Robust IgM responses following intravenous vaccination with Bacille Calmette-Guérin associate with prevention of Mycobacterium tuberculosis infection in macaques.

Development of an effective tuberculosis (TB) vaccine has suffered from an incomplete understanding of the correlates of protection against Mycobacterium tuberculosis (Mtb). Intravenous (i.v.) vaccination with Bacille Calmette-Guérin (BCG) provides nearly complete protection against TB in rhesus macaques, but the antibody response it elicits remains incompletely defined. Here we show that i.v. BCG drives superior antibody responses in the plasma and the lungs of rhesus macaques compared to traditional intradermal BCG administration. While i.v. BCG broadly expands antibody titers and functions, IgM titers in the plasma and lungs of immunized macaques are among the strongest markers of reduced bacterial burden. IgM was also enriched in macaques that received protective vaccination with an attenuated strain of Mtb. Finally, an Mtb-specific IgM monoclonal antibody reduced Mtb survival in vitro. Collectively, these data highlight the potential importance of IgM responses as a marker and mediator of protection against TB.

BCG vaccination alters the epigenetic landscape of progenitor cells in human bone marrow to influence innate immune responses.

Although the Bacille-Calmette-Guérin (BCG) vaccine is used to prevent tuberculosis, it also offers protection against a diverse range of non-mycobacterial infections. However, the underlying protective mechanisms in humans are not yet fully understood. Here, we surveyed at single-cell resolution the gene expression and chromatin landscape of human bone marrow, aspirated before and 90 days after BCG vaccination or placebo. We showed that BCG alters both the gene expression and epigenetic profiles of human hematopoietic stem and progenitor cells (HSPCs). Changes in gene expression occurred primarily within uncommitted stem cells. By contrast, changes in chromatin accessibility were most prevalent within differentiated progenitor cells at sites influenced by Kruppel-like factor (KLF) and early growth response (EGR) transcription factors and were highly correlated (r > 0.8) with the interleukin (IL)-1ß secretion capacity of paired peripheral blood mononuclear cells (PBMCs). Our findings shed light on BCG vaccination's profound and lasting effects on HSPCs and its influence on innate immune responses and trained immunity.

A noninvasive BCG skin challenge model for assessing tuberculosis vaccine efficacy.

We report here on the characterisation in mice of a noninvasive bacille Calmette-Guérin (BCG) skin challenge model for assessing tuberculosis (TB) vaccine efficacy. Controlled human infection models (CHIMs) are valuable tools for assessing the relevant biological activity of vaccine candidates, with the potential to accelerate TB vaccine development into the clinic. TB infection poses significant constraints on the design of a CHIM using the causative agent Mycobacterium tuberculosis (Mtb). A safer alternative is a challenge model using the attenuated vaccine agent Mycobacterium bovis BCG as a surrogate for Mtb, and intradermal (skin) challenge as an alternative to pulmonary infection. We have developed a unique noninvasive imaging system based on fluorescent reporters (FluorBCG) to quantitatively measure bacterial load over time, thereby determining a relevant biological vaccine effect. We assessed the utility of this model to measure the effectiveness of 2 TB vaccines: the currently licenced BCG and a novel subunit vaccine candidate. To assess the efficacy of the skin challenge model, a nonlinear mixed-effects models was built describing the decline of fluorescence over time. The model-based analysis identified that BCG vaccination reduced the fluorescence readout of both fluorophores compared to unvaccinated mice (p < 0.001). However, vaccination with the novel subunit candidate did not alter the fluorescence decline compared to unvaccinated mice (p > 0.05). BCG-vaccinated mice that showed the reduced fluorescent readout also had a reduced bacterial burden in the lungs when challenged with Mtb. This supports the fluorescence activity in the skin as a reflection of vaccine induced functional pulmonary immune responses. This novel noninvasive approach allows for repeated measurements from the challenge site, providing a dynamic readout of vaccine induced responses over time. This BCG skin challenge model represents an important contribution to the ongoing development of controlled challenge models for TB.

Multiple genetic loci influence vaccine-induced protection against Mycobacterium tuberculosis in genetically diverse mice.

