The Recombinant Bacille Calmette–Guérin Vaccine VPM1002: Ready for Clinical Efficacy Testing

Abstract

The only licensed vaccine against tuberculosis (TB), bacille Calmette–Guérin (BCG), protects against severe extrapulmonary forms of TB but is virtually ineffective against the most prevalent form of the disease, pulmonary TB. BCG was genetically modified at the Max Planck Institute for Infection Biology to improve its immunogenicity by replacing the urease C encoding gene with the listeriolysin encoding gene from Listeria monocytogenes. Listeriolysin perturbates the phagosomal membrane at acidic pH. Urease C is involved in neutralization of the phagosome harboring BCG. Its depletion allows for rapid phagosome acidification and promotes phagolysosome fusion. As a result, BCGΔureC::hly (VPM1002) promotes apoptosis and autophagy and facilitates release of mycobacterial antigens into the cytosol. In preclinical studies, VPM1002 has been far more efficacious and safer than BCG. The vaccine was licensed to Vakzine Projekt Management and later sublicensed to the Serum Institute of India Pvt. Ltd., the largest vaccine producer in the world. The vaccine has passed phase I clinical trials in Germany and South Africa, demonstrating its safety and immunogenicity in young adults. It was also successfully tested in a phase IIa randomized clinical trial in healthy South African newborns and is currently undergoing a phase IIb study in HIV exposed and unexposed newborns. A phase II/III clinical trial will commence in India in 2017 to assess efficacy against recurrence of TB. The target indications for VPM1002 are newborn immunization to prevent TB as well as post-exposure immunization in adults to prevent TB recurrence. In addition, a Phase I trial in non-muscle invasive bladder cancer patients has been completed, and phase II trials are ongoing. This review describes the development of VPM1002 from the drawing board to its clinical assessment.

Full text

September 2017 | Volume 8 | Article 11471
REVIEW
published: 19 September 2017
doi: 10.3389/fimmu.2017.01147
Frontiers in Immunology | www.frontiersin.org
Edited by:
Norbert Reiling,
Forschungszentrum Borstel (LG),
Germany
Reviewed by:
Mario M. D’Elios,
University of Florence, Italy
Sunil Joshi,
Old Dominion University,
United States
*Correspondence:
Stefan H. E. Kaufmann
kaufmann@mpiib-berlin.mpg.de
Specialty section:
This article was submitted to
Microbial Immunology,
a section of the journal
Frontiers in Immunology
Received: 05July2017
Accepted: 30August2017
Published: 19September2017
Citation:
NieuwenhuizenNE, KulkarniPS,
ShaligramU, CottonMF,
RentschCA, EiseleB, GrodeL and
KaufmannSHE (2017) The
Recombinant Bacille Calmette–
Guérin Vaccine VPM1002: Ready for
Clinical Efficacy Testing.
Front. Immunol. 8:1147.
doi: 10.3389/fimmu.2017.01147
The Recombinant Bacille Calmette–
Guérin Vaccine VPM1002: Ready for
Clinical Efficacy Testing
Natalie E. Nieuwenhuizen1, Prasad S. Kulkarni2, Umesh Shaligram2, Mark F. Cotton3,
Cyrill A. Rentsch4,5, Bernd Eisele6, Leander Grode6 and Stefan H. E. Kaufmann1*
1 Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany, 2 Serum Institute of India Pvt. Ltd.,
Pune, India, 3 Stellenbosch University, Tygerberg, South Africa, 4 Department of Urology, University Hospital Basel, Basel,
Switzerland, 5 Swiss Group for Clinical Cancer Research (SAKK), Bern, Switzerland, 6 Vakzine Projekt Management GmbH,
Hannover, Germany
The only licensed vaccine against tuberculosis (TB), bacille Calmette–Guérin (BCG),
protects against severe extrapulmonary forms of TB but is virtually ineffective against
the most prevalent form of the disease, pulmonary TB. BCG was genetically modi-
fied at the Max Planck Institute for Infection Biology to improve its immunogenicity by
replacing the urease C encoding gene with the listeriolysin encoding gene from Listeria
monocytogenes. Listeriolysin perturbates the phagosomal membrane at acidic pH.
Urease C is involved in neutralization of the phagosome harboring BCG. Its depletion
allows for rapid phagosome acidification and promotes phagolysosome fusion. As a
result, BCGΔureC::hly (VPM1002) promotes apoptosis and autophagy and facilitates
release of mycobacterial antigens into the cytosol. In preclinical studies, VPM1002 has
been far more efficacious and safer than BCG. The vaccine was licensed to Vakzine
Projekt Management and later sublicensed to the Serum Institute of India Pvt. Ltd., the
largest vaccine producer in the world. The vaccine has passed phase I clinical trials
in Germany and South Africa, demonstrating its safety and immunogenicity in young
adults. It was also successfully tested in a phase IIa randomized clinical trial in healthy
South African newborns and is currently undergoing a phase IIb study in HIV exposed
and unexposed newborns. A phase II/III clinical trial will commence in India in 2017 to
assess efficacy against recurrence of TB. The target indications for VPM1002 are new-
born immunization to prevent TB as well as post-exposure immunization in adults to
prevent TB recurrence. In addition, a Phase I trial in non-muscle invasive bladder cancer
patients has been completed, and phase II trials are ongoing. This review describes the
development of VPM1002 from the drawing board to its clinical assessment.
Keywords: tuberculosis, bacille Calmette–Guérin, VPM1002, vaccine, listeriolysin, immune response
Abbreviations: AIM2, absent in melanoma 2; BCG, bacille Calmette–Guérin; CFUs, colony forming units; CTL, human
cytotoxic lymphocyte; DC, dendritic cell; EPI, expanded program of immunization; GBP, guanylate binding protein; HEU,
HIV-exposed uninfected; IgG, immunoglobulin G; LC3, microtubule-associated protein light chain 3; LLO, listeriolysin O;
MHC, major histocompatibility complex; Mtb, Mycobacterium tuberculosis; NLRP3, NLR family pyrin domain-containing 3;
NMIBC, non-muscle-invasive bladder cancer; rBCG, recombinant BCG; RD1, Region of Dierence 1; SCID, severe combined
immunodeciency; STING, stimulator of interferon genes; TB, tuberculosis; TCM, central memory Tcells; TEM, eector memory
Tcells; TFH, follicular Tcells; , T helper cell.

FIGURE 1 | Schematic overview of the development of the VPM1002 vaccine candidate. Clinical trials are labeled by their ClinicalTrials.gov Identifier number.
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INTRODUCTION
Infection with Mycobacterium tuberculosis (Mtb) led to 10.4
million recorded cases of tuberculosis (TB) in 2015, with 1.8
million recorded deaths [World Health Organization (WHO)
report 2016]. e current therapy involves 6–9 months of
antibiotics, with the emergence of multiple drug resistant
strains being a continuing obstacle. An attenuated form of the
bovine Mycobacterium species, Mycobacterium bovis bacille
Calmette–Guerin (BCG) has been in clinical use since 1921
and remains the only licensed vaccine against TB. BCG partially
protects against TB meningitis and disseminated TB in infants
and has non-specic immunostimulatory eects (1), which
reduce general infant mortality by enhancing responses to
other infectious diseases (2, 3). However, in all age groups, BCG
does not adequately protect against pulmonary TB, the most
prevalent form of disease and the route of disease transmission.
In addition, BCG can cause severe adverse eects in immuno-
compromised individuals (4) and hence is contraindicated in
HIV-infected individuals, the group that is most vulnerable to
TB. However, in the absence of an alternative, BCG continues
to be used in the immunization programs of several countries.
To overcome these issues, several TB vaccine candidates are
under development (5). One of the most advanced among them
is BCG ΔureC::hly (VPM1002) (6).
