key: cord-0798035-6c2f9by5
authors: Moorlag, Simone J C F M; Taks, Esther; ten Doesschate, Thijs; van der Vaart, Thomas W; Janssen, Axel B; Müller, Lisa; Ostermann, Philipp; Dijkstra, Helga; Lemmers, Heidi; Simonetti, Elles; Mazur, Marc; Schaal, Heiner; ter Heine, Rob; van de Veerdonk, Frank L; Bleeker-Rovers, Chantal P; van Crevel, Reinout; ten Oever, Jaap; de Jonge, Marien I; Bonten, Marc J; van Werkhoven, Cornelis H; Netea, Mihai G
title: Efficacy of Bacillus Calmette-Guérin vaccination against respiratory tract infections in the elderly during the Covid-19 pandemic
date: 2022-03-05
journal: Clin Infect Dis
DOI: 10.1093/cid/ciac182
sha: 9dcd48d1fdcb4389ef79e2343e2d19c364968078
doc_id: 798035
cord_uid: 6c2f9by5

BACKGROUND: Older age is associated with increased severity and death from respiratory infections, including coronavirus disease 2019 (Covid-19). The tuberculosis vaccine Bacille Calmette-Guérin (BCG) may provide heterologous protection against non-tuberculous infections, and has been proposed as a potential preventive strategy against Covid-19. METHODS: In this multicenter, placebo-controlled trial, we randomly assigned elderly individuals (60 years or older, n=2014) to intracutaneous vaccination with BCG (n=1008) or placebo (n=1006). The primary endpoint was the cumulative incidence of respiratory tract infections that required medical intervention, during 12 months of follow-up. Secondary endpoints included the incidence of Covid-19, and the effect of BCG vaccination on the cellular and humoral immune responses. RESULTS: The cumulative incidence of respiratory tract infection requiring medical intervention was 0.029 in the BCG-vaccinated group and 0.024 in the control group (subdistribution hazard ratio [SHR], 1.26; 98.2% confidence interval [CI], 0.65 to 2.44). 51 and 48 individuals tested positive for SARS-CoV-2 by PCR in the BCG and placebo group, respectively (SHR, 1.053; 95% CI, 0.71 to 1.56). No difference was observed in the frequency of adverse events. BCG vaccination was associated with enhanced cytokines responses after influenza, and partially also after SARS-CoV-2 stimulation. In patients diagnosed with Covid-19, antibody responses after infection were significantly stronger if the volunteers had previously received BCG. CONCLUSIONS: BCG-vaccination had no effect on the incidence of respiratory tract infections, including SARS-CoV-2 infection, in elderly volunteers. However, BCG vaccination improved cytokine responses stimulated by influenza and SARS-CoV-2, and induced stronger antibody titers after Covid-19 infection.

The elderly are at high risk for developing severe respiratory tract infections (RTIs).

Protection against respiratory disease by vaccination is associated with a decreased risk of infection and death in elderly individuals, but protection is frequently incomplete due to ageassociated decline in immune function, also termed 'immunosenescence'. [1] The elderly also account for the majority of severe coronavirus disease 2019 (Covid- 19) cases and associated deaths. [2] While specific vaccines give the best chance as preventive strategy against infection, they are not immediately available when a new pathogen emerges. Therefore, additional strategies to lower infection-related morbidity and mortality in a new outbreak are needed.

The Bacille Calmette-Guérin (BCG) vaccine not only protects against tuberculosis, but also induces broad effects on host defense, offering protection against a wide range of other infections. [3] In countries with high infection pressure, BCG vaccination in infants is associated with a reduction in all-cause neonatal mortality, mainly attributed to a reduced incidence of RTIs and sepsis. [4] These effects are thought to be mediated by heterologous lymphocyte activation and induction of trained immunity, a de facto immunological memory of innate immune cells. [5] A recent randomized clinical trial suggested a protective effect of BCG revaccination on the incidence of RTIs in hospitalized elderly from Greece [6] , but whether similar effects occur in BCG-naïve elderly is unknown.

