterial and virus strains
BCG vaccine (Bacillus Calmette-Guérin)Biological samples

Chemicals, peptides, and recombinant proteins13

Critical commercial assays
AntiCoV-ID™ IgG ELISASARS-CoV-2 Protein Microarray: 2-in-1 protein and peptide assay
Point of Care SARS-CoV-2 testing (PCR, Rapid Antigen test, Antibody
test)

Akston BioSciences,
Beverly, MA
CDI Labs, Baltimore, MD.
(http://cdi.bio)
Quest Diagnostics,
Secaucus, NJ

SKU: 600016

StudyTrax, Macon, GA,
USA

StudyTrax

CDICOV2-001.0
N/A

Deposited data

Experimental models: Cell lines

Experimental models: Organisms/strains

Oligonucleotides

Recombinant DNA

Software and algorithms
StudyTrax Electronic Data Capture System for Clinical Research14

OtherBD Vacutainer Serum Blood Collection Tubes (Red Top)BD Vacutainer Push Button Blood Collection Set

Becton and Dickinson,
Franklin Lakes, NJ
Becton and Dickinson,
Franklin Lakes, NJ

REF 366430
REF 367344

METHOD DETAILS
Study design and participants
We assessed the safety and efficacy of ≥3 BCG vaccinations (Tokyo-172 strain) versus placebo
for prevention of COVID-19 and other infectious diseases in a community-based, single-center,
Phase 2/3 randomized, double-blind, placebo-controlled clinical trial. The trial duration was 15
months, starting at the beginning of the SARS-CoV-2 pandemic in the US (01/01/2020) andending with the US launch of COVID-19 vaccines in April 2021 (Fig. 1A). All participants only
received the placebo or BCG vaccine and did not receive COVID-19 antigen specific vaccines.
Clinical trial participants were volunteers ages 18-50 years old with a co-morbid condition, type1 diabetes (Fig. 1B). The current trial was a parallel study using already enrolled double-blindedparticipants in a multi-dose BCG vaccine trial assessing its 5-year impact on other outcomes
unrelated to infections (US FDA Investigational New Drug [IND]#16434; Massachusetts General
Hospital [MGH] protocol #2013P002633). At the three-year time point of the original study, thisparallel study on COVID-19 protection was initiated, while the original trial continued in a
double-blinded fashion. The original clinical trial and this parallel clinical trial were preregistered in the clinicaltrials.gov NCT site as: NCT02081326.
The protocol for the current COVID-19 prevention trial was approved as an amendment
to the original FDA IND and MGH protocols as well as a new MGH protocol #2020P001462. A
study synopsis is provided (Figure S6). The current trial and the original trial were performed inaccordance with ethical principles that have their origin in the Declaration of Helsinki and areconsistent with International Conference on Harmonization (ICH)/Good Clinical Practice,applicable regulatory requirements, and the MGH policy on Bioethics. Informed consent was
written for the intervention; informed consent could be oral for the COVID-19 symptom
surveys.
A total of 150 subjects were recruited for the original trial. All subjects were recruitedwith a requirement to have no previous or current tuberculosis and no history of BCG
vaccinations, even during childhood. All subjects were US born and followed from the beginningof the original trial for all infection diseases (viral, bacterial, parasitic, etc) and any sort ofantibiotic usage through a direct MedDra documentation system. The US has never offered
newborn or childhood BCG vaccinations. The original trial recruited 150 subjects (2:1
BCG:placebo) and at the start of this COVID-19 trial two years later, 144 subjects were enrolled
for this parallel trial design. The parallel infectious disease study design studied the off-target
effects of repeat BCG vaccinations for COVID-19 and infectious disease protection (96 BCG
15