VPM1002 is a recombinant BCG (rBCG) in which the urease
C gene has been replaced by the listeriolysin O (LLO) encoding
gene (hly) from Listeria monocytogenes (7). Urease C drives neu-
tralization of phagosomes containing mycobacteria by generation
of ammonia, thereby inhibiting phagolysosomal maturation and
contributing to the survival of mycobacteria inside the mac-
rophage (8, 9). Its depletion allows for rapid phagosome acidi-
cation, which promotes phagolysosome fusion and provides the
optimal pH for LLO stability (10). LLO is a cholesterol-dependant
cytolysin that forms transmembrane β-barrel pores in the phago-
lysosome membrane, allowing escape of L. monocytogenes into
the cytosol (10, 11). Its expression in VPM1002 results in the
release of antigens and bacterial DNA into the cytosol, triggering
autophagy, inammasome activation, and apoptosis. VPM1002
has demonstrated substantially increased immunogenicity, e-
cacy, and safety in preclinical studies, successfully passed Phase
I and II clinical trials, and will now enter a Phase II/III clinical
trial in India in 2017. is review summarizes the development,
preclinical, and clinical testing of VPM1002 (Figure1).

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DESIGN AND GENERATION OF VPM1002
e attenuation of BCG was achieved by passaging virulent M.
bovis in bile-containing medium for 13years in the laboratory
(12), during which time several genome segments were lost,
including a segment known as Region of Dierence 1 (RD1)
which encodes the unique mycobacterial ESX-1 type VII
secretion system (13, 14). ESX-1-dependent perturbation of
host cell membranes requires direct contact with pathogenic
mycobacteria such as Mtb, allowing the bacilli or their antigens
to egress the phagosome into the cytosol (15). Mtb antigens
are thus accessible to both the endocytic major histocompat-
ibility complex (MHC) class II antigen presentation pathway
and the MHC I antigen presentation pathway in the cytosol,
and consequently can stimulate CD4+ and CD8+ T-cell subsets,
respectively, both of which are required for optimal protection
against TB (16–21). In addition, ESX-1 dependent release of
Mtb DNA into the cytosol can be detected by host sensors,
leading to activation of NLR family pyrin domain-containing
3 (NLRP3) and absent in melanoma 2 inammasomes, release
of interferons, increased autophagy and apoptosis (22–25).
Induction of apoptosis in infected host cells generates vesicles
carrying mycobacterial antigens that can be phagocytosed
by bystander antigen presenting cells, mainly dendritic cells
(DCs) and tracked through MHC I antigen processing
pathways to stimulate CD8+ T cells in a process known as
cross-priming (26, 27). Mice with decient cross-presentation
due to the absence of annexin 1 show impaired Mtb-specic
CD8+ Tcells and are highly susceptible to TB (28). Lacking the
ESX-1 secretion system, BCG is restricted to the phagosome
of host cells, therefore its antigens and bacterial DNA do not
enter the cytosol and the antigens are primarily processed by
MHC class II pathways, stimulating CD4+ T cell responses
(13, 14, 29, 30). BCG induces only weak apoptosis and CD8+
Tcell responses (26). Furthermore, both BCG and Mtb inhibit
surface MHC II expression, as urease-dependent alkalinization
of the phagosome causes intracellular sequestration of MHC II
dimers, resulting in suboptimal CD4+ Tcell responses (31–33).
Phagosomal biology is therefore a clear target for interventions
aimed at enhancing Tcell responses against mycobacteria.
Originally, VPM1002 was designed to improve accessibility
of mycobacterial antigens to the MHC I pathway via cytosolic
egression of antigens mediated by LLO perturbation of phago-
somal membranes in order to improve induction of CD8+
Tcells by the parental BCG strain (34, 35). In addition, leakage
of phagolysosomal proteases such as cathepsins into the cytosol
could activate caspases, leading to apoptosis and subsequent
cross-presentation of mycobacterial antigens, which promotes
both MHC I and MHC II restricted T cell stimulation (36).
Studies with L. monocytogenes have shown that pore formation
by LLO also triggers many downstream eects such as activation
of the NLRP3 inammasome, induction of cytokine expression,
activation of kinases, triggering of endocytosis, histone modica-
tion and release of calcium from intracellular stores (37). An Hly
recombinant strain, hly+ rBCG+, was generated by integrating
the hly gene into BCG using the mycobacteria-Escherichia coli
shuttle vector pMV306 (34). LLO was detected in the membrane
structures, phagosomal space, and cytoplasmic vacuoles of
macrophages infected with BCG pMV306::hly, and intracel-
lular persistence of this strain was reduced compared with the
parental BCG strain. MHC I presentation of co-phagocytosed
soluble protein was improved in macrophages infected with this
strain compared to BCG (34) and an invitro human cytotoxic
Tlymphocyte (CTL) assay using cultured DCs and Tcells from
healthy human donors demonstrated that hly+ BCG infection
was better at inducing CTL responses than BCG infection (38).
In the next generation strain, deletion of ureC was performed
to ensure an optimal (acidic) pH for LLO stability; however,
absence of ureC also promotes MHCII tracking to the mac-
rophage surface (31), which would also stimulate CD4+ Tcell
responses. To generate ΔureC hly+ BCG, the chromosomal inte-
grative shuttle vector pMV306hyg-hly (8) was used to transform
M. bovis BCG ΔureC::aph, and hygromycin-resistant clones
were selected (35). e vaccine was licensed to Vakzine Projekt
Management, and named “VPM1002.” e resistance cassette
was subsequently successfully removed, although VPM1002
is equally sensitive to the antimycobacterial agents isoniazid,
rifampicin, and ethambutanol in the presence or absence of the
hygromycin resistance gene (39).
HOST CELL RESPONSES TO VPM1002
IN VITRO
Increased quantities of mycobacterial antigen were detected in
VMP1002 infected macrophages compared to BCG infected
macrophages (35), and mycobacterial DNA was detected only
in the cytosol of VPM1002 infected but not BCG infected
macrophages (29), indicating that expression of LLO in BCG
ΔureC::hly allows the escape of bacterial products to the cytosol,
presumably by perturbation of the phagosomal membrane. e
bacteria themselves do not escape to the cytosol, unlike Mtb
bacilli (29, 35). Infection of primary human and mouse mac-
rophages demonstrated increased apoptosis aer infection with
VPM1002 compared to both BCG and BCG::hly, demonstrating
the additional benet of urease C deletion (35). Membrane
disruption can facilitate the release of phagolysosomal proteases
such as cathepsins into the cytosol, which are known to induce
apoptosis (36, 40). Both the presence of mycobacterial proteins
in the cytosol and the induction of apoptosis by perforation of
the phagosomal membrane could cause increased tracking
of antigens to MHC I pathways (35). Apoptosis results in an
increase in both CD8+ and CD4+ Tcell responses in mycobacte-
rial infection, suggesting that DCs may transfer eerocytosed
antigens to the endocytic system (27, 36). e priming potential
of apoptotic vesicles isolated from BCG and VPM1002 infected
mouse macrophages was investigated in a co-culture system with
splenic DCs and Tcells, and VPM1002-infected apoptotic vesi-
cles induced more profound CD4+ and CD8+ Tcell responses
compared to those infected with BCG (41). Vesicles from
VPM1002 infected macrophages also induced higher produc-
tion of the T helper type ()17-polarizing cytokines interleukin
(IL)-6 and IL-23, and the immunoregulatory cytokine IL-10 by
bone marrow-derived DCs.

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Experiments in THP1 macrophages demonstrated that
VPM1002 infection leads to activation of multiple caspases (29).
e apoptotic eector caspases 3 and 7 were highly activated by
VPM1002 in comparison to BCG, as well as caspase 1, which
mediates pyroptosis, an inammatory form of cell death and
is an important regulator of the inammatory response (42).