Here we present the results of a multicenter, randomized, placebo-controlled trial to evaluate the safety and efficacy of BCG vaccination against RTIs, including SARS-CoV-2 infection, in individuals aged 60 years or older from a Western European country in which BCG vaccination has never been part of the standard immunization program.

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The study was a double blind, placebo-controlled, randomized clinical trial conducted in two university hospitals in the Netherlands from April 16, 2020, to May 14, 2021 

Participants were informed about the study by advertisements and could self-register. Eligible participants were randomly assigned in a 1:1 ratio to receive either 0.1 mL of BCG (Danish strain 1331, SSI, Denmark) or 0.1 mL saline placebo via intradermal injection, using a computer-generated dynamic randomization algorithm, stratified for hospital and age at randomization.

During the 12-month follow-up, participants used a mobile application (Research Follow App, Your Research, Huizen, the Netherlands) to fill in a daily questionnaire regarding symptoms and adverse events and a weekly questionnaire regarding Covid-19 testing, Covid-19 exposure, and visits to healthcare professionals (Table S1 ). Participants unable to use a smartphone completed paper-questionnaires, were called monthly and asked to contact the study team in case they had contacted a healthcare professional. Adherence to questionnaires was monitored and quantified. Participants were unblinded at the end of follow-up.

A c c e p t e d M a n u s c r i p t 6 

The initial primary endpoint 'cumulative incidence of SARS-CoV-2 related hospital admission' was changed upon approval by the ethical committee (before data were unblinded) due to the almost complete absence of outcomes consistent with the primary endpoint after four months of follow-up. The primary endpoint was changed into cumulative incidence of clinically relevant RTIs as defined as onset or sudden aggravation of preexisting symptoms, as reported by the participant, of at least one respiratory symptom (cough, throat ache, rhinorrhea or dyspnea) and one systemic symptom (fever, muscle ache, chills, fatigue) that required medical intervention within 5 days of the onset of symptoms. Following medical assessment in primary or secondary care, an intervention was defined as initiation of antibiotic, antiviral or corticosteroid treatment; adaptation in pulmonary maintenance medication; or hospitalization.

During the follow-up period, individuals in the Netherlands were encouraged to be tested for SARS CoV-2 in a publicly available testing facility in case of symptoms suggestive of Covid-19, using a polymerase chain reaction (PCR)-test on a nasopharyngeal swab. The endpoint documented SARS-CoV-2 infection required a self-reported positive PCR-test. Participants reporting any infection or positive PCR-test were contacted for confirmation and follow-up.

An overview of secondary endpoints is listed in Supplementary Materials.

A subgroup of participants that had not received a Covid-19 specific vaccine was asked to donate blood at the end of follow-up (month 12) for cellular and humoral immune assays.

Isolation and stimulation of peripheral blood mononuclear cells (PBMCs) were performed as previously described. [7] Cytokine levels were measured using ELISA (R&D systems, MN, USA). A SARS-CoV-2 fluorescent-microsphere-based multiplex immunoassay was used to A c c e p t e d M a n u s c r i p t 7 quantify antibody responses as previously described. [8] , [9] For experiment details see Supplementary Materials.

The study was designed as an endpoint-driven trial with a symmetrical group sequential design. The sample size was calculated using R package gsDesign version 3.0.1. We used a two-sided alpha of 0.05 and aimed for 90% power. Hence, we aimed to enrol 1000 subjects per arm.

The primary endpoint was reported as the cumulative incidence by treatment arm and analysed using a competing events analysis (Fine & Gray model) with time to event as dependent outcome, study arm as independent variable, and mortality as potential competing event. Stratification variables and moderate-to-strong predictors of the outcome were included as covariates. These included, site of enrolment, age (categorized as 60-69, 70-79 and ≥ 80 years), and the comorbidities cardiovascular disease (including hypertension), diabetes mellitus (type 1 and 2), and chronic pulmonary disease. The effect was reported as a hazard ratio with 98.2% CI for the primary endpoint and 95% CI for other endpoints. Data 

Between April 16 and May 14 2020, participants were randomized to vaccination with BCG (n=1008) or placebo (n=1006) (Figure 1 (Figures 2A and S2 ). In addition, the incidence of SARS-CoV 2 infection was 5.1% in the BCG group (n = 51) and 4.8% in the control group (n = 48), SHR of 1.053 (adjusted 95% CI, 0.71 to 1.56) ( Figure 2B ). No differences were observed in the incidence of RTIs irrespective of a medical intervention (n = 351 in the BCG group and n = 338 in the placebo group, SHR, 1.055; 95% CI, 0.91 to 1.23) ( Table 2) . Only 3 participants were hospitalized due to Covid-19 and one participant died of Covid-19 (Table 2) .