treated, 48 placebo treated). There was no patient attrition over the 15-month time course of
the COVID-19 study. The original exclusion criteria included positive purified protein derivative(PPD) test, positive T-spot test for tuberculosis, or born in a foreign country with mandatory
BCG vaccinations. The goal of these exclusion criteria was to prevent prior ongoing and durable
protection from Mycobacterium bovis (the origin of the BCG vaccine) or Mycobacterium
tuberculosis (TB) exposures causing long-term protection. Exclusion criteria also included no
active glucocorticoids treatment, chronic immunosuppressive medications, or currently livingwith an immunosuppressed individual to prevent an adverse event from the administration ofthis live vaccine. All subjects lived within the United States (Figure S5). The largest contingentof participants, 36, was from Massachusetts, the second largest, 16, was from New York, andthe next largest, 11, was from Texas. During this time in the US for this COVID-19 study (January
2020 - April 2021) the Delta variant was identified in May 2021 so this study addresses BCG
protection from all infectious diseases and resistance to predominately the Alpha variant of the
SARS-CoV-2 pandemic. Importantly both the diagnostic PCR testing and serology testing
performed to confirm COVID-19 disease, detected all genetic variants of the virus.
This trial had three types of oversight. At 6-month intervals, this trial had audits fromeither Massachusetts General Brigham (MGB) Division of Quality Management or from outside
auditors (Advanced Clinical Trials, Deerfield, IL). All trial data was processed by two unblindedstatisticians (D.S., H.Z.), and an independent data and safety monitoring board (DSMB) met at
6-month intervals to monitor subject safety and reporting compliance.
The MGH Research Pharmacy generated the randomization code for the administration
to study participants of either the BCG or the saline placebo vaccine. This randomization code
was held in the MGH pharmacy. All drug within the syringe had an identical appearing fluid for
the BCG and placebo vaccine in the 0.1cc volume. With this vaccine there are absolutely no
systemic symptoms after administration so blinding of study staff and also subjects collecting
post-vaccine adverse systemic events is easier than antigen specific vaccines that frequently
yield multi-day systemic reactions and sometimes missed workdays. A separate third party
study nurse collected systemic reactions to the vaccine (none reported) and also consulted withparticipants for any skin reactions. Skin reactions were kept in separate locked storage centerand not within the Studytrax or EPIC data management center. All investigators and study staff
remained blinded when studying both the primary and parallel study outcomes of COIVID-19
and infectious diseases. Subject and staff unblinding will occur at the end of the primary study.
Procedures
The BCG vaccine or saline placebo (both 0.1ml volume per dose) was administeredintradermally over a period of 2.5 to 3 years prior to the start of this parallel trial. Two BCG
doses were given 4 weeks apart in the beginning of year 1, and an identical booster dose wasgiven in year 2. Site staff were responsible for reporting all drug- and non-drug-related safety
information and were unaware of group assignments.
Monitoring for COVID-19 or infectious disease over the 15-month surveillance period
included emailed surveys (every 2 months), as well as surveys during every visit to our clinic.
The survey asked about COVID-19 and any other infections subjects had over the previous threemonths. It was completed by the patient via email, over the phone with research staff at the 6month visit time or online through Studytrax. Studytrax is an FDA compliant electronic data
16

capture system for clinical research. At each 6-month clinic visit 10 ml of blood was drawn. The
survey was also completed by the patient for any adult household member with infectious
symptoms. Participants also directly reached out to the clinic to report infections. Household
members’ blood for confirmation of SARS-CoV-2 infection was locally obtained by a national
based chain of blood drawing centers (Quest Diagnostics, Secaucus, NJ). The symptoms
questionnaire for study participants and infected household members was in compliance withFDA Guidance for Industry 51. For each symptom, the participants provided a severity score of 0
(none), 1 (mild), 2 (moderate) or 3 (severe). The length of the infectious illness in days was also
reported. From the individual symptom scores a Total Symptom Score and an Average
Symptom Score were tabulated. Then, multiplying by the length of illness, the Total Infectious
Symptom Index or the Average Infectious Symptom Index was assessed (Figure S3A).
For SARS-CoV-2 antibodies detected, the average and standard deviation of antibody
levels prior to the onset of COVID-19 pandemic (Pre-2020) was determined. The average of thelevels after the start of the pandemic (2020 and 2021 data) was also calculated. Using these
averages and the Pre-2020 standard deviation the Z-score per patient was then calculated. The
Z-score thus represents the difference in average pre-COVID and average COVID signal levels,
expressed as the number of standard deviations of pre-COVID. A Z-score of ≥3 was considered
to represent a statistically significant difference.
Outcomes
This trial had the following co-primary efficacy endpoints on potential benefits of multi-dose
BCG: prevention of symptomatic and molecularly-confirmed COVID-19 (see case criteria below);
and reduction of infectious disease (including COVID-19) symptomatology and severity by the
Total or Average Infectious Disease Index. The study synopsis is provided (Figure S6). These
outcomes conformed to other primary US based trials for the antigen-specific vaccines (PfizerBioNTech and Moderna trials). Secondary outcomes were average workdays missed, andindividual infection symptom severity, and household members’ total infectious symptom
index.
COVID-19 is defined according to FDA by the presence of at least one of 12 symptoms 51(headache, chills/shivering, diarrhea, nausea/vomiting, fatigue, shortness of breath, loss of
smell or taste, muscle aches, nasal congestion, cough, sore throat and fever) and molecularconfirmation. These symptoms, other than the loss or smell and taste, are symptoms of mostinfectious diseases, so the survey was used for both purposes.
Our criteria for symptomatic and molecularly confirmed COVID-19 had to be rigorous
while overcoming the problem that PCR testing was not available at the origin of this trial
and/or not equally accessible in all parts of the US. PCR testing only became widely available
seven months into the trial. In addition, PCR detects SARS-CoV-2 virus in a smaller time window
than molecular antibody confirmation through the measurement of host direct SARS-CoV-2
antibodies. Therefore for symptomatic and molecularly confirmed COVID-19 we required
subjects to exhibit one or more FDA-defined symptoms of COVID-19 1 together with positive
findings on ≥5 out of 10 assays (9 SARS-CoV-2 IgG antibody binding assays and a tenth assaythat included PCR testing for viral RNA) (Figure S1). A subject was deemed positive for aparticular SARS-CoV-2 antibody assay if the serology antibody Z-score was ≥ 3, i.e., at least 3

