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ArticleLate in the US pandemic, multi-dose BCG vaccinesprotect against COVID-19 and infectious diseases
Willem M.
Kühtreiber, Emma
R. Hostetter, Grace
E. Wolfe, ..., Joan
E. Braley, HuiZheng, Denise L.
Faustmandfaustman@mgh.harvard.edu

Highlights
A randomized Phase III trial
of BCG vaccines for
COVID-19 and infection
protection
Trial conducted late in US
pandemic in infectious
vulnerable type 1 diabetics
BCG vaccines provide
platform infectious diseaseprotection including
COVID-19
Commercial mRNA
vaccines in type 1 diabetics
do not appear to protectfrom COVID-19

Kühtreiber et al., iScience --,109881--, 2024 ª 2024 The
Author(s). Published by Elsevier
Inc.
https://doi.org/10.1016/
j.isci.2024.109881

Please cite this article in press as: Kühtreiber et al., Late in the US pandemic, multi-dose BCG vaccines protect against COVID-19 and infectious
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ArticleLate in the US pandemic, multi-dose
BCG vaccines protect against COVID-19
and infectious diseases
Willem M. Kühtreiber,1 Emma R. Hostetter,2 Grace E. Wolfe,2 Maya S. Vaishnaw,2 Rachel Goldstein,2Emily R. Bulczynski,2 Neeshi S. Hullavarad,2 Joan E. Braley,2 Hui Zheng,2 and Denise L. Faustman1,3,*
SUMMARY

The Bacillus Calmette-Guérin vaccine has many off-target benefits, including protection from diverse infectious diseases. As SARS-CoV-2 evolved, COVID-19 disease became more transmissible and less lethal.
In this Phase III double-blinded, placebo-controlled trial conducted late in the pandemic, we tested at-risk
US adults with type 1 diabetes if multi-dose BCG protected against COVID-19 and other infectious disease, co-primary outcomes. From April 2021 to November 2022, Tokyo-strain BCG vaccines provided significant protection against COVID-19 disease (p = 0.023) and strong platform protection against all infectious diseases (p < 0.0001). Over the course of the study, commercial COVID-19 vaccines were rolled out,
most of which were mRNA-based. In contrast to the protection afforded by BCG, as reported by others,
COVID-19 mRNA vaccine alone provided no protection from COVID-19 disease (p = 0.43). BCG vaccination efficacy was unaffected by concurrent COVID-19 vaccinations; COVID-19 vaccines neither helped norhindered BCG protection.
INTRODUCTION
Clinical trial and epidemiologic investigations conducted over the past decade have shown that the Bacillus Calmette-Guérin (BCG) vaccine,originally developed for tuberculosis protection, appears to have wide-ranging off-target benefits in the form of platform infectious diseaseprotection.1–14 The BCG vaccine, a live attenuated version of the mycobacterium that causes tuberculosis in cattle (Mycobacterium bovis),
confers durable protection. When administered to neonates, it can protect against tuberculosis for more than 40 years, and it can benefit
glucose metabolism for more than 70 years.15,16 Heralded as the safest vaccine in continuous use globally, the BCG vaccine is designatedan essential medicine by the World Health Organization.17 mRNA vaccines developed for COVID-19 have limitations due to narrow efficacy
against specific viral variants as well as short duration of protection against infection.18
As the SARS-CoV-2 pandemic unfolded, a profusion of international investigations sought to test whether BCG could protect against
development of COVID-19. Five randomized trials showed efficacy,13,19–23 whereas seven randomized trials showed no efficacy.24–29 . Furthermore, looking at the hard endpoint of death after COVID-19 infections, a post-hoc analysis of eight of the recent clinical trials found that BCG
groups had statistically significant less death than among placebo recipients.30 It is important to note the differences in the designs of thesetrials.31 All of the clinical trials showing no benefit had one or more features that might have diminished the likelihood of finding a BCG effect:
the trial enrolled subjects that had previous received neonatal BCG vaccination or previous exposure to tuberculosis (TB), all of which confer
long-term protection; the trials used a less potent strains of BCG; the trials used a single BCG dose, rather than multiple dose benefits; or thetrial had insufficient follow-up time from the vaccination date to the infection exposure data and therefore could not capture BCG’s efficacy(i.e., trial was weeks to months instead of the needed two or more years for many off-target effects). When the BCG vaccine was used successfully against autoimmune diseases in BCG-naı̈ve adults in Europe and the US, full efficacy was not reached until at least two years. The
time lag may be due to BCG’s potential mechanism of action involving the multi-year slow epigenetic reset of critical genes in the adult humaninnate and adaptive immune system, at least in adults.13,16,32–34
We now report on the second of two trials of multi-dose BCG protection against COVID-19 in a US population of infection vulnerable type1 diabetics. The earlier Phase II trial was conducted early in the US pandemic.13 Studying a US population has multiple advantages: it has notbeen vaccinated with neonatal BCG; it has low rates of endemic tuberculosis; and it is highly vulnerable to infectious diseases in general and to
COVID-19 in particular.35–37 In our earlier BCG study, an RCT Phase II trial, we excluded patients with a history of tuberculosis exposure andwith a history of neonatal BCG vaccination.13 The same exclusions apply to the current Phase III trial conducted late in the US pandemic, also a

1Massachusetts General Hospital and Harvard Medical School, Boston, MA 02192, USA
2Massachusetts General Hospital, Boston, MA 02192, USA
3Lead contact*Correspondence: dfaustman@mgh.harvard.edu
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ArticleFigure 1. Flow Diagram of the current Phase III BCG trial
The current Phase III clinical trial started on April 1, 2021 and continued for 19 months until near the end of the COVID-19 public health emergency in the UnitedStates on May 11, 2023, as defined by the US Centers for Disease Control and Prevention. The co-primary outcomes were BCG vaccine protection from COVID-19
disease and from other infectious diseases.

randomized, double-blinded, placebo-controlled evaluation. While the patient population and outcome measures are similar across the twotrials, there are three key differences: non-overlapping, consecutive time periods of study with different circulating SARS-CoV-2 variants (earlyvs. late pandemic); different diagnostics (antibody profiling vs. standard of care diagnostics); and different US availability of commercial
COVID-19 vaccines (none vs. multiple vaccines) (Figures 1, S1, and S2).
A Phase III randomized, double-blinded placebo-controlled trial was conducted in the United States over a 19-month period to test if
multi-dose, intradermal BCG offered protection from COVID-19 disease and protection from overall infectious disease, the co-primary outcomes (Figure 1). Randomization was 2:1 with two subjects receiving BCG for every enrolled placebo subject (Figure 1). The Phase III trial was
conducted late in the COVID-19 pandemic during sequential dominance of Beta, Gamma, Delta, and Omicron variants. As compared to theoriginal SARS-CoV-2 strain, these later variants are more transmissible and, in some cases, less virulent.38 During this trial, the US rollout of thecommercial COVID-19 vaccines unfolded gradually, consisting largely of mRNA vaccines. We therefore evaluated the efficacy of COVID-19
vaccines with and without concurrent BCG vaccination as an observational study.