First episodes of self-reported symptoms associated with infection (severity score ≥ 3) occurred in 647 (64.2%) of the participants in the BCG group and 611 (60.7%) in the control group (SHR, 1.11; 95% CI, 0.994 to 1.239). No significant differences were observed between the BCG and placebo group regarding the cumulative incidence of any of the selfreported symptoms. The total number of days on which participants reported symptoms (severity score ≥ 3) was 9183 days in the BCG group and 9630 days in the placebo group (RR, 0.99; 95% CI, 0.84-1.17). The total number of days of dyspnea was significantly lower in the BCG group (RR, 0.48; 95% CI, 0.26 to 0.88). No differences were found in the total number of days of any other self-reported symptom ( Table 2 ). In a post-hoc analysis of the A c c e p t e d M a n u s c r i p t 9 effect of BCG on the primary endpoint, incidence of SARS-CoV-2 infection and the incidence and duration of cough and dyspnea, no significant differences were observed among subgroups defined according to age, sex, prior-BCG-vaccination, or the presence of comorbidities (Table S2 -S7) .

Injection site reactions, fatigue, myalgia, fever and headache were more common in the BCG group on day 7 and 14 after vaccination as compared to placebo. Two participants developed a local abscess at the injection site, for which a conservative approach was followed and the abscesses resolved without any treatment. The incidence of SAEs was similar among the BCG and placebo group (Table S8) and were considered to be unrelated to the vaccine.

In a subgroup of participants (55 placebo and 50 BCG-vaccinated), we assessed ex-vivo cytokine responses to influenza A H1N1 California strain and SARS-CoV-2 Wuhan Hu-1 strain at end of the study (month 12). Production of pro-inflammatory cytokines by PBMCs stimulated with influenza was significantly higher in the BCG group compared to placebo: A c c e p t e d M a n u s c r i p t 10 difference was observed between the placebo and BCG group on the production of TNFα, IL-1 or IFN-γ after stimulation with SARS-CoV-2 ( Figure 3A and B).

In 30 participants (15 placebo and 15 BCG-vaccinated) who tested positive for SARS-CoV-2 between September 2020 and February 2021, IgG responses against SARS-CoV-2 stabilized trimeric spike (S) glycoprotein, the nucleocapsid (N) protein, and the receptor-binding domain (RBD) were quantified at the end of the study. Age, sex and time between positive test result and antibody measurement were balanced between BCG and placebo group (P = 0.52, P > 0.99, P = 0.27 respectively). Significantly higher concentrations of IgG antibodies against S-protein and RBD were found in BCG-vaccinated participants as compared to placebo (Mann-Whitney U, P < 0.05) ( Figure 3C ). Furthermore, significantly higher levels of IgM against S protein were observed in the BCG group as compared to placebo (Mann-Whitney U, P < 0.05) ( Figure S3 ).

The data presented in this study does not support the hypothesis that BCG vaccination reduces the incidence of RTIs in elderly living in a Western European country. Similarly, no effect was observed on the incidence of SARS-CoV-2 infection or any of the other secondary clinical endpoints. Exaggerated inflammatory reactions characterized by an increased concentration of circulating cytokines have been described to contribute to Covid-19 severity [11] . We argue that when BCG is administered to a healthy individual it will elicit a fast and robust immune response that reduces viremia and levels of systemic inflammation, as A c c e p t e d M a n u s c r i p t 11 has been demonstrated in a previous BCG-vaccination study involving young adults. [12] In support of this, results of this study indicate that BCG-vaccination of the elderly was safe, as no differences in the incidence of SAEs were observed between the BCG and placebo group.

