Available online at www.sciencedirect.com
ScienceDirect
Bridging the gap between vaccination with Bacille
Calmette-Gue´ rin (BCG) and immunological tolerance:
the cases of type 1 diabetes and multiple sclerosis
1,5 2,5 3,5
Giovanni Ristori , Denise Faustman , Giuseppe Matarese ,
1 1,4
Silvia Romano and Marco Salvetti
At the end of past century, when the prevailing view was that the sixties [1]. Throughout the next 30 years we and
treatment of autoimmunity required immune suppression, others obtained several results that reinforced the ratio-
experimental evidence suggested an approach of immune- nale, contradicting, at least in part, certain beliefs or
stimulation such as with the BCG vaccine in type 1 diabetes (T1D) paradigms on autoimmune disorders. In particular the
and multiple sclerosis (MS).Translating these basic studies into following evidence prompted us to translate this approach
clinical trials, we showed the following: BCG harnessed the in human neuroinflammation: the resistance to EAE
immune system to ‘permanently’ lower blood sugar, even in induction by the pre-immunization with diverse antigens
advanced T1D; BCG appeared to delay the disease progression (both non-self and auto-antigens) [2]; the immune devia-
in early MS; the effects were long-lasting (years after vaccination) tion toward a T helper 1 cytokine profile (considered
in both diseases.The recently demonstrated capacity of BCG to pathogenic) in multiple sclerosis (MS) patients with a
boost glycolysis may explain both the improvement of satisfactory response to interferon beta therapy [3]; the
metabolic indexes in T1D, and the more efficient generation of ‘physiological’ role of molecular mimicry, at least in T cell
inducible regulatory T cells, which counteract the autoimmune responses [4,5]; the safety of influenza and other vaccines
attack and foster repair mechanisms. in MS [6,7].
Addresses We studied the effect of BCG vaccine in patients with
1
Center for Experimental Neurological Therapies, Sant’Andrea Hospital, early MS. A single cross-over magnetic resonance imaging
Department of Neurosciences, Mental Health and Sensory Organs,
(MRI)-monitored trial [8] was performed on 14 partici-
Sapienza University of Rome, S. Andrea Hospital, Via di Grottarossa
pants. Comparing monthly MRI scans of pre-vaccination
1035, 00189 Rome, Italy
2 and post-vaccination period we found that MRI activity,
Immunobiology Department, Massachusetts General Hospital and
Harvard Medical School, Boston, MA 02129, USA expressed as gadolinium (Gd)-enhanced lesions plus new
3
Treg Cell Lab, Dipartimento di Medicina Molecolare e Biotecnologie and enlarging lesions in T2-weighted (T2W) images,
Mediche, Universita` di Napoli ‘Federico II’, IEOS-CNR, Via S. Pansini 5,
significantly dropped after BCG. No major adverse events
80131 Napoli, Italy
4 were reported [9 ].
IRCCS Istituto Neurologico Mediterraneo (INM) Neuromed,
86077 Pozzilli, IS, Italy
In this same cohort of MS patients we evaluated the
Corresponding authors:
effect of BCG vaccine on the evolution of new Gd-
Ristori, Giovanni ([email protected]),
enhancing lesions to hypointense lesions on T1-
Salvetti, Marco ([email protected])
5 weighted MRI images through three new MRI scans
Equal contribution.
over 24 months after vaccination [10]. This allowed to
Current Opinion in Immunology 2018, 55:89–96 quantify the so-called black holes (BH), expression of
irreversible tissue damage, to evaluate the vaccine influ-
This review comes from a themed issue on Autoimmunity
ence on axonal loss, that underlies the development of
Edited by Stetson
clinical disability [11]. Once again we compared the two
phases of the trial (the run-in versus the post-BCG
percentage of NEL that evolve into BH), and found a
https://doi.org/10.1016/j.coi.2018.09.016 significant result: new enhancing lesions at MRI per-
sisted less and less frequently evolved to black holes
0952-7915/ã 2018 Published by Elsevier Ltd.
after the BCG vaccine. This post-hoc analysis seemed to
indicate that vaccination with BCG might decrease the
tissue damage. Moreover, the dynamics of BCG effects
over time prompted us to hypothesize its long-term
benefit and to design subsequent studies on a longer
temporal scale.