Mycobacterium tuberculosis (M.tb.) infection leads to over 1.5 million deaths annually, despite widespread vaccination with BCG at birth. Causes for the ongoing tuberculosis endemic are complex and include the failure of BCG to protect many against progressive pulmonary disease. Host genetics is one of the known factors implicated in susceptibility to primary tuberculosis, but less is known about the role that host genetics plays in controlling host responses to vaccination against M.tb. Here, we addressed this gap by utilizing Diversity Outbred (DO) mice as a small animal model to query genetic drivers of vaccine-induced protection against M.tb. DO mice are a highly genetically and phenotypically diverse outbred population that is well suited for fine genetic mapping. Similar to outcomes in people, our previous studies demonstrated that DO mice have a wide range of disease outcomes following BCG vaccination and M.tb. challenge. In the current study, we used a large population of BCG-vaccinated/M.tb.-challenged mice to perform quantitative trait loci mapping of complex infection traits; these included lung and spleen M.tb. burdens, as well as lung cytokines measured at necropsy. We found sixteen chromosomal loci associated with complex infection traits and cytokine production. QTL associated with bacterial burdens included a region encoding major histocompatibility antigens that are known to affect susceptibility to tuberculosis, supporting validity of the approach. Most of the other QTL represent novel associations with immune responses to M.tb. and novel pathways of cytokine regulation. Most importantly, we discovered that protection induced by BCG is a multigenic trait, in which genetic loci harboring functionally-distinct candidate genes influence different aspects of immune responses that are crucial collectively for successful protection. These data provide exciting new avenues to explore and exploit in developing new vaccines against M.tb.

Lung-resident CD3-NK1.1+CD69+CD103+ Cells Play an Important Role in Bacillus Calmette-Guérin Vaccine-Induced Protective Immunity against Mycobacterium tuberculosis Infection.

Tissue-resident immune cells play important roles in local tissue homeostasis and infection control. There is no information on the functional role of lung-resident CD3-NK1.1+CD69+CD103+ cells in intranasal Bacillus Calmette-Guérin (BCG)-vaccinated and/or Mycobacterium tuberculosis (Mtb)-infected mice. Therefore, we phenotypically and functionally characterized these cells in mice vaccinated intranasally with BCG. We found that intranasal BCG vaccination increased CD3-NK1.1+ cells with a tissue-resident phenotype (CD69+CD103+) in the lungs during the first 7 d after BCG vaccination. Three months post-BCG vaccination, Mtb infection induced the expansion of CD3-NK1.1+CD69+CD103+ (lung-resident) cells in the lung. Adoptive transfer of lung-resident CD3-NK1.1+CD69+CD103+ cells from the lungs of BCG-vaccinated mice to Mtb-infected naive mice resulted in a lower bacterial burden and reduced inflammation in the lungs. Our findings demonstrated that intranasal BCG vaccination induces the expansion of CD3-NK1.1+CD69+CD103+ (lung-resident) cells to provide protection against Mtb infection.

Prevention of tuberculosis in macaques after intravenous BCG immunization.

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide1. The only available vaccine, BCG (Bacillus Calmette-Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission1,2. Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates (Macaca mulatta). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography-computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis.

Gaucher disease protects against tuberculosis.

Biallelic mutations in the glucocerebrosidase (GBA1) gene cause Gaucher disease, characterized by lysosomal accumulation of glucosylceramide and glucosylsphingosine in macrophages. Gaucher and other lysosomal diseases occur with high frequency in Ashkenazi Jews. It has been proposed that the underlying mutations confer a selective advantage, in particular conferring protection against tuberculosis. Here, using a zebrafish Gaucher disease model, we find that the mutation GBA1 N370S, predominant among Ashkenazi Jews, increases resistance to tuberculosis through the microbicidal activity of glucosylsphingosine in macrophage lysosomes. Consistent with lysosomal accumulation occurring only in homozygotes, heterozygotes remain susceptible to tuberculosis. Thus, our findings reveal a mechanistic basis for protection against tuberculosis by GBA1 N370S and provide biological plausibility for its selection if the relatively mild deleterious effects in homozygotes were offset by significant protection against tuberculosis, a rampant killer of the young in Europe through the Middle Ages into the 19th century.