RESULTSType 1 diabetic subjects’ baseline characteristics were similar in the BCG (n = 93) and placebo (n = 48) groups (Table 1). Participants were adultmen and women with similar sex distributions in the BCG arm (43% female and 57.0% male) and placebo arm (39.6% female and 60.4% male).
All participants were US citizens and, at the time of original enrollment, were negative for prior BCG vaccines, and were negative for tuberculosis as confirmed with a negative QuantiFERON Gold TB test.
The retention rate was excellent, with 100% of subjects (n = 141) completing the Phase III trial of 19 months’ duration. At the launch of thisPhase III clinical trial, all subjects had been randomized and vaccinated with 6 BCG or placebo vaccine (70% of subjects) or vaccinated with 5
BCG or placebo vaccines (30%) (Figure 1). The average number of days until the sixth and final vaccine was 124 G 16 days. The trial used theTokyo-174 strain of BCG. The median age at diabetes onset for all subjects was 26.2 G 1 year, and the average duration of type 1 diabetes was
17.9 years (Table 1).

BCG protects against COVID-19 disease, a co-primary outcome
Subjects with confirmed COVID-19 disease were identified over the 19 months of observations. The subjects were determined to have a
confirmed case of COVID-19 disease when all of the following conditions were met: reporting at least one COVID-19 symptom (as definedby the FDA Guidelines for Industry document;39 at least one day of illness; and testing positive in point of care testing for COVID-19 infection
using commercial assays (PCR or Rapid Antigen testing).

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Table 1. Characteristics of participants in phase III clinical trial

Characteristics of Participants

BCG (N = 93)

Placebo (N = 48)

Total (N = 141)Male

53 (57.0)

29 (60.4)

82 (58.2)

Female40 (43.0)

19 (39.6)

59 (41.8)

93 (66.0)

48 (34.0)

141 (100)

100%

100%

141 (100)

100%

100%

141 (100)

44.3 G 1.3 (43.3)

43.4 G 1.9 (40.5)

44.0 G 1.1 (42.3)

40.7 G 1.3 (39.6)

39.8 G 1.9 (36.7)

40.4 G 1.1 (38.9)

26.6 G 1.2 (25.9)

25.1 G 1.9 (23.4)

26.1 G 1.0 (25.0)

17.7 G 0.7 (17.2)

18.4 G 1.0 (16.2)

17.9 G 0.6 (17.2)

Pfizer52 (55.9)

22 (45.8)

74 (52.5)

Moderna23 (24.7)

11 (22.9)

34 (24.1)

Johnson & Johnson

1 (1.1)

6 (12.5)7 (5.0)None

17 (18.3)

9 (18.8)

26 (18.4)

Sex – no. (%)

Country – no. (%)
United States
Tuberculosis Status – (%)
Negative
History of BCG Vaccinations
Un- VaccinatedCurrent Age – yr
Average (Median)Age at Baseline Injection – yr
Average (Median)Age of Diabetes Onset – yr
Average (Median)
Duration of Diabetes – yr
Average (Median)
Vaccinations – no. (%)

During the 19 months of this clinical trial, COVID-19 disease occurred in 22 of 93 participants in the BCG group (23.7%) and in 20 out of 48
participants in the placebo group (41.7%) (Figure 2). A one-tailed Fisher’s exact test was significant at p = 0.023, showing that multiple BCG
vaccines protected type 1 diabetic subjects. BCG vaccine efficacy versus placebo, according to the formula in the STAR methods section,
was 43.2%.
We also stratified BCG protection from COVID-19 disease according to calendar year (2021 and 2022) (Figure 2B). For 2021, there were 10
out of 93 unique confirmed COVID-19-positive subjects in the BCG group (10.8%) and 7 out of 48 (14.6%) in the placebo group. BCG vaccine
efficacy was 26.3%, but the difference between groups was not significant (one-tailed Fisher’s exact test p = 0.342). For 2022 there were 13 outof 93 confirmed COVID-19-positive subjects in the BCG group (14.0%) and 14 out 48 (29.2%) in the placebo group. BCG vaccine efficacy was
52.1%, and the difference was significant (one-tailed Fisher’s exact test p = 0.015). Therefore, during predominance of Omicron, the mosttransmissible SARS-CoV-2 variant, BCG vaccinations provided more protection than placebo.
This is the second US trial of BCG’s efficacy in the type 1 diabetic population as it relates to infectious disease protection and COVID-19
protection. Although these were two separate trials with separate clinical trial protocols, separate consents, separate data and safety monitoring board (DSMB), separate time periods with different COVID-19 genetic variant exposures and with different primary outcomes, both
trials used the same randomized type 1 diabetic subjects residing in the United States. A cumulative timeline of COVID-19 events acrossthe two trials is presented in Figure 3. The Phase II trial (January 2020 to April 2021) covered 15 months, with BCG efficacy of 92% (p =0.004) (Figure 3A). The current Phase III trial (April 2021 - November 2022) shows BCG efficacy of 43.2% (p = 0.023) (Figure 3B). In this phasethe placebo group accumulated more COVID-19 events and at a faster rate during the latter part of the trial (Figure 3B). There was a clear stepup of cumulative events during Omicron-dominance, presumably corresponding to its higher transmissibility as compared to earlier variants.38 The statistical difference between the BCG and the placebo groups persisted (p = 0.023) (Figure 3B). Over the full 34 months of
the US COVID-19 pandemic, BCG shows an efficacy of 54.3% (p = 0.0007) (Figure 3C). The numbers at risk across the two trials are presented
in Figure S2B.