BCG-vaccinated individuals were not more frequently admitted to the hospital because of SARS-CoV-2 infection, indicating that recent BCG vaccination is not associated with increased Covid-19-related morbidity in elderly.

In contrast to our findings, several smaller studies performed in Indonesia, Japan, South Africa and Greece observed a protective effect of BCG on the incidence of RTIs in adults [6, [13] [14] [15] [16] . The difference in the effect of BCG between our study and the other reports can have several causes. One cause could have been the difference between first vaccination with BCG compared to revaccination: however, we found no indication of an effect of previous BCGvaccination in our study, although the number of the earlier vaccinated individuals was only around 25%. Another possible factor explaining these differences might be the dose of the vaccine, which was the standard dose used in our study, while Wardhana et al. vaccinated volunteers with BCG once a month for three following months resulting in a reduction of RTIs as compared to placebo [13] . Additionally, improved immune responses when repeated doses of BCG were given as opposed to a single BCG dose have been shown. [17] Furthermore, it is known that the immunogenicity of BCG is significantly influenced by the BCG vaccine strain used. [18] Other causes may include the different genetic backgrounds of the populations, or differences in the epidemiology of RTIs [19] during the lockdowns caused by the pandemic. Indeed, BCG has previously shown to strongly protect against influenza in mice[20], while incidence of influenza was extremely low during the study period. The most likely explanation, however, for the differences observed between earlier trials and the current study is that the immune pathways activated by BCG-induced trained immunity may be involved to a lesser extent for host defense against Covid-19: indeed, the cellular assays A c c e p t e d M a n u s c r i p t 12 performed in this study demonstrated a stronger amplification of the cytokine response to influenza virus in BCG-vaccinated volunteers, compared to the modest improved response to SARS-CoV-2 ( Figure 3 ). The lack of effect of BCG on the incidence of Covid-19 in a developed country is supported by results of the BCG-CORONA study which showed no effect of BCG on the incidence of Covid-19 among healthcare workers (ten Doesschate et al., submitted). It has to be underlined that no conclusions can be drawn on the impact of BCG on severity of Covid-19, for which larger studies or meta-analyses will be needed.

BCG vaccination has been earlier reported to boost the function of innate immune cells, which correlated with a decrease in experimental human viremia. [12] Studies on the use of intravesical BCG instillations for bladder cancer also found an increase in IL-6 production capacity, and increased protection against viral infections [21] [22] [23] . In line with this, BCGvaccinated volunteers in this study reacted with stronger monocyte-derived cytokine production (hallmark of trained immunity) upon influenza stimulation compared to placebovaccinated individuals, while T-cell derived IFN production was similar. In contrast, BCG vaccination resulted only in an increased IL-6 response after SARS-CoV-2 stimulation, while no significant increase in the production of IL-1, TNFα and IFN was observed. The absence of a strong trained immunity effect of BCG-vaccination on stimulation with SARS-CoV-2 (in contrast to influenza) may partially explain the lack of effect on susceptibility to Covid-19. It is important to point out that differences in cytokine production induced by BCG-vaccination in this study are in line with earlier investigations [12, 24] and of the same order of magnitude to other conditions in which inflammatory processes play an important role. [25] Interestingly, among BCG-vaccinated participants diagnosed with Covid-19, we observed higher concentrations of IgG antibodies compared to placebo-vaccinated volunteers. These production. [32] , [33] Future studies are warranted to investigate the effect of BCG on SARS-CoV-2 vaccines and strategies to optimally exploit the immunomodulatory effects of BCG.

There are also limitations of this study. First, it was not possible to completely blind participants, as the local scar often produced by BCG-vaccination is different from the response to placebo. However, no differences in loss to follow-up or administration of Covid-19 specific vaccines were observed between the groups. Second, we were not able to perform immune measurements and serology at baseline, due to the crisis situation of the first wave of M a n u s c r i p t 21 -Guérin) , CI (confidence interval), ICU (intensive care unit), NA (not applicable), RTI (respiratory tract infections), SHR (subdistribution hazard ratio), RR (risk ratio). SHRs are adjusted for stratification variables site and age category and for cardiovascular disease (including hypertension), diabetes, and chronic pulmonary disease.

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