BCG vaccine in neuroinflammation: multiple
sclerosis and beyond
The rationale for the use of adjuvant therapy in Experi- The suggestion found confirmation in a third study on
mental Allergic Encephalomyelitis (EAE) dates back to subjects with the first demyelinating episode (usually
www.sciencedirect.com Current Opinion in Immunology 2018, 55:89–96
90 Autoimmunity
referred to as clinically isolated syndrome—CIS), that is The mechanisms of action of BCG vaccine in MS are
considered, in the presence of a brain and spinal cord MRI not clear. However, clinical observations along with
compatible with MS, the onset of clinical disease [12]. In genetic and functional studies suggest a net beneficial
these parallel groups trials we met several significant end role of TNF in human neuroinflammation, that may be
points after a single vaccination with BCG in people with deficient in MS patients, and seems to be at variance
CIS: reduced disease activity with MRI monitoring, less with more complex effects seen in EAE [15]. In fact,
propensity to develop black holes at 18 months, lower diseases worsening occurred when therapies antagoniz-
occurrence of the second demyelinating event (that is ing tumor necrosis factor (TNF) were tried in MS [16],
conversion to clinically definite MS) at 60 months, with- contrary to other autoimmune disorders where anti-
out major adverse event due to vaccine (Figure 1) [13 ]. TNF is beneficial, but bears risks of demyelinating
episodes. Moreover, an MS-associated genetic variant
Underway currently is our trial with the BCG vaccine in directs increased expression of a soluble protein that
people with ‘radiologically isolated syndromes’ (RIS): a acts as an analogous of anti-TNF agents [17]; notably,
new subclinical entity, that occurs when an MRI of the this variant is not associated with autoimmune disorders
central nervous system shows results compatible with where TNF antagonism results beneficial. A potent
MS, but without any symptom or sign of disease [14]. TNF inducer, such as BCG vaccination, may be useful
The trial stemmed from reasoning that safe and manage- to rescue TNF deficit in MS, and this is in keeping with
able approaches, that can be used with the (biological) evidences coming from type 1 diabetes (see next
onset of the disease, are important unmet needs in MS. In section).
fact, the last-decade therapeutic progress for the overt
relapsing-remitting form was reached at the cost of safety, Another promising field for adjuvant approach seems to
quality of life and overtreatment. Being safe, cheap and be that of more common neurological disorders, such as
available, the BCG vaccine seemed appropriate as a front- Alzheimer’s and Parkinson disease, classically considered
line immune-modulatory approach for people with RIS. of degenerative nature. Recent evidences consistently
Figure 1
(a) 2 (b)
Placebo 1,0
BCG placebo + DMT BCG + DMT
0,8
1 T-1 hypontense
0,6
lesions including ‘black holes’ 0 Cumulative survival relapse free of
0,4 Mean changestotal in (±SEM)
0102030 40 50 60 Baseline-month 6 Baseline-month 12 Baseline-month 18 Follow-up (months)
Current Opinion in Immunology
Long-term effects of a single BCG vaccination in people with first demyelinating episode. (a) Mean change in the total T1-hypointense lesions,
including ‘black holes’ that express irreversible tissue damage, over 18 months. (b) Proportion of people free from the second clinical event (that
is not converting to clinically definite multiple sclerosis) over 60 months.
Current Opinion in Immunology 2018, 55:89–96 www.sciencedirect.com
Bridging the gap between vaccination with Bacille Calmette-Gue´ rin (BCG) and immunological tolerance Ristori et al. 91
showed a neuroinflammatory component in these dis- T cell maturation and proper education of T cells. Interrupt
eases, with a prevalent role for glial cells and innate of this pathway would be predicted to cause autoimmunity.