BCG protects against infectious diseases, the co-primary outcome
We analyzed all infectious disease events collected as adverse events by the MedDRA classification coding (Figure S3). For this clinical trial theobserved infectious diseases included viral, bacterial, and fungal events as well as COVID-19 disease itself. BCG provides broad-based protection against infectious diseases from the global literature, and we have previously shown this to be the case for type 1 diabetic subjects
during the Phase II trial.1–3,13

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ArticleFigure 2. BCG vaccines protect type 1 diabetics from COVID-19 disease(A) During the 19-month trial, multi-dose BCG vaccines protected type 1 diabetics against COVID-19 disease. During the Phase III trial period (April 1, 2021–
November 3, 2022), 22 of 93 subjects (23.7%) in the BCG group acquired COVID-19 disease (i.e., became COVID-19 positive), whereas 20 of 48 subjects(41.7%) in the placebo group became COVID-19 positive. The one-tailed Fisher’s exact test shows that this difference is significant (p = 0.023). BCG vaccine
efficacy was 43.2%, with efficacy defined by (p1 – p2)/p1 x 100, where p1 is the % COVID-positive in the placebo group and p2 is the % COVID-positive in
the BCG group.
(B) Percent of type 1 diabetics patients who became COVID-19-positive, according to year. In 2021 10 of 93 subjects (10.8%) in the BCG group became COVID-19
positive, whereas in the placebo group, 7 of 48 (14.6%) did. For 2022,13 (14.0%) in the BCG group became COVID-19-positive and 14 (29.2%) in the placebo groupdid. A one-tailed Fisher’s exact test for the 2021 data was not significant (p = 0.342), but for 2022 the difference between groups was significant (p = 0.015).

We therefore studied whether BCG vaccines in this Phase III trial period of 19 months continued to show broad protection after introduction of commercial COVID-19 vaccines late in the US pandemic. In the BCG group there were 44 infectious events, whereas there were
66 in the placebo group. The average number of Infections per Subject was lower in the BCG than placebo groups, with Poisson statistics
indicating a significant difference at p < 0.0001. A violin plot demonstrates more subjects in the placebo group had many more infectious
events as compared to the subjects in the BCG group (Figure 4B). Taking into account the difference in number of subjects in the BCG(n = 93) and placebo (n = 48) groups with the 2:1 randomization, the heatmap shows a breakdown of the various infectious events per
subject (Figure 4C). The darker shades of red for many of the infections indicates a higher infection rate in the placebo group than in
the BCG group. Taken together, these results indicate that BCG-treated subjects have fewer infectious events compared to placebotreated subjects.
We analyzed the accumulation of infectious events over time for the full 34 months of the Phase II and Phase III randomized double blindedclinical trials, a period covering almost the entire COVID-19 pandemic in the United States. The Consort Diagram places the previous Phase II
trial and current Phase III trial on a timeline of dominant SARS-CoV-2 variants (Figure 5A). To take account of the different sizes of BCG andplacebo groups (BCG n = 93; Placebo n = 48), we studied the cumulative rate of Infectious Events per Subject. For the current Phase III clinical
trial, the data demonstrates that subjects in the placebo group accumulate infectious diseases at a faster rate compared to the BCG group(Figure 5B). The combined total infectious disease events in the Phase II and Phase III clinical trials (34 months) shows strongly that BCGtreated subjects have a lower rate of infection across the whole period as compared to the placebo-treated subjects (Figure 5C; Poisson statistics p < 0.0001).
We also performed Andersen-Gill Cox Proportional Hazard Statistics to evaluate whether the length of time that subjects were vaccinatedwith COVID-19 vaccines had an effect (beneficial or detrimental) on overall infectious disease rates. This was done by including the number ofcalendar days for COVID-19 vaccination as a parameter. Using infectious disease data for both the Phase II and the Phase III clinical trials, theresulting p-value was nonsignificant (p = 0.154). The Hazards ratio was 0.998 (95% CI 0.995–1.001). We conclude that the amount of time that a
subject is treated with COVID-19 vaccines does not play a beneficial or harmful role in the prevention of infections afforded by BCG.

Commercial COVID-19 vaccines offer no protection of type 1 diabetics from COVID-19 disease
Although not a primary outcome of this Phase III clinical trial, it was important to evaluate the impact of the commercially available COVID-19
vaccines for type 1 diabetics in the absence or presence of BCG vaccination. During this trial, rollout of the commercial, predominantly mRNA
vaccines, occurred. Receipt of Pfizer, Moderna and Johnson & Johnson commercial vaccines in our study population is recorded in Table 1.
First, we quantified the efficacy of commercial COVID-19 vaccines in the absence of BCG vaccination as an observational outcome (Figure 6). Placebo recipients (non-BCG vaccinated) were divided into COVID-19-vaccinated and never-COVID-19-vaccinated cohorts. The number of subjects in each group with COVID-19 disease was tabulated. As was the case for the overall study, a subject was considered to have
COVID-19 disease when they had one or more COVID-19 symptoms as defined by the FDA,39 with a length of illness of at least one day, as well
as a positive test for COVID-19 at Point of Care via PCR or Rapid Antigen testing.
Out of a total of 48 placebo subjects, 39 received commercial COVID-19 vaccines and 9 never received COVID-19 vaccines. In the COVID19 vaccine-treated cohort, 17 (43.6%) had COVID-19, whereas in the never-COVID-19-vaccinated cohort 3 (33.3%) had COVID-19 (Figure 6A).

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Figure 3. BCG vaccines protect type 1 diabetics from COVID-19 disease events over nearly the entire US pandemic
Based on the start date of each COVID-19 event, the number of days since the start of either the Phase II trial or the current Phase III clinical trial were calculated foreach COVID-19 event in the BCG and placebo groups separately. The number of subjects in the BCG and placebo groups were different (Phase II, BCG n = 96 andplacebo n = 48; current Phase III, BCG n = 93 and placebo n = 48). The data were normalized relative to group size by expressing the cumulative number of
COVID-19 events as a rate per subject. The cumulative event rate per subject was then plotted in a step graph against elapsed time in days. (A) Cumulative
COVID-19 event rate per subject during Phase II trial (1/1/2020 to 4/1/2021). The placebo group experienced a much higher rate of COVID-19 events per
subject compared to the BCG group.13(B) Cumulative COVID-19 event rate per subject during Phase III trial (4/1/2021 to 11/3/2022). The placebo group experienced a much higher rate of COVID-19
events per subject as compared to the BCG group. The dotted vertical lines delineate the approximate periods during which various SARS-CoV-2 variants were
dominant in the United States. Note the sudden increase in COVID-19 event rates once the Omicron variant supplanted earlier ones.
(C) Phase II and Phase III trials combined. The dominant variants are delineated by the dotted vertical lines. Regardless of time period in the US pandemic, BCG
vaccines continuously protect from COVID-19 disease in the BCG-vaccinated vs. placebo type 1 diabetic population. The number at risk data for these figures isshown in Figure S2B.