Disparities in Tuberculosis Incidence by Race and Ethnicity Among the U.S.-Born Population in the United States, 2011 to 2021 : An Analysis of National Disease Registry Data.

BACKGROUND: Elevated tuberculosis (TB) incidence rates have recently been reported for racial/ethnic minority populations in the United States. Tracking such disparities is important for assessing progress toward national health equity goals and implementing change. OBJECTIVE: To quantify trends in racial/ethnic disparities in TB incidence among U.S.-born persons. DESIGN: Time-series analysis of national TB registry data for 2011 to 2021. SETTING: United States. PARTICIPANTS: U.S.-born persons stratified by race/ethnicity. MEASUREMENTS: TB incidence rates, incidence rate differences, and incidence rate ratios compared with non-Hispanic White persons; excess TB cases (calculated from incidence rate differences); and the index of disparity. Analyses were stratified by sex and by attribution of TB disease to recent transmission and were adjusted for age, year, and state of residence. RESULTS: In analyses of TB incidence rates for each racial/ethnic population compared with non-Hispanic White persons, incidence rate ratios were as high as 14.2 (95% CI, 13.0 to 15.5) among American Indian or Alaska Native (AI/AN) females. Relative disparities were greater for females, younger persons, and TB attributed to recent transmission. Absolute disparities were greater for males. Excess TB cases in 2011 to 2021 represented 69% (CI, 66% to 71%) and 62% (CI, 60% to 64%) of total cases for females and males, respectively. No evidence was found to indicate that incidence rate ratios decreased over time, and most relative disparity measures showed small, statistically nonsignificant increases. LIMITATION: Analyses assumed complete TB case diagnosis and self-report of race/ethnicity and were not adjusted for medical comorbidities or social determinants of health. CONCLUSION: There are persistent disparities in TB incidence by race/ethnicity. Relative disparities were greater for AI/AN persons, females, and younger persons, and absolute disparities were greater for males. Eliminating these disparities could reduce overall TB incidence by more than 60% among the U.S.-born population. PRIMARY FUNDING SOURCE: Centers for Disease Control and Prevention.

BCG-induced protection against Mycobacterium tuberculosis infection: Evidence, mechanisms, and implications for next-generation vaccines.

The tuberculosis (TB) vaccine Bacillus Calmette-Guérin (BCG) was introduced 100 years ago, but as it provides insufficient protection against TB disease, especially in adults, new vaccines are being developed and evaluated. The discovery that BCG protects humans from becoming infected with Mycobacterium tuberculosis (Mtb) and not just from progressing to TB disease provides justification for considering Mtb infection as an endpoint in vaccine trials. Such trials would require fewer participants than those with disease as an endpoint. In this review, we first define Mtb infection and disease phenotypes that can be used for mechanistic studies and/or endpoints for vaccine trials. Secondly, we review the evidence for BCG-induced protection against Mtb infection from observational and BCG re-vaccination studies, and discuss limitations and variation of this protection. Thirdly, we review possible underlying mechanisms for BCG efficacy against Mtb infection, including alternative T cell responses, antibody-mediated protection, and innate immune mechanisms, with a specific focus on BCG-induced trained immunity, which involves epigenetic and metabolic reprogramming of innate immune cells. Finally, we discuss the implications for further studies of BCG efficacy against Mtb infection, including for mechanistic research, and their relevance to the design and evaluation of new TB vaccines.

A century of BCG: Impact on tuberculosis control and beyond.

BCG turns 100 this year and while it might not be the perfect vaccine, it has certainly contributed significantly towards eradication and prevention of spread of tuberculosis (TB). The search for newer and better vaccines for TB is an ongoing endeavor and latest results from trials of candidate TB vaccines such as M72AS01 look promising. However, recent encouraging data from BCG revaccination trials in adults combined with studies on mucosal and intravenous routes of BCG vaccination in non-human primate models have renewed interest in BCG for TB prevention. In addition, several well-demonstrated non-specific effects of BCG, for example, prevention of viral and respiratory infections, give BCG an added advantage. Also, BCG vaccination is currently being widely tested in human clinical trials to determine whether it protects against SARS-CoV-2 infection and/or death with detailed analyses and outcomes from several ongoing trials across the world awaited. Through this review, we attempt to bring together information on various aspects of the BCG-induced immune response, its efficacy in TB control, comparison with other candidate TB vaccines and strategies to improve its efficiency including revaccination and alternate routes of administration. Finally, we discuss the future relevance of BCG use especially in light of its several heterologous benefits.