Inammasomes are multi-protein complexes composed of
intracellular sensors and caspase 1. ey control activation of
caspase 1, which in turn cleaves the precursors of the cytokines
IL-1β and IL-18 into their active forms (43). VPM1002 infection
increased production of IL-1β and IL-18, which was dependent
on AIM2 inammasome activation but not on NLRP 1 and 3
inammasome activation. Furthermore, VPM1002 induced
increased levels of the autophagy marker microtubule-associated
protein light chain 3 in an AIM2- and stimulator of interferon
genes (STING)-dependent manner. e AIM2 inammasome
senses cytosolic DNA and is involved in the induction of caspase
1-dependent pyroptosis (44, 45), while STING acts as an essential
adaptor protein in the induction of autophagy by cytosolic DNA
(25). Autophagy, a protein degradation process induced by stress
conditions such as infection, promotes the delivery of cytosolic
antigens to MHC tracking pathways (46, 47). It has also been
shown to contribute to innate immunity against mycobacteria
and other intracellular pathogens (48, 49). While autophagy
was originally thought to be non-specic, it is now known that
it can selectively target intracellular pathogens in a process
known as xenophagy that involves ubiquitination of pathogen
proteins or pathogen-containing endosomes (50). Intriguingly,
gene expression of guanylate-binding proteins (GBPs) was also
elevated in VPM1002 infected THP-1 macrophages compared
to BCG infected macrophages. Interferon-inducible GBPs have
multiple roles in inammasome activation, autophagy, and lysis
of pathogen-containing vacuoles and can even directly target
the pathogens themselves (51–54). Whether they play a role in
the translocation of mycobacterial components from the phago-
some into the cytosol during VMP1002 infection remains to be
determined.
Disruption of the VPM1002-containing phagosome mem-
brane by LLO and release of mycobacterial DNA into the cytosol
appears to have eects in inducing immune responses that are
similar to the eects of ESX-1 activity in Mtb or M. marinum.
ESX-1 of M. marinum stimulates autophagosome formation and
recruitment to the vacuole; however, unlike LLO it also inhibits
autophagic ux, thereby preventing bacterial degradation (49).
Testing of vaccine candidates expressing ESX-1 such as Mtb
Δppe25-pe19 (55) and BCG expressing ESX-1 of M. marinum
(BCG:ESX-1Mmar) (56) demonstrated that ESX-1 was critical for
enhancing innate immune responses via phagosome rupture.
BCG:ESX-1Mmar induced the cGas/STING/TBK1/IRF-3/type I
interferon axis and promoted AIM2 and NLRP3 inammasome
activation, resulting in increased frequencies of antigen-specic
CD8+ and CD4+ T cells and increased protection against Mtb
compared to BCG (56), while Mtb Δppe25-pe19 also led to
enhanced protection. ESX-1 may induce protective immunity
by an additional mechanism, as ESAT6 is required for rapid,
non-cognate IFN-γ production by CD8+ Tcells, mediated by the
NLRP3/caspase-1/IL-18 axis (57).
PRECLINICAL EFFICACY AND SAFETY
Aerosol challenge of vaccinated BALB/c or C57BL6 mice with
100–200 colony-forming units (CFUs) of Mtb H37Rv or a clini-
cal isolate of the Beijing/W genotype family demonstrated that
VPM1002 immunization has signicantly greater protective
ecacy than the parental BCG strain, with bacterial loads in
the lungs typically reduced by one to two logs in late stages
of infection (35, 58–61). In a low dose infection (30 CFU),
VPM1002 led to an almost 1000-fold reduction of Mtb in the
lungs compared to naïve mice at day 200 aer infection (35).
Homologous boosting with VPM1002 did not improve protec-
tion compared to a single immunization (60). However, a post-
exposure vaccination model using antibiotics for an extended
period and then allowing bacterial regrowth demonstrated that
mice with subclinical TB had lower bacterial burdens when
vaccinated with VPM1002 compared to BCG, suggesting that
VPM1002 could also be considered for use as a post-exposure
vaccine (60).
e safety prole of VPM1002 has been evaluated in animal
models including mice, guinea pigs, rabbits, and non-human
primates (6). In RAG1-/- immunodecient mice lacking mature
T and Bcells, bacterial loads were not signicantly dierent in
lungs and spleen aer vaccination with VPM1002 compared
to BCG (35). However, VPM1002 demonstrated substantially
lower virulence in severe combined immunodeciency mice,
most likely due to the reduced intracellular persistence of
this strain (35, 61). Aer immunization of wildtype BALB/c
or C57BL6 mice, VPM1002 was more rapidly cleared from
the draining lymph nodes than BCG and disseminated less
to the spleens, where it was also quickly cleared (59, 61).
Dissemination to the lungs was observed in BCG vaccinated
but not VPM1002 vaccinated mice. Enhanced adaptive
immune responses aer VPM1002 vaccination are therefore
likely to play a role in the reduced dissemination of VPM1002
in immunocompetent mice. Overall, the data demonstrate
increased safety and protective ecacy of VPM1002 compared
to parental BCG in mice.
In guinea pigs and non-human primates, the safety of
VPM1002 was comparable to that of BCG (6, 39). As the pri-
mary target population for vaccination against TB is newborns,
the safety proles of VPM1002 and BCG were also compared in
newborn rabbits (39). No dissemination to tissues was observed
aer VPM1002 administration, and the body weight gain was not
aected during the 90days observation period, whereas the body
weight was reduced in the BCG vaccinated group compared to
the saline control group. No premature mortality was observed in
either group. e preclinical safety of VPM1002 is thus supported
by a large body of evidence.
ANALYSIS OF IMMUNE RESPONSES
TO VPM1002 IN MICE
Analysis of gene expression in mice early aer immunization
with VPM1002 demonstrated that, as in THP-1 cells, expres-
sion of IL-18 and IL-1β was increased, as well as expression
of IFN-inducible genes such as Tmem173 (STING), Gbp’s ,

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and other GTPases (29, 61). Apoptosis was increased in the
lymph nodes of VPM1002 immunized mice compared with
BCG immunized mice by day 14 (61). Immunization with
VMP1002 induced both type 1 and type 17 cytokine responses
in mice, whereas BCG induced type 1 responses only (58). Aer
restimulation with PPD, levels of IFN-γ, IL-17, IL-2, IL-6, and
GM-CSF were increased in lung cells isolated from VPM1002
immunized mice compared to those from BCG immunized
mice, and splenocytes from VPM1002-vaccinated mice also
produced more IL-17. Furthermore, percentages of γδ Tcells
producing IFN-γ and IL-17 were increased aer vaccination
with VPM1002 (58). Seven days aer Mtb challenge, IL-2+TNF+
double cytokine producing cells were increased in the lungs of
VPM1002-immunized mice compared with BCG-vaccinated
mice, suggesting recall responses, because newly generated
Tcells take 12–14days to reach the lungs during Mtb infection
(62). IL-2+TNF+ CD4+ Tcells typically show a central memory
phenotype (TCM) (21), and further studies demonstrated that
VPM1002 immunization indeed induces higher frequencies
of TCM than immunization with BCG (59, 61). Ag85B-specic
CD4+ TCM were signicantly increased in the draining lymph
nodes of VPM1002-vaccinated compared to BCG-vaccinated
mice at day 14 (59).
Bacille Calmette–Guérin induces eector memory CD4+
T (TEM) cells that can control acute infection but appears to
induce insucient numbers of TCM cells for long-term protec-
tion (21). Transfer studies demonstrated that TCM cells from
VPM1002 infected mice conferred protection against TB infec-
tion whereas TEM, T follicular helper (TFH), and naïve Tcells did
not, at least at the numbers of cells tested (59). ese ndings
concur with other studies in which TCM cells were associated
with protection (21). While TEM cells appear early aer infection
and provide protection by the secretion of eector cytokines
such as IFN-γ and TNF-α, TCM cells proliferate in the LN and
generate new pools of TEM cells aer re-exposure to antigen (59,
63, 64). e TCM cells generated by subcutaneous vaccination
with VPM1002 or BCG were found to reside over the long term
in the secondary lymphoid organs, rather than in the lung, and
to be recruited to the lungs aer Mtb challenge (58, 59). Waning
of BCG-induced immunity correlates with a decline in Tcell
functions such as cytokine production and CTL activity and
an increase in terminally dierentiated, dysfunctional Tcells
(65). us, systemic maintenance of TCM populations over the
long term and the rapid recruitment of TCM cells to the lung
following Mtb infection remains a key goal in the develop-
ment of more eective vaccine candidates (59). VPM1002 also
induced an increase in mycobacteria-specic immunoglobulin
G levels aer vaccination compared to BCG, and a concomitant
increase in CXCR5-expressing TFH cells (59, 61), which have
been associated with decreased lung pathology (66) and stimu-
late germinal center Bcell responses (63). Passive transfer of
serum from VPM1002- or BCG-immunized mice on the day of
Mtb infection and thrice weekly did not reduce bacterial load at
day 14 (59), but growing evidence suggests that antibodies may
play a role in protection against Mtb (67–71). Overall, increased
protection conferred by VPM1002 immunization in the mouse
model was associated with increased numbers of TCM and TFH
cells, increased 17 responses, earlier recruitment of Tcells
to the lungs following Mtb challenge and increased levels of
anti-mycobacterial antibodies (58, 59, 61).