immune system [18]. In this context a neuroprotective If there was perhaps too little TNF or at least sluggish
action of BCG vaccine emerged in experimental models TNF-mediated signaling in the immune system, could
of neurodegenerative diseases. Two works in models of something as simple of adding back TNF be therapeutic
PD suggested a BCG-mediated immune stimulation, that for T1D? Indeed, TNF selectively killed autoreactive T
may limit deleterious microglial response to a neuronal cells causing T1D and multiple sclerosis and also in other
insult [19 ], may promote T regulatory protective autoimmune diseases such a multiple sclerosis antigen
responses [20]. Other two works in models of AD eluci- presenting defects in HLA class I have been reported
dated additional mechanisms of neuroprotection: activa- [36–39]. The data started to accumulate that this inflam-
tion of tumor necrosis factor receptor 2 (TNFR2), that matory cytokine, TNF might be therapeutic. Animal mod-
block neuroinflammation and promote neuronal survival els of autoimmunity also demonstrated the therapeutic
[21], as well as recruitment of inflammation-resolving benefit of TNF. Disease causing cytotoxic T cells, har-
monocyte (producing anti-inflammatory cytokines and vested from the spleens of diabetes affect mice, normally
neurotrophic factors) at the choroid plexus and perivas- transfer disease to naı¨ve animals. If these splenocytes were
cular spaces of plaque pathology [22]. Along the same line first in vitro treated with TNF, disease transfer was pre-
of research several works showed a beneficial effect of vented [36]. TNF itself or TNF induction with the BCG
neonatal BCG vaccination on neurodevelopment and on vaccine was found to prevent onset as well as reverse even
counteracting neurobehavioral impairment and neuroin- end-stage disease in the NOD mouse model when admin-
flammation due to secondary immune challenge in adult istered in vivo [40,41]. This suggested that autoimmune
mice [23,24]. diseases needed TNF, not immunosuppression, as a ther-
apeutic approach (Figure 2). As further refinements of
BCG and type 1 diabetes these experiments continued, TNF or TNF stimulation
Our 20-year journey towards using BCG to treat type through the TNFR2 receptor was the central pathway for
1 diabetes (T1D) started off in a paradoxical direction. disease modification [42,43].
Studying both human andmousemodels, we discovered T-
lymphocyte (T cell) imbalances, both an overabundance of Translating these basic studies into clinical trials of T1D
pathogenic cytotoxic T cells (CD8 CTLs), and a paucity of could be accomplished by two routes: manufacture and
suppressive T-regulatory (CD4 Treg) cells. Importantly start a TNF drug program (TNFR2 agonistic antibodies
these immune cell imbalances could be corrected with were not available at the time) or develop and recycle a
tumor necrosis factor (TNF), a well-known pro-inflamma- safe 100 year-old drug, BCG, an inactive version of
tory cytokine with immune-stimulatory properties. At the Mycobacteria bovis originally developed to protect from
time, however, the prevailing view was that treatment of tuberculosis and a known inducer of TNF. Because of
T1D and other forms of autoimmunity required immuno- monetary limitations, we decided to launch a T1D trial
suppression, not immunostimulation, the same belief that with BCG as a method to induce and restore TNF.
also dominated the multiple sclerosis field.
In 2012, we published the Phase I results from multi-
Through basic science, we discovered that the antigen- dosing BCG in long-term T1Ds (>10 years disease dura-
presenting cells of T1D patients had interruptions in CD8 tion) [44 ]. This was a safety trial that also studied
T cell education due to a paucity of self-peptides being important biomarkers of efficacy. We established that
generated for the HLA class I groove, a process controlled BCG was safe in this patient population, and showed
by intracellular processing in antigen-presenting cells [25 consistent trends with the proposed mechanism of action
,26,27]. This was some of early T1D data implementing of BCG: death of autoreactive T cells, induction of Tregs,
CD8 T cells in the disease; prior studies thought CD4 T and minor increase of C-peptide secretion (a marker of
cells were the central cells in T1D. The intracellular insulin production by the pancreas), suggesting some
antigen presenting process normally utilized the TAP restored activity [44 ]. In support of BCG in T1D, an
and proteasome genes and this presentation of self antigens epidemiology study from Turkey has since showed that
was altered in T1D. Interestingly these HLA class I pre- multi-dosing BCG may not only be beneficial in T1D
senting genes were located in the high-risk HLA region treatment, but also in its diabetes prevention. Participants
[28–33]. Our research suggested that HLA class I presen- who received greater than 2 childhood vaccines of BCG
tation ofself-proteins was necessary for tolerance [30].With had diminished lifelong risk for developing T1D [45].