Impact of National Public-Private Mix and Medical Expense Support Program to Control Tuberculosis in South Korea: An Interrupted Time Series Analysis.

Tuberculosis (TB) remains a major threat to global public health. Various measures at the national level have been implemented to control TB, and no evidence with long-term effectiveness has yet been evaluated on TB control programs. We confirmed the long-term effectiveness of the TB control programs in reducing overall burden in South Korea using interrupted time series analysis. Our finding suggests that, along with the public-private mix, relieving the economic burden of people with TB may complement achieving the End TB Strategy. For countries currently developing strategies for TB control, results may provide important insights in effective TB control.

Review of Current Tuberculosis Human Infection Studies for Use in Accelerating Tuberculosis Vaccine Development: A Meeting Report.

Tools to evaluate and accelerate tuberculosis (TB) vaccine development are needed to advance global TB control strategies. Validated human infection studies for TB have the potential to facilitate breakthroughs in understanding disease pathogenesis, identify correlates of protection, develop diagnostic tools, and accelerate and de-risk vaccine and drug development. However, key challenges remain for realizing the clinical utility of these models, which require further discussion and alignment among key stakeholders. In March 2023, the Wellcome Trust and the International AIDS Vaccine Initiative convened international experts involved in developing both TB and bacillus Calmette-Guérin (BCG) human infection studies (including mucosal and intradermal challenge routes) to discuss the status of each of the models and the key enablers to move the field forward. This report provides a summary of the presentations and discussion from the meeting. Discussions identified key issues, including demonstrating model validity, to provide confidence for vaccine developers, which may be addressed through demonstration of known vaccine effects (eg, BCG vaccination in specific populations), and by comparing results from field efficacy and human infection studies. The workshop underscored the importance of establishing safe and acceptable studies in high-burden settings, and the need to validate >1 model to allow for different scientific questions to be addressed as well as to provide confidence to vaccine developers and regulators around use of human infection study data in vaccine development and licensure pathways.

Estimating the Potential Public Health Value of BCG Revaccination.

An upcoming trial may provide further evidence that adolescent/adult-targeted BCG revaccination prevents sustained Mycobacterium tuberculosis infection, but its public health value depends on its impact on overall tuberculosis morbidity and mortality, which will remain unknown. Using previously calibrated models for India and South Africa, we simulated BCG revaccination assuming 45% prevention-of-infection efficacy, and we evaluated scenarios varying additional prevention-of-disease efficacy between +50% (reducing risk) and -50% (increasing risk). Given the assumed prevention-of-infection efficacy and range in prevention-of-disease efficacy, BCG revaccination may have a positive health impact and be cost-effective. This may be useful when considering future evaluations and implementation of adolescent/adult BCG revaccination.

US Guidelines Fall Short on Short-Course Tuberculosis-Preventive Therapy.

The provision of tuberculosis-preventive therapy (TPT) to vulnerable populations is critical for global control. Shorter-course TPT regimens are highly effective and improve completion rates. Despite incorporation of 1 month of rifapentine and isoniazid into global guidelines, current US TPT guidelines do not include this as a recommended regimen, but should.

Comparison of QuantiFERON Gold In-Tube Versus Tuberculin Skin Tests on the Initiation of Tuberculosis Preventive Therapy Among Patients Newly Diagnosed With HIV in the North West Province of South Africa (the Teko Study): A Cluster Randomized Trial.