CLINICAL TRIALS WITH VPM1002: A STEP
TOWARD A SAFER, MORE EFFICACIOUS
TB VACCINE
Human data on VPM1002 are available from three clinical trials,
all performed with the original hygromycin-resistant strain of
VPM1002. Two Phase I studies were performed in healthy adult
volunteers, and one Phase IIa study was conducted in healthy
newborn infants, one of the intended target populations. In
the rst Phase I clinical trial (ClinicalTrials.gov Identier:
NCT00749034) conducted in Germany, healthy Caucasian
adult males with (W) or without (WO) a history of BCG vac-
cination received VPM1002 randomized to three escalating
doses (N=30W+30WO) or BCG at the standard vaccine dose
(N= 10W + 10WO) and were followed for 6 months. Single
vaccination with VPM1002 up to 5× 10e5 CFU was safe and
well tolerated. e immunogenicity of VPM1002 as measured
by IFN-γ release by stimulated Tcells was dose dependent. Both
VPM1002 and BCG induced multifunctional CD4+ and CD8+
T cell subsets, which are thought to play a role in protection
against TB (72–74), with VPM1002 showing an earlier increase
in double and triple cytokine producing Tcells which remained
at heightened levels throughout the study (7). Furthermore, only
VPM1002 induced serum antibodies against mycobacterial anti-
gens (7), echoing preclinical studies in which VPM1002 induced
higher levels of mycobacteria-specic antibodies than BCG in
mice (59, 61). In the second Phase I clinical trial (ClinicalTrials.
gov Identier: NCT01113281), performed in South Africa, 24
healthy male and female adults with a history of BCG immuni-
zation, predominantly from the indigenous African population,
were vaccinated with VPM1002. e study showed that a single
vaccination with VPM1002 is safe, well tolerated and elicits a
profound immune response in an African adult population (6).
e Phase IIa clinical trial (ClinicalTrials.gov Identier:
NCT01479972) was the rst investigation of VPM1002 in
newborns (75). It was conducted in Cape Town, South Africa,
a region with a high TB burden. Forty-eight HIV-unexposed,
newborn infants were vaccinated with either VPM1002 (n=36)
or BCG (n=12) through an open label, randomized, controlled
design. Polyfunctional CD4+ and CD8+ Tcell responses were
similar between the groups, and both groups had increased
IFN-γ responses aer 7 h PPD stimulation at all measured
time points post vaccination compared to baseline. Both vac-
cines induced IL-17 responses; though, unlike BCG, VPM1002
induced increased proportions of CD8+ IL-17+ Tcells at day 14
and month 6 time points compared to the baseline. e incidence
of abscess formation was lower for VPM1002 compared to BCG.
us, VPM1002 was safe, well tolerated, and immunogenic in
newborn infants.
In addition, a Phase IIb clinical trial is currently ongoing in
South Africa (ClinicalTrials.gov Identier: NCT02391415). is
trial is a double-blind, randomized, controlled study to evaluate

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Frontiers in Immunology | www.frontiersin.org September 2017 | Volume 8 | Article 1147
the safety and immunogenicity of VPM1002 in comparison with
BCG in HIV-exposed uninfected (HEU) and HIV-unexposed,
BCG-naive newborn infants. e inclusion of HEU infants in the
trial is important, as this group comprises 30% of the newborns
requiring BCG vaccination in South Africa, and they may be at
higher risk of Mtb infection than HIV-unexposed infants. e
proportion of HEU may vary in dierent countries. Previous
work from Brazil suggests that HEU infants have poorer T-cell
proliferation and lower levels of IFN-γ production compared to
HIV-unexposed infants (76). Enrollment of 416 infants has been
completed and follow-up is in progress. Follow-up will continue
for 12months, as opposed to 6months in NCT01479972, ena-
bling collection of preliminary ecacy data.
In addition to its development as a vaccine for newborns,
VPM1002 is also being assessed as a post-exposure vaccine for
adults, since preclinical studies in mice demonstrated that it
reduced bacterial loads in a post exposure model (60). A phase
II/III trial has received regulatory approval by the Indian authori-
ties. Once ethics committee approvals are received for all sites,
the trial will commence across India (ClinicalTrials.gov Identier:
NCT03152903). e study will be conducted in 2000 adults who
were TB patients, but received drug treatment and were cured of
disease. In such populations, there is a high risk of recurrence
(including re-infection and relapse), especially within 12months
aer completing treatment. e multi-centric, placebo-controlled,
randomized, controlled study will assess whether VPM1002 can
prevent such TB recurrence over a 1-year follow-up period.
Currently, no intervention is licensed for this indication, includ-
ing BCG, which means there is clearly an unmet medical need.
e study will also expand the safety database on VPM1002.
EVALUATION OF VPM1002 AS A BLADDER
CANCER THERAPY
Bladder cancer is the ninth most common cancer in the world,
and is four times more common in men than in women (77). e
main risk factors for developing bladder cancer include smoking,
Schistosoma infection (bilharzia), and exposure to industrial
chemicals (77, 78). Tumors can be non-muscle invasive, i.e., con-
ned to the mucosa of the bladder wall, or muscle-invasive. More
than seventy percent of bladder cancers are detected while they
are still non-muscle invasive (79). Due to its immunostimulatory
properties, repeated intravesical BCG instillation is the standard
adjuvant treatment for intermediate to high-risk non-muscle-
invasive bladder cancer (NMIBC) aer transurethral resection of
the tumors (80–82). BCG therapy reduces the risk of recurrence
and the progression to muscle invasive bladder cancer. e repeated
instillations require much higher doses and volumes of BCG than
vaccination against TB does, and some patients have adverse
events that lead to discontinuation of the therapy (83, 84). Adverse
events include fever, bladder irritation, decreased bladder capacity,
incontinence, hematuria, u-like symptoms and in approximately
5% of cases, BCG infection (85, 86). Patients undergoing traumatic
catheterization are at risk for intraluminal BCG dissemination,
resulting in a potentially lethal systemic infection (87).
e precise immune mechanisms by which BCG promotes
anti-tumor activity in bladder cancer are not completely resolved,
but it is well-established that the ability of BCG to promote 1
responses is important, as well as the recruitment of neutrophils
and innate lymphocytes including natural killer cells (82, 88,
89). Activation of immune cells may lead to elimination of the
urothelial cancerous cells that have internalized BCG (82, 90).
Increased CD4+ T cell responses have been measured during
BCG therapy, and BCG was shown to promote secretion of both
1- and 2-type cytokines (88, 91–93). A positive response to
BCG therapy (no recurrence or evidence of disease during follow-
up examinations) has been associated with an intratumoral 2
predisposition (increased GATA3) and decreased concentrations
of IL-10, combined with a 1 functional phenotype indicated by
increased levels of 1-related inammatory metabolites (88). In
another study, increased regulatory Tcells and tumor-associated
macrophages in the tumor microenvironment were also associ-
ated with non-responsiveness, while increased GATA3+ and CD4+
Tcells were associated with responders (88, 94). BCG Connaught
conferred greater 5-year recurrence-free survival than BCG Tice
and induced stronger 1 type responses, BCG-specic CD8+
Tcells and Tcell recruitment to the bladder (93). Genetic analysis
demonstrated several dierences between the two strains, includ-
ing the absence of RD15 in BCG Connaught (93).