proteasomedysfunction, additionalintracellularprocessing
defects were also detected in T1D. NFkB activation, a Our 12-week-long Phase 1 trial showed that C-peptide
proteasome-dependent process for T cell education path- production was not increased in a clinically meaningful
ways, was also dysfunctional [34,35]. TNF is the cytokine way, although seeing any restored C-peptide in T1D with
that normallystimulatestheNFkB signaling pathway.This this long standing disease was unusual. We asked, had we
TNF-stimulated pathway promotes antigen presentation, followed our patients long enough? We subsequently
www.sciencedirect.com Current Opinion in Immunology 2018, 55:89–96 92 Autoimmunity
Figure 2
BCG CTL Treg TL Treg CTL CTL CTL TNF CTL Treg
CTL CTL CTL
Treg Treg
Treg
Treg UnhealthyTreg
Autoimmune target tissue CTL Cytotoxic T cells
CTL Cytotoxic T cell Treg Activated TNFR2 Treg
CTL
Current Opinion in Immunology
Immune mechanisms for the action of TNF induction through BCG. BCG through direct TNF induction or TNFR2 agonism on infected antigen
presenting cells can directly and selectively kill cytotoxic T cells and proliferate Treg cells, thus re-establish immune tolerance.
learned from the multiple sclerosis trials by Ristori et al. that beneficial clinical effects in patients with long standing
BCG’s benefit, while slow to start, showed measurable diabetes. Before this time, immune interventions were
disease improvement over the next 2–3 years. With this exclusively tried in only new onset T1D, a clinical setting
in mind, we re-opened our Phase I trial to follow our T1D thought easier to reverse.
patients for another 8 years. Similar to the timeframe with
multiple sclerosis, we found that, by year 3, blood sugar Immunometabolic effects of BCG in
began to drop and hemoglobin A1c (HbA1c) gradually autoimmunity
returned to near normal levels. This slow-to-materialize, BCG, as therapeutic opportunity to induce immunologi-
but remarkable, correction of blood sugars towards normal cal tolerance, is a very attractive one. At mechanistic level,
by BCG persisted for 5 more years [46 ]. Repeat BCG despite compelling experimental evidence suggests the
vaccines were a powerful way to harness the immune capacity of BCG to affect immune responses, the cellular
system and alter metabolism to ‘permanently’ lower blood and molecular determinants controlling this process are
sugars, even in advanced TID. In 2018, we showed that the still not fully understood. Originally, BCG was considered
mechanism for better blood sugar control by BCG was due a powerful tool to stimulate immunity against Mycobacte-
to a systemic shift in metabolism from oxidative phosphor- rium tuberculosis (MTB); indeed BCG infection is charac-
ylation to aerobic glycolysis, a metabolic state with accel- terized by a classical strong induction of Th1/Th17
erated transport and utilization of serum sugar for energy. responses in immunized individuals, able to exert protec-
BCG therapy in human autoimmunity thus opened up the tion against MTB infection, particularly in very young
exciting possibility of a safe and an effective vaccine for individuals. The induction of this type of response is in
resetting the immune system even in existing T1D disease ‘apparent contrast’ with the capacity of BCG to protect
states. The BCG vaccine confirmed and verified an entirely from induction of mouse EAE and the development of
novel way to regulate blood sugars. Fully enrolled and T1D in the NOD mouse, both models of disease caused
ongoing human clinical trials continue to test the validity by pro-inflammatory Th1/Th17 switches [47,48].
of BCG lowering of blood sugars in large patient popula-
tions and unlike most immune intervention trials is T1D, A possible explanation for this ‘apparent paradox’ comes
the BCG vaccine was potent enough to have these from recent studies linking immunometabolic pathways
Current Opinion in Immunology 2018, 55:89–96 www.sciencedirect.com
Bridging the gap between vaccination with Bacille Calmette-Gue´ rin (BCG) and immunological tolerance Ristori et al. 93
to the capacity of BCG to induce glycolysis in T cells and glycolysis appears to be necessary to generate iTreg cells
innate immune cells such as monocytes/macrophages from Tconv cells [50 ,51 ]. Specifically, this process
[49 ]. Glycolysis fuels proliferation and differentiation appeared tightly linked with the timing and strength of
+
of CD4 T cells towards Th1 and Th17 responses, but it is T cell receptor (TCR) engagement; indeed only TCR
also necessary to generate specific populations of induc- stimulation determined a consistent engagement of gly-
ible regulatory T cells (iTreg) cells from conventional T colysis able to sustain the appropriate induction and
(Tconv) cells in humans [50 ]. While differentiated Treg maintenance overtime of the Foxp3 gene, whose expres-
cells show a mixed metabolic profile being able to engage sion is crucial for Treg cell maintenance and suppressive
both glycolysis and lipid oxidation, engagement of functions [50 ,51 ,52 ].