BACKGROUND: Tuberculosis (TB) preventive therapy (TPT) reduces the risk of TB disease in people with human immunodeficiency virus (HIV), yet uptake has been suboptimal in many countries. We assessed whether QuantiFERON Gold In-Tube (QGIT) during routine HIV care increased TB infection (TBI) testing and TPT prescriptions. METHODS: This parallel-arm, 1:1 cluster-randomized controlled trial compared the standard-of-care tuberculin skin test to QGIT in South Africa. We enrolled consenting, TPT-eligible adults diagnosed with HIV ≤30 days prior and used intention-to-treat analyses for the outcomes: proportion of patients with documented TBI results, proportion with documented TPT, and time from enrollment to outcomes. FINDINGS: We enrolled 2232 patients across 14 clinics from November 2014 to May 2017 (58% in intervention clinics). At 24 months of follow-up, more participants in intervention clinics had TBI results (69% vs 2%, P < .001) and TPT prescriptions (45% vs 30%, P = .13) than control clinics. Controlling for baseline covariates, intervention clinics had 60% (95% confidence interval, 51-68; P < .001) more participants with TBI results and 12% (95% confidence interval, -6 to 31; P = .18) more with TPT prescriptions. Among participants with results, those in intervention clinics received results and TPT faster (intervention: median of 6 and 29 days after enrollment vs control: 21 and 54 days, respectively). INTERPRETATION: In this setting, QGIT in routine HIV care resulted in more patients with TBI results. Clinicians also initiated more people with HIV on TPT in QGIT intervention clinics, and did so more quickly, than the control arm. CLINICAL TRIALS REGISTRATION: NCT02119130.

Human Disease due to Mycobacterium bovis Linked to Free-Ranging Deer in Michigan.

BACKGROUND: A unique enzootic focus of Mycobacterium bovis in free-ranging deer was identified in northern lower Michigan in 1994, with subsequent evidence of transmission to local cattle herds. Between 2002 and 2017, 3 Michigan deer hunters with M. bovis disease were previously reported. We present 4 additional human cases linked to the zoonotic focus in deer, utilizing genomic epidemiology to confirm close molecular associations among human, deer and cattle M. bovis isolates. METHODS: Identification of human tuberculosis (TB) cases with cultures of M. bovis was provided from the Michigan Department of Health and Human Services (MDHHS) tuberculosis database. Clinical review and interviews focused on risk factors for contact with wildlife and cattle. Whole genome sequences of human isolates were compared with a veterinary library of M. bovis strains to identify those linked to the enzootic focus. RESULTS: Three confirmed and 1 probable human case with M. bovis disease were identified between 2019 and 2022, including cutaneous disease, 2 severe pulmonary disease cases, and human-to-human transmission. The 3 human isolates had 0-3 single-nucleotide polymorphisms (SNPs) with M. bovis strains circulating in wild deer and domestic cattle in Michigan. CONCLUSIONS: Spillover of enzootic M. bovis from deer to humans and cattle continues to occur in Michigan. Future studies should examine the routes of transmission and degree of risk to humans through expanded epidemiological surveys. A One Health approach linking human, veterinary and environmental health should address screening for TB infection, public education, and mitigation of transmission.

Long-Term Protective Effect of Tuberculosis Preventive Therapy in a Medium/High Tuberculosis Incidence Setting.

BACKGROUND: The duration of the protective effect of tuberculosis preventive therapy (TPT) is controversial. Some studies have found that the protective effect of TPT is lost after cessation of therapy among people with human immunodeficiency virus (HIV) in settings with very high tuberculosis incidence, but others have found long-term protection in low-incidence settings. METHODS: We estimated the incidence rate (IR) of new tuberculosis disease for up to 12 years after randomization to 4 months of rifampin or 9 months of isoniazid, among 991 Brazilian participants in a TPT trial in the state of Rio de Janeiro, with an incidence of 68.6/100 000 population in 2022. The adjusted hazard ratios (aHRs) of independent variables for incident tuberculosis were calculated. RESULTS: The overall tuberculosis IR was 1.7 (95% confidence interval [CI], 1.01- 2.7) per 1000 person-years (PY). The tuberculosis IR was higher among those who did not complete TPT than in those who did (2.9 [95% CI, 1.3-5.6] vs 1.1 [.4-2.3] per 1000 PY; IR ratio, 2.7 [1.0-7.2]). The tuberculosis IR was higher within 28 months after randomization (IR, 3.5 [95% CI, 1.6-6.6] vs 1.1 [.5-2.1] per 1000 PY between 28 and 143 months; IR ratio, 3.1 [1.2-8.2]). Treatment noncompletion was the only variable associated with incident tuberculosis (aHR, 3.2 [95% CI, 1.1-9.7]). CONCLUSIONS: In a mostly HIV-noninfected population, a complete course of TPT conferred long-term protection against tuberculosis.