Because approximately 30–40% of patients do not respond to
BCG therapy and others suer from adverse events, rBCG tech-
nology has been tested for improving the ecacy and tolerability
of BCG in bladder cancer therapy (82). rBCGs that have been
modied to express immunostimulatory molecules, cytokines,
or antigens have been tested in mice for their capacity to induce
stronger and more specic immune responses. VPM1002 is cur-
rently being evaluated in SAKK 06/14, a Phase I/II trial for immu-
notherapy in patients with NMIBC (ClinicalTrials.gov Identier:
NCT02371447). e phase I part of the trial has been completed
in Switzerland. Intravesical application of VPM1002BC dem-
onstrated that the product is safe and well tolerated in NMIBC
patients. e recommended phase II dose has been established as
1–19.2×10e8 CFUs of VPM1002BC. e phase II part has been
approved by the Swiss and German regulatory authorities and is
currently ongoing in both countries.
OUTLOOK
e available preclinical and clinical data reveal that VPM1002
is immunogenic and may be better than BCG in terms of safety.
VPM1002 could be a safe, well-tolerated and ecacious alterna-
tive to the BCG vaccine in the future. With an annual capacity
of 100 million doses, Serum Institute of India Pvt. Ltd. can meet
the global demand for a BCG vaccine and is well poised to supply
the new vaccine if ecacy trials are successful. While this vaccine
progresses through ecacy trials, next-generation derivatives are
being designed and tested in preclinical models aimed at optimiz-
ing ecacy and/or safety (61, 95). Furthermore, VPM1002 is
currently being tested in goats by the Friedrich Loeer Institute
in Germany for the prevention of M. caprae infection (Menge etal.
unpublished data). Infections with M. caprae and M. bovis, closely
related species of the same clade that cause TB in goats and cattle,
respectively, are of agricultural importance, and can potentially be
transmitted to humans (96, 97).

7
Nieuwenhuizen et al. The rBCG Vaccine VPM1002
Frontiers in Immunology | www.frontiersin.org September 2017 | Volume 8 | Article 1147
Almost 100 years aer the rst immunization with BCG, a
rBCG vaccine candidate is ready for clinical ecacy testing. is
marks a major step forward in the long journey that began when
the recombinant vaccine was constructed in the late 1990s and
tested in dierent animal models to determine its safety and
protective eect.
AUTHOR CONTRIBUTIONS
NN, LG, and SK wrote and reviewed the manuscript. All other
authors (BE, PK, US, CR, and MC) reviewed the manuscript.
ACKNOWLEDGMENTS
e authors thank Souraya Sibaei for her help in preparing the
manuscript and Diane Schad for excellent graphics work. is
work was supported by e European Union’s Seventh Framework
Programme (EU FP7) ADITEC (HEALTH-F4-2011-280873); by
the EU Horizon 2020 project TBVAC 2020 (grant no. 643381);
e Bill & Melinda Gates Foundation (BMGF) GC6-2013, #OPP
1055806 and #OPP 1065330; the Bundesministerium für Bildung
und Forschung (BMBF) project “Infect Control 2020” (grant no.
03ZZ0806A).
REFERENCES
1. Bekkering S, Blok BA, Joosten LA, Riksen NP, van Crevel R, Netea MG.
In vitro experimental model of trained innate immunity in human primary
monocytes. Clin Vaccine Immunol (2016) 23(12):926–33. doi:10.1128/
cvi.00349-16
2. Aaby P, Kollmann TR, Benn CS. Nonspecic eects of neonatal and infant
vaccination: public-health, immunological and conceptual challenges. Nat
Immunol (2014) 15(10):895–9. doi:10.1038/ni.2961
3. Kandasamy R, Voysey M, McQuaid F, de Nie K, Ryan R, Orr O, etal. Non-
specic immunological eects of selected routine childhood immunisations:
systematic review. BMJ (2016) 355:i5225. doi:10.1136/bmj.i5225
4. Talbot EA, Perkins MD, Silva SF, Frothingham R. Disseminated bacille
Calmette-Guerin disease aer vaccination: case report and review. Clin Infect
Dis (1997) 24(6):1139–46. doi:10.1086/513642
5. Kaufmann SH, Lange C, Rao M, Balaji KN, Lotze M, Schito M, etal. Progress
in tuberculosis vaccine development and host-directed therapies – a state
of the art review. Lancet Respir Med (2014) 2(4):301–20. doi:10.1016/
S2213-2600(14)70033-5
6. Kaufmann SH, Cotton MF, Eisele B, Gengenbacher M, Grode L,
Hesseling AC, etal. e BCG replacement vaccine VPM1002: from drawing
board to clinical trial. Expert Rev Vaccines (2014) 13(5):619–30. doi:10.1586/
14760584.2014.905746
7. Grode L, Ganoza CA, Brohm C, Weiner J III, Eisele B, Kaufmann SH.
Safety and immunogenicity of the recombinant BCG vaccine VPM1002 in
a phase 1 open-label randomized clinical trial. Vaccine (2013) 31(9):1340–8.
doi:10.1016/j.vaccine.2012.12.053
8. Reyrat JM, Berthet FX, Gicquel B. e urease locus of Mycobacterium
tuberculosis and its utilization for the demonstration of allelic exchange in
Mycobacterium bovis bacillus Calmette-Guerin. Proc Natl Acad Sci U S A
(1995) 92(19):8768–72. doi:10.1073/pnas.92.19.8768
9. Gordon AH, Hart PD, Young MR. Ammonia inhibits phagosome-ly-
sosome fusion in macrophages. Nature (1980) 286(5768):79–80.
doi:10.1038/286079a0
10. Hamon MA, Ribet D, Stavru F, Cossart P. Listeriolysin O: the Swiss army knife
of Listeria. Trends Microbiol (2012) 20(8):360–8. doi:10.1016/j.tim.2012.04.006
11. Shaughnessy LM, Hoppe AD, Christensen KA, Swanson JA. Membrane
perforations inhibit lysosome fusion by altering pH and calcium in
Listeria monocytogenes vacuoles. Cell Microbiol (2006) 8(5):781–92.
doi:10.1111/j.1462-5822.2005.00665.x
12. Luca S, Mihaescu T. History of BCG vaccine. Maedica (Buchar) (2013)
8(1):53–8.
13. Brodin P, Majlessi L, Marsollier L, de Jonge MI, Bottai D, Demangel C, etal.
Dissection of ESAT-6 system 1 of Mycobacterium tuberculosis and impact on
immunogenicity and virulence. Infect Immun (2006) 74(1):88–98. doi:10.1128/
IAI.74.1.88-98.2006
14. Simeone R, Bottai D, Brosch R. ESX/type VII secretion systems and their
role in host-pathogen interaction. Curr Opin Microbiol (2009) 12(1):4–10.
doi:10.1016/j.mib.2008.11.003
15. Conrad WH, Osman MM, Shanahan JK, Chu F, Takaki KK, Cameron J,
etal. Mycobacterial ESX-1 secretion system mediates host cell lysis through
bacterium contact-dependent gross membrane disruptions. Proc Natl Acad Sci
U S A (2017) 114(6):1371–6. doi:10.1073/pnas.1620133114
16. Muller I, Cobbold SP, Waldmann H, Kaufmann SH. Impaired resistance
to Mycobacterium tuberculosis infection aer selective invivo depletion of
L3T4+ and Lyt-2+ Tcells. Infect Immun (1987) 55(9):2037–41.