17

standard deviations higher than during the pre-COVID period (baseline). Serum was obtained in
all cases within 3 months of the infection for the detection of IgG antibodies.
Statistical Analysis
The sample size and power analysis of this trial were based on the primary randomized Phase II
clinical trial that started 2-3 years prior to this parallel trial focusing on COVID-19. Vaccine
efficacy is defined by (p1 – p2)/p1 x 100, where p1 is the % Covid-positive in the placebo groupand p2 is the % Covid-positive in the BCG group. For comparison to published Covid vaccine
trials such as the mRNA trials, we also calculated the posterior probability that the vaccine
efficacy is greater than 30% using a Bayesian Beta binomial model fitted using WinBUGS67. We
used a non-informative beta prior with parameters (1,1) and took 5000 MCMC samples tocompute the posterior probability for efficacy to be greater than 30%. The 95% credible interval
for the posterior probability for efficacy to be >30% is 98.9-100%. Further details are provided
in the Figure legends and these method mirror past studies on COVID-19 vaccine efficacy68.
Average antibody levels were compared using Student’s T-testing in Prism (Graphpad Software,
San Diego, CA) or Microsoft Excel. The number of patients positive in the BCG versus thePlacebo cohort were compared using Fisher’s Exact Test (available online athttps://www.graphpad.com/quickcalcs/contingency1/). Differences in the symptoms scores
between participants and household members were compared between the BCG-treated groupand the placebo-treated group using a Two-Sample Wilcoxon test. Statistics were consideredsignificant at p<0.05.
This trial is registered as ClinicalTrials.gov number, NCT 16434

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24

Figure 1.

A

Study Diagram
IND#16434 in T1D:
Double-blind RCT~ 2-3 years~ 2-3 years≥ 3 BCG or Placebo
Vaccines

Expected Completion:
2023

COVID-19 Pandemic:
Start January 2020

15 Month
Surveillance Period

BCG Treated(n = 96)

Antibody Levels

SARS-CoV-2
Infection Testing

PCR

Placebo Treated(n = 48)

Evaluation for
COVID-19 andInfectious Disease
Symptoms

Patient Reporting
Bi-monthly Survey

COVID-19 and Infectious
Disease in Household
Members

BCG COVID Trial CompletedApril 2021

B

Characteristics of Participants
Sex – no. (%)
Male
FemaleCountry – no. (%)
United States
Current Age – yr
Average (Median)Age at Baseline Injection – yr
Average (Median)Age of Diabetes Onset – yr
Average (Median)
Duration of Diabetes – yr
Average (Median)BCG (N = 96)

Placebo (N= 48)

Total (N = 144)

55 (38.2)
41 (28.5)

29 (20.1)
19 (13.2)

84 (58.3)
60 (41.7)

96 (66.7)

48 (33.3)

144 (100)

44.5 ± 1.3 (43.1)

43.6 ± 1.9 (40.7)

43.8 ± 1.1 (42)39.3 ± 1.3 (38)38.4 ± 1.9 (35.5)

39 ± 1.0 (37)26.8 ± 1.2 (26.3)

25.1 ± 1.9 (23.4)

25.8 ± 1.0 (25)

17.7 ± 0.7 (17)18.6 ± 1.0 (16.5)

18 ± 0.6 (17)25

Figure 2.