While the percent positive for COVID-19 in the never-vaccinated group trended lower, the difference was not statistically significant (onetailed Fisher’s exact test p = 0.43).
Since different SARS-CoV-2 variants were dominant during 2021 (Beta, Gamma and Delta) as compared to 2022 (mainly Omicron) andcommercial COVID-19 vaccines have short duration of efficacy and different efficacy depending on the variant, we also analyzed the approximate two-year time period as separate years (Figure 6B). No statistical significance was reached in either year. In 2021, 12.8% of the COVID-19
vaccinated subjects were COVID-19-positive versus 22.2% of the never-COVID-19-vaccinated subjects. The one-tailed Fisher’s exact test

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ArticleFigure 4. BCG vaccines protect type 1 diabetic subjects against infectious diseases(A) The bar plot shows the average rate and SEM across all infectious disease events in BCG (blue) versus placebo (orange) subjects. Averages were calculated
and then expressed as a rate (average events/subject) to take account of the different numbers of participants in each group. Patient numbers per group: BCG(n = 93) vs. placebo (n = 48). Total number of infectious events in the BCG group is 44 vs. placebo group is 66. Average rate/subject: BCG, 0.47; Placebo, 1.38.
Poisson statistics indicate that the difference between BCG and placebo groups is significant at p < 0.0001. Data are represented as mean G SEM.
(B) The Violin plot shows the density of the number of Infectious Events per Subject during the entire 19-month trial period. The distribution in the orange plot
(placebo) shows greater number compared to the blue plot (BCG). This is also evident from the higher median in the orange plot (white dotted lines indicate themedian).
(C) Heatmap of BCG versus placebo groups shows the number of various types of infectious events per patient over the entire Phase III trial. The darker red colors
in the placebo group indicate that this group tended to have a higher number of infection events as compared to the BCG group. A detailed listing of the data inthis heatmap is shown in Figure S3. ****p < 0.0001.

showed no significant difference, p = 0.39. In 2022, 30.8% of the COVID-19 vaccinated subjects were COVID-19-positive versus 22.2% of neverCOVID-19-vaccinated subjects. The one-tailed Fisher’s exact test was also not significant, p = 0.47. These observational results indicate thatthe commercially available COVID-19 vaccines do not appear to protect type 1 diabetes against COVID-19 disease, at least during the time
period under study.

BCG affords broad protection of type 1 diabetic subjects against infectious diseases irrespective of COVID-19 vaccine
Another way to study the impact of commercial COVID-19 vaccines is to analyze infectious disease outcomes (which include COVID-19). This
analysis is by treatment group (BCG vs. placebo) and by time periods within this Phase III trial: pre-COVID-19 vaccines, post-COVID-19 vaccines, and pre- and post-COVID-19 vaccines (Figure 7). Since the commercial COVID-19 vaccines were received at random or at individualized
times, we calculated the number of months that each subject was pre-COVID-19 vaccine (i.e., time before receiving the COVID vaccine) andpost-COVID-19 vaccine (i.e., time after receiving the vaccine) and then expressed the data as the rate of Infectious Events per Subject and perMonth. We performed Poisson statistics for Figure 7C (Pre- + Post-COVID-vaccines; Poisson distribution) since this encompassed a constanttime period for each subject (581 days). In the three time periods of analysis per Subject, the rate of COVID-19 disease in the BCG group was

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Figure 5. Regardless of the time frame of the US pandemic, BCG vaccines continuously protect type 1 diabetics from all infectious diseases
Based on the start date of each event, the number of days since the start of the Phase II or the current Phase III trial was calculated for each Infectious Event in the
BCG vs. placebo groups separately. The number of subjects in the BCG group and the placebo group were different (Phase II, BCG n = 96 and Placebo n = 48;
Phase III, BCG n = 93; Placebo n = 48). Therefore, the data were normalized relative to the group size by expressing the cumulative number of infectious events asa rate per subject. The Cumulative Event Rate per Subject was then plotted in a step graph against elapsed time in days. For a list of types of Infectious Events
observed, and the total number of infections per subject please see Figure 4B. The names of infectious disease events are listed in Figure S3. Note that thisincludes, but is not limited to, COVID-19 events. (A) Consort diagram shows the timeline of the Phase II and Phase III BCG trials in type 1 diabetic subjects,
in relation to the dominant SARS-CoV-2 variant.
(B) Cumulative Infectious Event rate per Subject during the Phase III trial (4/1/2021 to 11/3/2022). The placebo group experienced a much higher rate of Infectious
Events per Subject compared to the BCG group. The dotted vertical lines delineate the approximate periods of predominance of SARS-CoV-2 variants in the
United States. (Poisson statistics p < 0.0001).
(C) Phase II and Phase III trial periods combined. The dominant variants are delineated by dotted vertical lines. (Poisson statistics p < 0.0001). We also performed
an Andersen-Gill Cox proportional hazard statistic on these data to see the positive or negative impact of COVID-19 vaccines on BCG protective effects. With
calendar days of COVID-19 vaccinations as a variable we showed that there was no significant benefit or harm of COVID-19 vaccines on the prevention of overall
BCG prevention of infectious diseases (p = 0.253). (C) Phase II and Phase III trial periods combined. The dominant variants are delineated by dotted vertical lines.
(Poisson statistics p < 0.0001). Andersen-Gill Cox proportional hazard statistics with calendar days of COVID-19 vaccination as a variable was also not significant(p = 0.154).

always low and was always lower than that in the placebo group, regardless of whether the subjects had received a COVID-19 vaccine(p < 0.001). This analysis is consistent with the earlier one (Figure 6) that the commercial COVID-19 vaccines did not protect type 1 diabetics
from COVID-19. The data also supports the conclusion that concurrent COVID-19 vaccines did not harm BCG recipients.