17. Kaufmann SH. Tuberculosis vaccine development: strength lies in tenacity.
Trends Immunol (2012) 33(7):373–9. doi:10.1016/j.it.2012.03.004
18. Sharpe S, White A, Sarfas C, Sibley L, Gleeson F, McIntyre A, etal. Alternative
BCG delivery strategies improve protection against Mycobacterium tubercu-
losis in non-human primates: protection associated with mycobacterial anti-
gen-specic CD4 eector memory T-cell populations. Tuberculosis (Edinb)
(2016) 101:174–90. doi:10.1016/j.tube.2016.09.004
19. Chen CY, Huang D, Wang RC, Shen L, Zeng G, Yao S, etal. A critical role
for CD8 Tcells in a nonhuman primate model of tuberculosis. PLoS Pathog
(2009) 5(4):e1000392. doi:10.1371/journal.ppat.1000392
20. Ritz N, Hanekom WA, Robins-Browne R, Britton WJ, Curtis N.
Inuence of BCG vaccine strain on the immune response and protec-
tion against tuberculosis. FEMS Microbiol Rev (2008) 32(5):821–41.
doi:10.1111/j.1574-6976.2008.00118.x
21. Lindenstrom T, Knudsen NP, Agger EM, Andersen P. Control of chronic
Mycobacterium tuberculosis infection by CD4 KLRG1- IL-2-secreting central
memory cells. J Immunol (2013) 190(12):6311–9. doi:10.4049/jimmunol.1300248
22. Stanley SA, Johndrow JE, Manzanillo P, Cox JS. e type I IFN response
to infection with Mycobacterium tuberculosis requires ESX-1-mediated
secretion and contributes to pathogenesis. J Immunol (2007) 178(5):3143–52.
doi:10.4049/jimmunol.178.5.3143
23. Dorhoi A, Nouailles G, Jorg S, Hagens K, Heinemann E, Pradl L, et al.
Activation of the NLRP3 inammasome by Mycobacterium tuberculosis is
uncoupled from susceptibility to active tuberculosis. Eur J Immunol (2012)
42(2):374–84. doi:10.1002/eji.201141548
24. Wassermann R, Gulen MF, Sala C, Perin SG, Lou Y, Rybniker J, et al.
Mycobacterium tuberculosis dierentially activates cGAS- and inam-
masome-dependent intracellular immune responses through ESX-1. Cell Host
Microbe (2015) 17(6):799–810. doi:10.1016/j.chom.2015.05.003
25. Watson RO, Manzanillo PS, Cox JS. Extracellular M. tuberculosis DNA targets
bacteria for autophagy by activating the host DNA-sensing pathway. Cell
(2012) 150(4):803–15. doi:10.1016/j.cell.2012.06.040
26. Schaible UE, Winau F, Sieling PA, Fischer K, Collins HL, Hagens K, et al.
Apoptosis facilitates antigen presentation to Tlymphocytes through MHC-I
and CD1 in tuberculosis. Nat Med (2003) 9(8):1039–46. doi:10.1038/nm906
27. Winau F, Weber S, Sad S, de Diego J, Hoops SL, Breiden B, etal. Apoptotic
vesicles crossprime CD8 Tcells and protect against tuberculosis. Immunity
(2006) 24(1):105–17. doi:10.1016/j.immuni.2005.12.001
28. Tzelepis F, Verway M, Daoud J, Gillard J, Hassani-Ardakani K, Dunn J,
et al. Annexin1 regulates DC eerocytosis and cross-presentation during
Mycobacterium tuberculosis infection. J Clin Invest (2015) 125(2):752–68.
doi:10.1172/JCI77014
29. Saiga H, Nieuwenhuizen N, Gengenbacher M, Koehler AB, Schuerer S,
Moura-Alves P, et al. e recombinant BCG ΔureC::hly vaccine targets the
AIM2 inammasome to induce autophagy and inammation. J Infect Dis
(2015) 211(11):1831–41. doi:10.1093/infdis/jiu675
30. Pedrazzini T, Hug K, Louis JA. Importance of L3T4+ and Lyt-2+ cells in the
immunologic control of infection with Mycobacterium bov is strain bacillus
Calmette-Guerin in mice. Assessment by elimination of Tcell subsets invivo.
J Immunol (1987) 139(6):2032–7.

8
Nieuwenhuizen et al. The rBCG Vaccine VPM1002
Frontiers in Immunology | www.frontiersin.org September 2017 | Volume 8 | Article 1147
31. Sendide K, Deghmane AE, Reyrat JM, Talal A, Hmama Z. Mycobacterium
bovis BCG urease attenuates major histocompatibility complex class II
tracking to the macrophage cell surface. Infect Immun (2004) 72(7):4200–9.
doi:10.1128/IAI.72.7.4200-4209.2004
32. Wang Y, Curry HM, Zwilling BS, Lafuse WP. Mycobacteria inhibition of IFN-
gamma induced HLA-DR gene expression by up-regulating histone deacetyl-
ation at the promoter region in human THP-1 monocytic cells. J Immunol
(2005) 174(9):5687–94. doi:10.4049/jimmunol.174.9.5687
33. Fulton SA, Reba SM, Pai RK, Pennini M, Torres M, Harding CV, et al.
Inhibition of major histocompatibility complex II expression and antigen pro-
cessing in murine alveolar macrophages by Mycobacterium bov is BCG and the
19-kilodalton mycobacterial lipoprotein. Infect Im mun (2004) 72(4):2101–10.
doi:10.1128/IAI.72.4.2101-2110.2004
34. Hess J, Miko D, Catic A, Lehmensiek V, Russell DG, Kaufmann SH.
Mycobacterium bovis bacille Calmette-Guerin strains secreting listeriolysin
of Listeria monocytogenes. Proc Natl Acad Sci U S A (1998) 95(9):5299–304.
doi:10.1073/pnas.95.9.5299
35. Grode L, Seiler P, Baumann S, Hess J, Brinkmann V, Nasser Eddine A, et al.
Increased vaccine ecacy against tuberculosis of recombinant Mycobacterium
bovis bacille Calmette-Guerin mutants that secrete listeriolysin. J Clin Invest
(2005) 115(9):2472–9. doi:10.1172/JCI24617
36. Divangahi M, Desjardins D, Nunes-Alves C, Remold HG, Behar SM.
Eicosanoid pathways regulate adaptive immunity to Mycobacterium tubercu-
losis. Nat Immunol (2010) 11(8):751–8. doi:10.1038/ni.1904
37. Podobnik M, Marchioretto M, Zanetti M, Bavdek A, Kisovec M, Cajnko MM,
etal. Plasticity of listeriolysin O pores and its regulation by pH and unique
histidine [corrected]. Sci Rep (2015) 5:9623. doi:10.1038/srep09623
38. Conradt P, Hess J, Kaufmann SH. Cytolytic T-cell responses to human
dendritic cells and macrophages infected with Mycobacterium bovis BCG and
recombinant BCG secreting listeriolysin. Microbes Infect (1999) 1(10):753–64.
doi:10.1016/S1286-4579(99)80077-X
39. Velmurugan K, Grode L, Chang R, Fitzpatrick M, Laddy D, Hokey D, etal.
Nonclinical development of BCG replacement vaccine candidates. Vaccines
(Basel) (2013) 1(2):120–38. doi:10.3390/vaccines1020120
40. Leist M, Jaattela M. Triggering of apoptosis by cathepsins. Cell Death Dier
(2001) 8(4):324–6. doi:10.1038/sj.cdd.4400859
41. Farinacci M, Weber S, Kaufmann SH. e recombinant tuberculosis vaccine
rBCG ΔureC::hly(+) induces apoptotic vesicles for improved priming of
CD4(+) and CD8(+) Tcells. Vaccine (2012) 30(52):7608–14. doi:10.1016/j.
vaccine.2012.10.031
42. Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: mechanistic
description of dead and dying eukaryotic cells. Infect Immun (2005)
73(4):1907–16. doi:10.1128/IAI.73.4.1907-1916.2005
43. Martinon F, Mayor A, Tschopp J. e inammasomes: guardians of
the body. Annu Rev Immunol (2009) 27:229–65. doi:10.1146/annurev.
immunol.021908.132715
44. Fernandes-Alnemri T, Yu JW, Datta P, Wu J, Alnemri ES. AIM2 activates the
inammasome and cell death in response to cytoplasmic DNA. Nature (2009)
458(7237):509–13. doi:10.1038/nature07710
45. Hornung V, Ablasser A, Charrel-Dennis M, Bauernfeind F, Horvath G,
Carey DR, etal. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-acti-
vating inammasome with ASC. Nature (2009) 458(7237):514–8. doi:10.1038/
nature07725
46. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell (2008)
132(1):27–42. doi:10.1016/j.cell.2007.12.018
47. Jagannath C, Lindsey DR, Dhandayuthapani S, Xu Y, Hunter RL Jr, Eissa NT.
Autophagy enhances the ecacy of BCG vaccine by increasing peptide pre-
sentation in mouse dendritic cells. Nat Med (2009) 15(3):267–76. doi:10.1038/
nm.1928
48. Deretic V, Saitoh T, Akira S. Autophagy in infection, inammation and immu-
nity. Nat Rev Immunol (2013) 13(10):722–37. doi:10.1038/nri3532
49. Cardenal-Munoz E, Arafah S, Lopez-Jimenez AT, Kicka S, Falaise A,
Bach F, etal. Mycobacterium marinum antagonistically induces an autophagic
response while repressing the autophagic ux in a TORC1- and ESX-1-
dependent manner. PLoS Pathog (2017) 13(4):e1006344. doi:10.1371/journal.