Community-Wide Universal HIV Test and Treat Intervention Reduces Tuberculosis Transmission in Rural Uganda: A Cluster-Randomized Trial.

BACKGROUND: Human immunodeficiency virus (HIV) treatment reduces tuberculosis (TB) disease and mortality; however, the population-level impact of universal HIV-test-and-treat interventions on TB infection and transmission remain unclear. METHODS: In a sub-study nested in the SEARCH trial, a community cluster-randomized trial (NCT01864603), we assessed whether a universal HIV-test-and-treat intervention reduced population-level incident TB infection in rural Uganda. Intervention communities received annual, population-level HIV testing and patient-centered linkage. Control communities received population-level HIV testing at baseline and endline. We compared estimated incident TB infection by arms, defined by tuberculin skin test conversion in a cohort of persons aged 5 and older, adjusting for participation and predictors of infection, and accounting for clustering. RESULTS: Of the 32 trial communities, 9 were included, comprising 90 801 participants (43 127 intervention and 47 674 control). One-year cumulative incidence of TB infection was 16% in the intervention and 22% in the control; SEARCH reduced the population-level risk of incident TB infection by 27% (adjusted risk ratio = 0.73; 95% confidence interval [CI]: .57-.92, P = .005). In pre-specified analyses, the effect was largest among children aged 5-11 years and males. CONCLUSIONS: A universal HIV-test-and-treat intervention reduced incident TB infection, a marker of population-level TB transmission. Investments in community-level HIV interventions have broader population-level benefits, including TB reductions.

Twice-Daily Dolutegravir-Based Antiretroviral Therapy With 1 Month of Daily Rifapentine and Isoniazid for Tuberculosis Prevention.

BACKGROUND: One month of daily rifapentine + isoniazid (1HP) is an effective, ultrashort option for tuberculosis prevention in people with human immunodeficiency virus (HIV). However, rifapentine may decrease antiretroviral drug concentrations and increase the risk of virologic failure. AIDS Clinical Trials Group A5372 evaluated the effect of 1HP on the pharmacokinetics of twice-daily dolutegravir. METHODS: A5372 was a multicenter, pharmacokinetic study in people with HIV (≥18 years) already on dolutegravir-containing antiretroviral therapy with HIV RNA <50 copies/mL. Participants received daily rifapentine/isoniazid (600 mg/300 mg) for 28 days as part of 1HP. Dolutegravir was increased to 50 mg twice daily during 1HP, and intensive pharmacokinetic sampling was performed on day 0 (before 1HP) and on the final day of 1HP treatment. RESULTS: Thirty-two participants (41% female; 66% Black/African; median [Q1, Q3] age, 42 [34, 49] years) were included in the pharmacokinetic analysis; 31 had HIV RNA <50 copies/mL at the end of 1HP dosing. One participant had an HIV RNA of 160 copies/mL at day 28, with HIV RNA <50 copies/mL upon repeat testing on day 42. The median (Q1, Q3) dolutegravir trough concentration was 1751 ng/mL (1195, 2542) on day 0 versus 1987 ng/mL (1331, 2278) on day 28 (day 28:day 0 geometric mean ratio, 1.05 [90% confidence interval, .93-1.2]; P = .43). No serious adverse events were reported. CONCLUSIONS: Dolutegravir trough concentrations with 50 mg twice-daily dosing during 1HP treatment were greater than those with standard-dose dolutegravir once daily without 1HP. These pharmacokinetic, virologic, and safety data provide support for twice-daily dolutegravir use in combination with 1HP for tuberculosis prevention. CLINICAL TRIALS REGISTRATION: NCT04272242.