B

BCG Efficacy Against Symptomatic COVID-19
BCG(n=96)

Placebo(n=48)

Surveillancetime(months)

VaccineEfficacy

COVID-19 confirmed by
Point of Care and
SARS-CoV-2 Antibodies

1(1.0%)

6(12.5%)

15

92%

0.99

COVID-19 confirmed by
PCR only0(0.0%)

5(10.4%)

15

100%

0.99

Diagnostics Used to Confirm COVID-19 Infections

100%

20 I.

Posterior
Probability(Vaccine Efficacy >30%)

BCG
Placebo80%

II.

p-values

15

I.
II.
III.
IV.
V.

10

Cumulative COVID-19 Disease, %A

50
20 III.

0.0007
0.089
0.009
0.001
0.024

p-values
0.004
0.0003
0.009
0.009
0.029

VI.
VII.
VIII.
IX.
X.

91.7% IV.

93.8%

V.

90%

VI.

87.5%

94.4%

83.3% IX.

90%

X.

91.7%

15
10
5
0
20 VII.

VIII.

15
10
5
0

0

5

10

15 0

5

10

15 0

5

10

15 0

5

10

15

Time since January 2020, months

26

Figure 3.

A

Change in antibody levels for subjects reporting COVID-like symptoms (Z-scores)
Z-score
≥3
<3

BCG

40
30
20
10

Placebo

Symptomatic Subjects

Z-score≥50

0
III IV
IV V
V VI
VI VII
VII VIII
VIII IXIX
II IIII III

SARS-CoV-2 Antibody
Binding Sites

IX
II IIII III
III IV
IV V
V VI
VI VII
VII VIII
VIII IXSARS-CoV-2 Antibody
Binding Sites

COVID-19 confirmed by RT-PCR only
20

Cumulative COVID-19
Confirmed by PCR, %

B

BCG

15

Placebo10

Efficacy: 100%p-value: 0.0036

5
0
0

5

10

Time since January 2020,
months15

27

Figure 4.

Infections per Patient

0.4
0.3
0.2
0.1
0.0
0.0

Infectious Disease Index

C

250

Before and during current trial

BCG

0.60

Placebo
✱✱

0.40
0.20
0.00

1.5
0.5
1.0
Time since January 2020, years

D

Infectious Disease Index

Pretrial(~ 30 months)

Current Trial(15 months)

Average Workdays Missed

4

✱

✱

200
150
100
50
0

E

B

Current Clinical Trial
0.5✱

Missed Work during
Infections, days

Cumulative Infections
per PatientA

Average

Total

3

BCG
Placebo2
1
0

Individual Infection Symptom Severity
BCG

Headache
Chills/Shivering

Placebo

Diarrhea
Nausea/Vomiting
Fatigue
Shortness of Breath
Loss of Smell/Taste
Muscle Aches
Nasal Congestion
Cough
Sore Throat
Fever

0

1
2
Average Symptom Score

3 0

40
20
30
10
Patients with Symptoms, %28

Total Infectious Symptom Index
300

Trial Participant - Household Member

A
Household Members Symptom Index

Figure 5.

250
200
150
100
50
0
0

B

BCG

200

Placebo

150
100
50
0

-100
-200
-300
0
20
40
100 200 300
Household member Symptom index

100
200
300
Trial Participants Symptom Index

Symptom Severity Comparison

Type 1 Diabetics
Placebo
BCG

HouseholdMembers

Headache
Chills/Shivering
Diarrhea
Nausea/Vomiting
Fatigue
Shortness of Breath
Loss of Smell/Taste
Muscle Aches
Nasal Congestion
Cough
Sore Throat
Fever

Score 0Score 1
Score 2
Score 3

0

20

40

60

80 100 0

20

40

60

80 100 0

20

40

60

80 100Percentage, %29