ppat.1006344
50. Shibutani ST, Saitoh T, Nowag H, Munz C, Yoshimori T. Autophagy and
autophagy-related proteins in the immune system. Nat Immunol (2015)
16(10):1014–24. doi:10.1038/ni.3273
51. Kravets E, Degrandi D, Ma Q, Peulen TO, Klumpers V, Felekyan S, etal.
Guanylate binding proteins directly attack Toxoplasma gondii via supramo-
lecular complexes. Elife (2016) 5:e11479. doi:10.7554/eLife.11479
52. Meunier E, Dick MS, Dreier RF, Schurmann N, Kenzelmann Broz D,
Warming S, etal. Caspase-11 activation requires lysis of pathogen-contain-
ing vacuoles by IFN-induced GTPases. Nature (2014) 509(7500):366–70.
doi:10.1038/nature13157
53. Kim BH, Chee JD, Bradeld CJ, Park ES, Kumar P, MacMicking JD. Interferon-
induced guanylate-binding proteins in inammasome activation and host
defense. Nat Immunol (2016) 17(5):481–9. doi:10.1038/ni.3440
54. Man SM, Place DE, Kuriakose T, Kanneganti TD. Interferon-inducible
guanylate-binding proteins at the interface of cell-autonomous immunity and
inammasome activation. J Leukoc Biol (2017) 101(1):143–50. doi:10.1189/
jlb.4MR0516-223R
55. Sayes F, Pawlik A, Frigui W, Groschel MI, Crommelynck S, Fayolle C, etal.
CD4+ T cells recognizing PE/PPE antigens directly or via cross reactivity
are protective against pulmonary Mycobacterium tuberculosis infection. PLoS
Pathog (2016) 12(7):e1005770. doi:10.1371/journal.ppat.1005770
56. Groschel MI, Sayes F, Shin SJ, Frigui W, Pawlik A, Orgeur M, etal. Recombinant
BCG expressing ESX-1 of Mycobacterium marinum combines low virulence
with cytosolic immune signaling and improved TB protection. Cell Rep (2017)
18(11):2752–65. doi:10.1016/j.celrep.2017.02.057
57. Kupz A, Zedler U, Staber M, Perdomo C, Dorhoi A, Brosch R, etal. ESAT-
6-dependent cytosolic pattern recognition drives noncognate tuberculosis
control invivo. J Clin Invest (2016) 126(6):2109–22. doi:10.1172/JCI84978
58. Desel C, Dorhoi A, Bandermann S, Grode L, Eisele B, Kaufmann SH.
Recombinant BCG DeltaureC hly+ induces superior protection over parental
BCG by stimulating a balanced combination of type 1 and type 17 cytokine
responses. J Infect Dis (2011) 204(10):1573–84. doi:10.1093/infdis/jir592
59. Vogelzang A, Perdomo C, Zedler U, Kuhlmann S, Hurwitz R, Gengenbacher M,
et al. Central memory CD4+ T cells are responsible for the recombinant
bacillus Calmette-Guerin ΔureC::hly vaccine’s superior protection against
tuberculosis. J Infect Dis (2014) 210(12):1928–37. doi:10.1093/infdis/jiu347
60. Gengenbacher M, Kaiser P, Schuerer S, Lazar D, Kaufmann SH. Post-exposure
vaccination with the vaccine candidate bacillus Calmette-Guerin ΔureC::hly
induces superior protection in a mouse model of subclinical tuberculosis.
Microbes Infect (2016) 18(5):364–8. doi:10.1016/j.micinf.2016.03.005
61. Gengenbacher M, Nieuwenhuizen N, Vogelzang A, Liu H, Kaiser P,
Schuerer S, etal. Deletion of nuoG from the vaccine candidate Mycobacterium
bovis BCG ΔureC::hly improves protection against tuberculosis. MBio (2016)
7(3):e679–616. doi:10.1128/mBio.00679-16
62. Griths KL, Ahmed M, Das S, Gopal R, Horne W, Connell TD, et al.
Targeting dendritic cells to accelerate T-cell activation overcomes a bottleneck
in tuberculosis vaccine ecacy. Nat Commun (2016) 7:13894. doi:10.1038/
ncomms13894
63. Chevalier N, Jarrossay D, Ho E, Avery DT, Ma CS, Yu D, et al. CXCR5
expressing human central memory CD4 T cells and their relevance for
humoral immune responses. J Immunol (2011) 186(10):5556–68. doi:10.4049/
jimmunol.1002828
64. Knudsen NP, Olsen A, Buonsanti C, Follmann F, Zhang Y, Coler RN, etal.
Dierent human vaccine adjuvants promote distinct antigen-independent
immunological signatures tailored to dierent pathogens. Sci Rep (2016)
6:19570. doi:10.1038/srep19570
65. Nandakumar S, Kannanganat S, Posey JE, Amara RR, Sable SB. Attrition
of T-cell functions and simultaneous upregulation of inhibitory markers
correspond with the waning of BCG-induced protection against tuberculosis
in mice. PLoS One (2014) 9(11):e113951. doi:10.1371/journal.pone.0113951
66. Slight SR, Rangel-Moreno J, Gopal R, Lin Y, Fallert Junecko BA, Mehra S, etal.
CXCR5(+) T helper cells mediate protective immunity against tuberculosis.
J Clin Invest (2013) 123(2):712–26. doi:10.1172/JCI65728
67. Li H, Wang XX, Wang B, Fu L, Liu G, Lu Y, et al. Latently and uninfected
healthcare workers exposed to TB make protective antibodies against
Mycobacterium tuberculosis. Proc Natl Acad Sci U S A (2017) 114(19):5023–8.
doi:10.1073/pnas.1611776114
68. Lu LL, Chung AW, Rosebrock TR, Ghebremichael M, Yu WH, Grace PS, etal.
A functional role for antibodies in tuberculosis. Cell (2016) 167(2):433–43.
e14. doi:10.1016/j.cell.2016.08.072
69. Zimmermann N, ormann V, Hu B, Kohler AB, Imai-Matsushima A,
Locht C, et al. Human isotype-dependent inhibitory antibody responses

9
Nieuwenhuizen et al. The rBCG Vaccine VPM1002
Frontiers in Immunology | www.frontiersin.org September 2017 | Volume 8 | Article 1147
against Mycobacterium tuberculosis. EMBO Mol Med (2016) 8(11):1325–39.
doi:10.15252/emmm.201606330
70. Prados-Rosales R, Carreno L, Cheng T, Blanc C, Weinrick B, Malek A, etal.
Enhanced control of Mycobacterium tuberculosis extrapulmonary dissemina-
tion in mice by an arabinomannan-protein conjugate vaccine. PLoS Pathog
(2017) 13(3):e1006250. doi:10.1371/journal.ppat.1006250
71. Achkar JM, Chan J, Casadevall A. Bcells and antibodies in the defense against
Mycobacterium tuberculosis infection. Immunol Rev (2015) 264(1):167–81.
doi:10.1111/imr.12276
72. Seder RA, Darrah PA, Roederer M. T-cell quality in memory and protection:
implications for vaccine design. Nat Rev Immunol (2008) 8(4):247–58.