Figure 3
Autoimmunity (a) Damage
Th1 cells
Treg cells Tconv cells
Central nervous system Glycolysis
Damage
Proliferating Tconv Auto Ag-TCR stimulation Th17 cells Low induction of Treg cells and consequent higher generation of pathologic effector Th subsets Pancreatic-cells
Immune tolerance (b)
Treg cells
Tconv cells
High glycolysis Th17 cells
Central nervous system
Th1 cells
Auto Ag-TCR Proliferating stimulation Tconv cells
BCG Induction of Treg cells vaccination Control of pathogenic effector Th subsets Pancreatic-cells
Current Opinion in Immunology
Hypothetical pro-tolerogenic capacity of BCG vaccination via enhancement of glycolysis. (a) In autoimmune disorders such as MS and T1D there
is a reduced engagement of glycolysis in Tconv cells which leads to reduced iTreg cell induction and consequent enhancement of pro-pathogenic
Th1/Th17 responses. (b) BCG vaccination is able to boost engagement of glycolysis in Tconv cells upon TCR activation and consequently
increase iTreg cell generation which in turn control pro-pathogenic Th1/Th17 responses.
deleterious microglial response to a neuronal insul
www.sciencedirect.com Current Opinion in Immunology 2018, 55:89–96
94 Autoimmunity
2. Fiori P, Ristori G, Cacciani A, Buttinelli C, Falcone M, Di Giovanni S,
It is also interesting to note that autoimmune disorders
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a ‘double edged’ process in which while effector
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and inflammatory processes that consistently engage
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+
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Bacille Calmette-Gue` rin (BCG) in multiple sclerosis. Neurology
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it supports the broad rationale that a benign exposure to This was the first paper on BCG vaccine use in multiple sclerosis: a
reduction of disease activity at cerebral magnetic resonance imaging was
microbes (especially the ‘old friends’ that have co-
evident.
evolved with human beings for millennia) [54] may help
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antagonize conditions sharing chronic inflammation and
Salvetti M, Ristori G: The effect of Bacille Calmette-Guerin on
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at least in some autoimmune disorders, adds an intriguing
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12. Miller D, Barkhof F, Montalban X, Thompson A, Filippi M: Clinically
Conflict of interest statement isolated syndromes suggestive of multiple sclerosis, part I:
natural history, pathogenesis, diagnosis, and prognosis.
Nothing declared.
Lancet Neurol 2005, 4:281-288.
13. Ristori G, Romano S, Cannoni S, Visconti A, Tinelli E, Mendozzi L,
Acknowledgements Cecconi P, Lanzillo R, Quarantelli M, Buttinelli C et al.: Effects of
Bacille Calmette-Guerin after the first demyelinating event in
We thank Fortunata Carbone for figures.
the CNS. Neurology 2014, 82:41-48.
This paper showed that in patients with early multiple sclerosis (first
Fundings: GM is supported by the European Research Council demyelinating episode) BCG vaccine may delay the second episode over
“menTORingTregs” grant no. 310496, the Fondazione Italiana Sclerosi a 5-year period, suggesting long-term action of a single vaccination.
Multipla grant no. 2016/R/18 and the Telethon grant no. GGP17086. GR is
14. Okuda DT, Mowry EM, Beheshtian A, Waubant E, Baranzini SE,
supported by Fondazione Italiana Sclerosi Multipla, grant no. 2016/S/
Goodin DS, Baranzini SE, Goodin DS, Hauser SL, Pelletier D:
1. DLF is supported by The Iacocca Foundation.
Incidental MRI anomalies suggestive of multiple sclerosis: the
radiologically isolated syndrome. Neurology 2009, 72:800-805.
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Current Opinion in Immunology 2018, 55:89–96 www.sciencedirect.com