DISCUSSION
This randomized, double-blinded placebo-controlled Phase III trial conducted late in the US pandemic shows that multidose BCG vaccines ina vulnerable type 1 diabetic population protect from COVID-19 disease and infectious diseases. This was a time of sequential dominance of

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ArticleFigure 6. COVID-19 vaccines (Pfizer, Moderna, J&J) do not protect type 1 diabetic subjects from COVID-19 disease(A) To assess the efficacy of the COVID-19 vaccines by themselves in our type 1 diabetic subjects, we excluded BCG as a potential confounding factor andanalyzed only the placebo (No BCG) cohort. The placebo type 1 diabetic subjects were divided into COVID-19 vaccinated and never COVID-19 vaccinatedgroups. Out of a total of 48 subjects in the placebo (No BCG) arm, 39 were COVID-vaccinated and 9 were never COVID-vaccinated. We then counted thenumber of subjects that were diagnosed with COVID-19 disease. In the COVID-vaccine-treated cohort, 17 out of 39 subjects (43.6%) were COVID-19 positive,
whereas in the never vaccinated cohort 3 out of 9 (33.3%) tested positive. While the percent positive in the never vaccinated group trended lower, thedifference was not statistically significant (one-tailed Fisher’s exact test p = 0.43).
(B) Comparison of effectiveness of COVID-19 vaccines in 2021 versus 2022. We quantified the percentage of subjects with COVID-19 disease. This observational
analysis only included placebo subjects not given the BCG vaccine. No statistical significance was reached in either year (one-tailed Fisher’s exact test: 2021,
p = 0.39; 2022, p = 0.47), confirming that the commercially available COVID-19 vaccines did protect type 1 diabetic subjects from COVID-19.

Beta, Gamma, Delta, and Omicron SARS-CoV-2 variants, all more transmissible and some less virulent than earlier strains.38 Based on observational data, we also found no efficacy of commercial mRNA vaccines for this infectious disease-vulnerable type 1 diabetic population.
Finally, receipt of COVID-19 vaccination had no bearing on the efficacy of concurrent BCG vaccination.
This is the second successful test of the BCG vaccine in the United States. The two back-to-back trials—a Phase II and Phase III trial—sharesome similarities and some differences (Figure S2). Both used the Tokyo 174 BCG strain given as a multi-dose intradermal regimen, and both
excluded a history of neonatal BCG and prior TB. The Phase II trial occurred at the start of the pandemic when approved diagnostics were not
yet widely available. COVID-19 disease was confirmed through COVID-19 antibody profiling.13 The current Phase III clinical trial used point ofcare COVID-19 diagnostics for detecting and confirming COVID-19. The Phase II trial showed near total protection of the BCG group fromCOVID-19 disease (92% vs. placebo; p = 0.0036) with the less transmissible but highly virulent SARS-CoV-2 variants early in the pandemic (theoriginal and Alpha variant). The Phase III trial revealed somewhat lower protection of BCG vaccines against COVID-19 disease late in the
pandemic with the more transmissible SARS-CoV-2 variants (43% vs. placebo; p = 0.023), but still with continuing statistical significance. Taken
together, the two trials show that BCG vaccines provided continuous protection of type 1 diabetic subjects for nearly the entire COVID-19
pandemic in the United States. BCG protection is agnostic to the genetic variants of COVID-19 and even protected the vaccinated subjects
from Omicron, the most transmissible variant.38
Both Phase II and Phase III RCT trials also found platform protection of the BCG vaccines (vs. placebo) against other infectious diseases(p < 0.01, p < 0.0001, respectively). All clinical trial recipients received multiple doses of BCG and in both trials the participants had received >3
BCG vaccines over a 2-year period prior to the start of monitoring for infectious disease risk in either the randomized double blinded Phase II
or Phase III clinical trial.
It is appreciated that in naive adult populations, the BCG vaccine administered as an intradermal vaccine takes at least two years to achieve
full protection for a number of off-target effects.13,16,32 The Phase II clinical trial monitored subjects for 15 months, while the second clinical
trial monitored subjects for the next 18 months. The Phase III trial coincided with the US rollout of commercial anti-COVID-19 vaccines predominantly of the mRNA type. It enabled observational evaluation of mRNA efficacy as well as impact on concurrent BCG vaccine. The analysis found neither benefit nor harm of the antigen-specific vaccine technology in type 1 diabetic subjects (p = 0.43), as has also been reported
by others.40,41
We attribute the success of our Phase II and III trials to several methodological features. First, the follow-up time exceeded two years. BCG
vaccines in US adults take two years to realize infectious disease protection or other off-target benefits such as protection from autoimmunity
in naive previously non-vaccinated adults.16,32 Shortly after vaccination and within weeks, BCG stimulates innate immunity that certainly isrelated to the introduction of bacterial foreign products.42 For off-target effects in adults, BCG causes changes in DNA methylation of genesof the innate and adaptive immune system and of metabolism pathways, and signaling pathways become rewired in a time frame that correlates with the slow but durable clinical improvements.34,43–45 Second, we excluded individuals with a history of BCG or TB exposure. Protection from past exposure of this kind probably lasts decades, and thus would tend to obscure finding a benefit from a recent BCG vaccine.
Third, we used a potent strain of BCG (Tokyo 174). Like the use of BCG for bladder cancer, various strains of BCG have very different efficacy,
and the Tokyo 174 strain continues to show strong off-target effects.46 Fourth, we used multiple BCG doses. Animal and human studies show