doi:10.1038/nri2274
73. Wilkinson KA, Wilkinson RJ. Polyfunctional Tcells in human tuberculosis.
Eur J Immunol (2010) 40(8):2139–42. doi:10.1002/eji.201040731
74. Lindenstrom T, Agger EM, Korsholm KS, Darrah PA, Aagaard C, Seder RA,
etal. Tuberculosis subunit vaccination provides long-term protective immu-
nity characterized by multifunctional CD4 memory Tcells. J Immunol (2009)
182(12):8047–55. doi:10.4049/jimmunol.0801592
75. Loxton AG, Knaul JK, Grode L, Gutschmidt A, Meller C, Eisele B, etal. Safety
and immunogenicity of the recombinant Mycobacterium bovis BCG vaccine
VPM1002 in HIV-unexposed newborn infants in South Africa. Clin Vaccine
Immunol (2017) 24(2):e439–516. doi:10.1128/CVI.00439-16
76. Mazzola TN, da Silva MT, Abramczuk BM, Moreno YM, Lima SC,
Zorzeto TQ, et al. Impaired bacillus Calmette-Guerin cellular immune
response in HIV-exposed, uninfected infants. AIDS (2011) 25(17):2079–87.
doi:10.1097/QAD.0b013e32834bba0a
77. Antoni S, Ferlay J, Soerjomataram I, Znaor A, Jemal A, Bray F. Bladder cancer
incidence and mortality: a global overview and recent trends. Eur Urol (2017)
71(1):96–108. doi:10.1016/j.eururo.2016.06.010
78. Sanli O, Dobruch J, Knowles MA, Burger M, Alemozaar M, Nielsen ME, etal.
Bladder cancer. Nat Rev Dis Primers (2017) 3:17022. doi:10.1038/nrdp.2017.22
79. Sexton WJ, Wiegand LR, Correa JJ, Politis C, Dickinson SI, Kang LC. Bladder
cancer: a review of non-muscle invasive disease. Cancer Control (2010)
17(4):256–68. doi:10.1177/107327481001700406
80. Fuge O, Vasdev N, Allchorne P, Green JS. Immunotherapy for bladder cancer.
Res Rep Urol (2015) 7:65–79. doi:10.2147/RRU.S63447
81. Babjuk M, Bohle A, Burger M, Capoun O, Cohen D, Comperat EM, etal.
EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder:
update 2016. Eur Urol (2017) 71(3):447–61. doi:10.1016/j.eururo.2016.05.041
82. Zheng YQ, Naguib YW, Dong Y, Shi YC, Bou S, Cui Z. Applications of
bacillus Calmette-Guerin and recombinant bacillus Calmette-Guerin in
vaccine development and tumor immunotherapy. Expert Rev Vaccines (2015)
14(9):1255–75.
83. Mostad AH, Palou Redorta J, Sylvester R, Witjes JA. erapeutic options
in high-risk non-muscle-invasive bladder cancer during the current world-
wide shortage of bacille Calmette-Guerin. Eur Urol (2015) 67(3):359–60.
doi:10.1016/j.eururo.2014.11.031
84. Chou R, Selph S, Buckley DI, Fu R, Grin JC, Grusing S, etal. Intravesical
therapy for the treatment of nonmuscle invasive bladder cancer: a systematic
review and meta-analysis. J Urol (2017) 197(5):1189–99. doi:10.1016/j.
juro.2016.12.090
85. Xie J, Codd C, Mo K, He Y. Dierential adverse event proles associated with
BCG as a preventive tuberculosis vaccine or therapeutic bladder cancer vaccine
identied by comparative ontology-based VAERS and literature meta-analy-
sis. PLoS One (2016) 11(10):e0164792. doi:10.1371/journal.pone.0164792
86. Gonzalez-Del Vecchio M, Ruiz-Serrano MJ, Gijon P, Sanchez-Somolinos M,
de Egea V, Garcia de Viedma D, etal. Dierences between a probable and
proven BCG infection following intravesical instillations: 16 years experience
in a tertiary care hospital. Diagn Microbiol Infect Dis (2016) 85(3):338–43.
doi:10.1016/j.diagmicrobio.2016.04.006
87. Lukacs S, Tschobotko B, Szabo NA, Symes A. Systemic BCG-osis as a rare side
eect of intravesical BCG treatment for supercial bladder cancer. Case Rep
Urol (2013) 2013:821526. doi:10.1155/2013/821526
88. Pichler R, Gruenbacher G, Culig Z, Brunner A, Fuchs D, Fritz J, etal.
Intratumoral 2 predisposition combines with an increased 1
functional phenotype in clinical response to intravesical BCG in bladder
cancer. Cancer Immunol Immunother (2017) 66(4):427–40. doi:10.1007/
s00262-016-1945-z
89. Suttmann H, Riemensberger J, Bentien G, Schmaltz D, Stockle M, Jocham
D, etal. Neutrophil granulocytes are required for eective bacillus Calmette-
Guerin immunotherapy of bladder cancer and orchestrate local immune
responses. Cancer Res (2006) 66(16):8250–7. doi:10.1158/0008-5472.
CAN-06-1416
90. Sapre N, Corcoran NM. Modulating the immune response to bacillus
Calmette-Guerin (BCG): a novel way to increase the immunotherapeutic
eect of BCG for treatment of bladder cancer? BJU Int (2013) 112(6):852–3.
doi:10.1111/bju.12261
91. Ponticiello A, Perna F, Maione S, Stradolini M, Testa G, Terrazzano G, etal.
Analysis of local T lymphocyte subsets upon stimulation with intravesical
BCG: a model to study tuberculosis immunity. Respir Med (2004) 98(6):509–
14. doi:10.1016/j.rmed.2003.12.003
92. Biot C, Rentsch CA, Gsponer JR, Birkhauser FD, Jusforgues-Saklani H,
Lemaitre F, et al. Preexisting BCG-specic T cells improve intravesical
immunotherapy for bladder cancer. Sci Transl Med (2012) 4(137):137ra72.
doi:10.1126/scitranslmed.3003586
93. Rentsch CA, Birkhauser FD, Biot C, Gsponer JR, Bisiaux A, Wetterauer C,
etal. Bacillus Calmette-Guerin strain dierences have an impact on clinical
outcome in bladder cancer immunotherapy. Eur Urol (2014) 66(4):677–88.
doi:10.1016/j.eururo.2014.02.061
94. Bahria-Sediki IB, Yous N, Paul C, Chebil M, Cherif M, Zermani R, etal.
Clinical signicance of T-bet, GATA-3, and Bcl-6 transcription factor
expression in bladder carcinoma. J Transl Med (2016) 14(1):144. doi:10.1186/
s12967-016-0891-z
95. Gengenbacher M, Vogelzang A, Schuerer S, Lazar D, Kaiser P, Kaufmann SH.
Dietary pyridoxine controls ecacy of vitamin B6-auxotrophic tuberculosis
vaccine bacillus Calmette-Guerin ΔureC::hly Deltapdx1 in mice. MBio (2014)
5(3):e1262–1214. doi:10.1128/mBio.01262-14
96. de la Fuente J, Diez-Delgado I, Contreras M, Vicente J, Cabezas-Cruz A, Tobes
R, etal. Comparative genomics of eld isolates of Mycobacterium bovis and
M. caprae provides evidence for possible correlates with bacterial viability and
virulence. PLoS Negl Trop Dis (2015) 9(11):e0004232. doi:10.1371/journal.
pntd.0004232
97. Brosch R, Gordon SV, Marmiesse M, Brodin P, Buchrieser C, Eiglmeier
K, et al. A new evolutionary scenario for the Mycobacterium tuberculosis
complex. Proc Natl Acad Sci U S A (2002) 99(6):3684–9. doi:10.1073/
pnas.052548299
Conict of Interest Statement: SK and LG are co-inventors/patent holders of
BCG ΔureC::hly (VPM1002). BE and LG are working for the Vakzine Projekt
Management GmbH who is involved with the development of VPM1002. PK
and US are employed by Serum Institute of India Pvt. Ltd., which manufactures
VPM1002. NN, CR, and MC declare that they have no conicts of interest.
Copyright © 2017 Nieuwenhuizen, Kulkarni, Shaligram, Cotton, Rentsch, Eisele,
Grode and Kaufmann. is is an open-access article distributed under the terms
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