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Figure 7. BCG affords broad protection of type 1 diabetic subjects against infectious diseases irrespective of COVID-19 vaccines
Since type 1 diabetic subjects were vaccinated with commercial COVID-19 vaccines at different timepoints during this current Phase III clinical trial, the number ofmonths pre-COVID-19-vaccine and post-COVID-19 vaccine were calculated for BCG (n = 93) and placebo (n = 48) groups separately. The cumulative number ofInfectious Events in BCG vs. placebo pre-COVID-19 vaccine and post-COVID-19 vaccines were then determined and the rate of Infectious Events per Subject andper Month was calculated. Never-COVID-19-vaccinated subjects were considered as pre-COVID-vaccine for the entire duration of the Phase III clinical trial.
Differences between the BCG and placebo groups were determined using Poisson statistics.
(A) Infectious Events rate per Subject per Month before treatment with commercial COVID-19 vaccines is much higher in the placebo (No BCG) group ascompared to the BCG group.
(B) Infectious Events rate per Subject per Month after treatment with commercial COVID-19 vaccines is also much higher in the placebo (No BCG) group ascompared to the BCG group.
(C) Infectious Events rate per Subject per Month over the entire course of the Phase III trial, i.e., both pre- and post-COVID-vaccines, show the same pattern.
Andersen-Gill Cox Proportional Hazard statistics showed that there was no significant effect of COVID-19 vaccines on the time on the prevention or
acceleration of overall infectious diseases (p = 0.253). Overall, these results indicate that BCG broadly protects type 1 diabetes against infectious diseases,
irrespective of presence or absence of commercial COVID-19 vaccines. Further, commercial COVID-19 vaccines did not interfere with platform BCG vaccine
protection from COVID-19 and overall infectious disease.

that multi-dosing is best for metabolic changes and T cell changes through epigenetic reprogramming in both the adaptive and innate immune system.34,43–45 Fifth, we studied an infectious disease-vulnerable population of type 1 diabetics,35 which facilitated finding an effect.
Similarly, other positive BCG trials for COVID-19 were in high-risk populations.21,22 In contrast, three negative trials studied health care
workers,25,27,29 a healthy population often resistant to infections and lacking immune compromise.
We are not the first to report that diabetic subjects, including type 1 diabetic subjects, are not adequately protected against COVID-19 by
the mRNA vaccines.40,41 These studies show that diabetic subjects, as a group, are at risk of breakthrough COVID-19 infection post-COVIDvaccination. Type 1 diabetic subjects, more specifically, are likely to have an even worse risk of breakthrough infection, an inference based on
research showing that type 1 diabetics vs. type 2 diabetics are at least twice as likely to die from COVID-19.47 Given the lack of efficacy ofmRNA vaccination for type 1 diabetics, there is still an unmet need for this patient population, especially as relates to new and emerging
SARS CoV-2 variants. Given the high rate of infectious diseases in the vulnerable type 1 diabetic population, there is still an unmet needfor this patient population for a platform infectious disease protection.
The lack of efficacy of mRNA vaccines reported here is buttressed by research showing that mRNA vaccines in type 1 diabetic subjects
do not elicit any T cell responses.48 The finding fits with other data that type 1 diabetic subjects do not have the ability to mount an appropriate immune response using T cells of the adaptive immune response. Mechanistic data from our laboratory show that BCG’s benefit for
COVID-19 and infectious disease may stem from its strengthening the T cell response through de-methylation of many of the key T cell
receptor (TCR) genes.34 The data show that type 1 diabetic subjects ordinarily have low expression of the TCR complex resulting from

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Please cite this article in press as: Kühtreiber et al., Late in the US pandemic, multi-dose BCG vaccines protect against COVID-19 and infectious
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iScience
Articleover-methylation of the TCR genes as well as the associated CD3 protein of the TCR complexes. BCG corrects this defect over a three-year
period.
In the infectious disease field, there is a pressing need for new antibiotics or vaccines to meet the pace of emerging infectious diseases.
While mRNA vaccines are a leap forward for most individuals based on alacrity of vaccine production and biological response, the protection
is short-term (a matter of months) in the face of viral genetic drift.49 BCG vaccines appear to offer a platform opportunity but with caveat that
benefits may not be manifest for a minimum of 2 years. Nevertheless, BCG offers the prospect of near lifelong protection.50

Limitations of the study
This trial was conducted in an infectious disease-vulnerable population, type 1 diabetic subjects, and therefore findings may not apply to nondiabetic patient populations or healthy populations. This clinical trial was small in size, but it had no drop-outs. Also, this long trial in a randomized double-blinded clinical trial format allowed subject exposure to many SARS-CoV-2 variants and infectious disease exposures. This
longer in time clinical trial format is an alternative trial design compared to mRNA vaccine trials that enroll large numbers of patients but withshorter follow-up of months instead of years to capture the specific viral variant the mRNA vaccine was designed for.
Another limitation of this study is reliance on observational data regarding efficacy of mRNA vaccines, whether alone or with concurrent
BCG vaccination. Observational data cannot be used to infer cause and effect. Further, we do not know if the lack of mRNA vaccine efficacy
against COVID-19 reported here is due to the underlying disease state (i.e., type 1 diabetes) or due to exposure to new viral variants thatemerged over the course of study.
Another limitation is that we did not genetically sequence the COVID-19 variants from infected cohorts but used the US-based epidemiology time frame to document exposure. Finally, this was a US-based trial, so findings may not necessarily apply to other countries.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY

B Lead contact
B Materials availability
B Data and code availabilityd EXPERIMENTAL MODEL AND SUBJECT DETAILSd METHOD DETAILSB Procedures
B Outcomesd QUANTIFICATION AND STATISTICAL ANALYSISd OVERSIGHT OF THIS CLINICAL TRIAL
d ADDITIONAL RESOURCES

SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.109881.

ACKNOWLEDGMENTS
This research work was supported by The Iacocca Foundation, Boston, MASS.
We thank Miriam Davis, PhD, for her fine editorial editing skills. We also sincerely thank all the patients in this study who kept all their visitseven during the height of the pandemic.

AUTHOR CONTRIBUTIONS
W.M.K. and D.L.F. conceived the study and participated in the study design. D.L.F. was the PI on the study and overall ran the clinical trial. The
clinical trial management team interacted with the clinical trial subjects, performed clinical trial management, scheduled patients, performed
blood draws, etc. and was composed of E.R.H., G.E.W., M.S.V., R.G., E.R.B., N.S.H., J.E.B., and D.L.F. W.M.K. prepared the blinded vaccines
using the pharmacy-generated randomization codes. W.M.K. and H.Z. performed the data analysis. W.M.K. and D.L.F. wrote the manuscript.
All authors read, revised, and approved the final manuscript for publication.

DECLARATION OF INTERESTSNo author or author family member owns the study drug nor does any author or family member consult for the drug producer or have ownership interests in BCG Japan Laboratories. None of this clinical trial work was financially supported by the maker/manufacturer/owner of thisdrug, BCG Japan Laboratories. No Author has any ownership rights to the study drug. No authors receive consulting or research support fromBCG Japan Laboratories.

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Received: December 12, 2023
Revised: March 13, 2024
Accepted: April 29, 2024
Published: May 22, 2024
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Please cite this article in press as: Kühtreiber et al., Late in the US pandemic, multi-dose BCG vaccines protect against COVID-19 and infectious
diseases, iScience (2024), https://doi.org/10.1016/j.isci.2024.109881

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ArticleOPEN ACCESS

STAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCESOURCEIDENTIFIER

Japan BCG Laboratory, Tokyo, Japan

Japan 174 strain

Quest Diagnostics, Secaucus, NJ

N/AStudyTrax, Macon, GA, USA

StudyTrax

Bacterial and virus strains
BCG vaccine (Bacillus Calmette-Guérin)
Critical commercial assays
Point of Care SARS-CoV-2 testing (PCR, Rapid Antigen test, Antibody test)
Software and algorithms
StudyTrax Electronic Data Capture System for Clinical Research

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,
MD, PhD (dfaustman@mgh.harvard.edu).

Materials availability
This study did not generate new unique reagents.

Data and code availability
 All data reported in this paper will be shared by the lead contact upon request.
 This paper does not report original code.
 Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Late in the COVID-19 pandemic we conducted this Phase III US based clinical trial to determine the safety and efficacy of up to 6 doses of BCG
vaccines versus placebo for the prevention of COVID-19 and other infectious diseases. A total of 141 participants were enrolled in this Phase III
clinical trial evaluating the efficacy of the BCG vaccine in protecting infectious disease-vulnerable, type 1 diabetic subjects from April 2021 toNovember 2022 (Figure 1). A total of 141 participants also finished the trial; there was no patient attrition. This was a randomized, doubleblinded clinical trial with a 2:1 enrollment. During this 19-month course, the US population was no longer exposed to the early original
SARS-CoV-2 virus and Alpha variant but instead was predominantly exposed to the Beta, Gamma, Delta and Omicron variants (Figure 1).
Both the incidence of COVID-19 disease and other infectious diseases was determined using the methods described below. All participantsat the time of enrollment met the criteria of having no previous or current tuberculosis, no history of BCG vaccinations, even during childhood,and being born in the US. Throughout the trial, subjects were monitored for any infectious diseases (viral, bacterial, parasitic, etc.) using a
direct MedDRA documentation system. It is important to note that newborn or childhood BCG vaccinations have never been offered in
the US. Starting in early 2021, gradually mRNA vaccines became available in the US and although not a primary outcome of this trial, it
was possible in the placebo group to see if the combined use of either the two mRNA-based vaccines from Pfizer-BioNTech and Moderna,
as well as a Viral Vector vaccine from J&J/Janssen had any efficacy or harm in this infectious disease-vulnerable population. The use of thesecommercial vaccines in male and female placebo subjects is shown in Table 1.
At the start of this current infectious disease related clinical trial, subjects were volunteers with a mean age of 44 G 1.1 years with co-morbid
type 1 diabetes of long-standing origins, mean duration 17.9 G 0.06 (Table 1). The current Phase III clinical trial was a US-based randomized
clinical trial using already enrolled double-blinded subjects in an ongoing double blinded trial looking at the impact of the multi-dose BCG
vaccines on metabolic outcomes (US FDA IND #16434; MGH protocol #2003P002633). This trial is registered in the clinicaltrials.gov NCT site
as: NCT02081326. The randomized type 1 diabetic subjects receiving BCG or placebo vaccines had also been used early in the pandemic,
prior to mRNA vaccines for COVID-19 disease and infectious disease incidences study and this data is published (Figure 1).13 This earlier study
was terminated when the two way comparisons of the written protocol comparing BCG to placebo were no longer a valid trial with the roll outof the commerical mRNA vaccines.
The protocol, outcomes, data analysis methods, methods for COVID-19 diagnosis, non-overlapping trial length, COVID-19 genetic variant
exposures and consents for the current COVID-19 prevention trial were approved as a new study by MGH and the FDA and filed as an amendment to the original FDA IND and MGH protocols. This trial was labeled a Phase III clinical trial. A study synopsis is provided (Figure S1). The

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Please cite this article in press as: Kühtreiber et al., Late in the US pandemic, multi-dose BCG vaccines protect against COVID-19 and infectious
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iScience
Articlesubjects in this Phase III clinical trial and the earlier Phase II clinical trial were overlapping and were all type 1 diabetic subjects. Figure S2 shows
the differences in the two clinical trials. This trial was performed in accordance with ethical principles in the Declaration of Helsinki and areconsistent with the International Conference on Harmonization/Good Clinical Practice. At the start of this Phase III clinical trial 30% of subjects(BCG and placebo) had received 5 BCG/placebo vaccines and 70% of subjects had received 6 BCG/placebo vaccines. For the subjects with
only 5 vaccines at the time of consent, the average number of days until that sixth vaccine was 124 G 16 days. For the subjects with only 5vaccines at the time of consent, the average number of days since the last BCG vaccine was 241 days. This data is calculated from the protocol
approval date of April 2021.
The initial exclusion criteria for the trial included a positive purified protein derivative (PPD) test, a positive T-spot test for tuberculosis performed at screening; or being born in a foreign country with mandatory BCG vaccinations. These criteria were implemented to ensure thatparticipants did not have existing long-term protection against Mycobacterium bovis (the source of the BCG vaccine) or Mycobacterium
tuberculosis (TB) due to previous exposures. Additionally, individuals actively undergoing high dose glucocorticoid treatment; taking chronic
immunosuppressive medications; or living with an immunosuppressed person were also excluded to prevent adverse events from the administration of the live vaccine. All participants resided in the United States, with the largest number (36) coming from Massachusetts, followed by
16 from New York and 11 from Texas.
Both MGH and FDA approved COVID-19 survey were sent through email at two month intervals and the patients were also seen in theclinic every 6 months. The COVID-19 survey was under the FDA Guidance recommendations. There was good compliance. Also these subjects would also sometimes email or phone the clinic with any illness updates and reporting at non-clinic appointment times. For this trial, PCR
was approved so this method was used to confirm COVID-19 disease.
This trial had three levels of oversight. At 6-month to yearly intervals, audits were conducted either by the Massachusetts General Brigham(MGB) Division of Quality Management or by external auditors (Advanced Clinical Trials, Deerfield, IL). Unblinded statisticians (H.Z.) processed all trial data, and an independent DSMB met every six months to a year to ensure subject safety and compliance with reporting.
The randomization code for administering either the BCG vaccine or placebo saline to study participants was generated by the MGH
Research Pharmacy and securely held by them. Both the BCG and Placebo preparations appeared identical in the syringe, making it easier
to blind the study staff and participants. Unlike antigen-specific vaccines that often cause multi-day systemic reactions, site soreness andmissed workdays, the BCG vaccine did not induce any systemic or local symptoms immediately after administration. The BCG vaccine inadults administered as an intradermal vaccine after 4 weeks can induce a skin reaction that was blinded to study staff. Throughout the study,
investigators and study staff remained blinded to both the primary study outcomes and the parallel study outcomes related to COVID-19 andinfectious diseases. The statistical analysis team after November 2022 was unblinded to tabulate the data presented in this paper.

METHOD DETAILS
Procedures
We assessed late in the US COVID-19 pandemic, the safety and efficacy of 6 doses of BCG vaccines of the Tokyo-172 strain for COVID-19 andother infectious diseases. This was a community-based, but broadly US recruited randomized double blinded placebo controlled clinical trial.
The BCG vaccine or saline placebo (both 0.1 mL volume per dose) was administered intradermally starting 2.5–3 years prior to this phase of
the COVID-19 pandemic (Figure 1). The first two BCG or Placebo doses were given 4 weeks apart and subsequently at yearly intervals additional booster doses of BCG or Placebo were administered, for a total of up to 6 doses over a period of 60 months (5 years) (Figure 1). Site staff
were responsible for reporting all drug- and non-drug-related safety information and were blinded to group assignments.
In our published Phase II clinical trial, we used antibody profiling, a technology called the COVID Scan assay (CDI Labs, Baltimore, MD) to
identify COVID-19 infected subjects based on the presence of arrays of anti-SARS-CoV-2 antibodies.13 This method had been used because
there were no COVID-19 commercial diagnostic methods. With this Phase III clinical trial commercial COVID-19 diagnostics were used to diagnose COVID-19 disease. We therefore relied on Point of Care based PCR and/or Rapid Antigen testing to confirm COVID-19 in subjects
that presented with one or more COVID-19 symptoms as defined by the FDA Guidance Document39 and at least one day of illness (seebelow). It is important to additionally note that antibody scanning diagnostic methods were also no longer feasible with the rollout of
COVID-19 vaccines; indeed the vaccines can mimic the actual disease (see Figure S4).
COVID-19 is defined according to FDA by the presence of at least one of 12 symptoms (ref. 1) including headache, chills/shivering, diarrhea, nausea/vomiting, fatigue, shortness of breath, loss of smell or taste, muscle aches, nasal congestion, cough, sore throat and fever). 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 indays was also reported. From the individual symptom scores an Average Symptom Score were tabulated. Then, multiplying by the length ofillness, the Average Infectious Symptom Index was assessed.
During the trial period the launch of the mRNA vaccines happened, so an analysis of the efficacy of these licensed vaccines products in theabsence of BCG vaccination was also conducted.

Outcomes
We determined if multi-dosing with BCG-Japan (Tokyo-172) vaccine can protect high-risk T1D subjects from COVID-19, both in the presence
and in the absence of COVID-19 commerical vaccines. We also determined if BCG-Japan can protect from other infectious diseases in thepresence of COVID-19 vaccines. Finally, we also looked at the efficacy of the COVID-19 vaccines to protect the high-risk T1D subjects from

14

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Please cite this article in press as: Kühtreiber et al., Late in the US pandemic, multi-dose BCG vaccines protect against COVID-19 and infectious
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OPEN ACCESSCOVID-19 disease in the absence of BCG. We did not study severe COVID-19 disease as measured by hospitalizations or deaths because theperiod under study saw to some extent less lethal viral variants.
The study synopsis is provided (Figure S1). These outcomes conformed to the primary US based trials for the commercial COVID-19 vaccines (Pfizer-BioNTech and the Moderna clinical trials). For this trial we reported adverse events as they relate to infectious diseases. There
were no moderate or severe adverse events related to the BCG vaccines. In contrast to the commerical COVID-19 vaccines, the administrationof the BCG vaccines, even with multi-dosing, does not induce arm sourness, fever, fatigue, or other flu like symptoms in the 48 h after administration thus assisting in the blinding of the clinical trial. For this report, the full skin reaction logs belong to a time period not overlapping withthis clinical trial since most had received their full series. For vaccine safety for this clinical trial, no reports of a vaccine site composed ofredness, swelling and/or a scar greater that 3 cms was reported.

QUANTIFICATION AND STATISTICAL ANALYSIS
Vaccine efficacy is defined by (p1 – p2)/p1 3 100, where p1 is the % COVID-19 positive in the placebo group and p2 is the % COVID-19 positivein the BCG group. The number of COVID-19 positive patients in the BCG versus the placebo cohorts were compared using Fisher’s exact test
(available online at https://www.graphpad.com/quickcalcs/contingency1/). To satisfy the requirements for this test, each subject was only
counted once, even if the subject experienced more than one COVID-19 event. To be able to include multiple events per subject we createdcharts of rate of events per subject and used Poisson distribution statistics for counting scenarios that included the whole current trial period.
A similar approach was also used to compare all infectious events (both COVID-19 and non-COVID-19) between the cohorts. Statistics wereconsidered significant at p < 0.05.

OVERSIGHT OF THIS CLINICAL TRIAL
This trial was approved by the MGH human studies and by the US Food and Drug Administration with external audits at yearly intervals conducted by Advanced Clinical. The safety of BCG was monitored by an independent Data and Safety Monitoring Board (DSMB). The investigators designed the trial. The funders and the drug supplier Japan Laboratories had no role in the collection, analysis or interpretation of thedata or in the preparation, review or approval of the manuscript.

ADDITIONAL RESOURCES
This trial is listed on clinicaltrials.gov (https://clinicaltrials.gov/ct2/show/NCT02081326).

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