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12, and TNF-α; and increases expression of co-stimulant molecules in APCs surfaces leading to maturation
and activation of high affinity T cells [214] . After internalization by phagocytes, Lm is capable to escape from
phagolysosomes using its virulence factor called listeriolysin O (LLO) [215] . It works as a hemolysin that
perforates the phagosomal membranes of the bacterium could escape into the cytosol. Once in the cytosol,
they can replicate and secrete its antigens [216] . This mechanism makes antigen processing and presentation
+
to be via both class I and II MHC molecules [217] inducing potent specific responses from both T-CD4 and
+
T-CD8 cells [218] .
These features of Lm have been studied with genetic engineering looking for recombinant strains capable
to secrete tumor antigens [219] . They could be employed as live vectors through vaccines to potentiate
cellular response and overcome immunotolerance towards certain types of cancers [131] . This could be
achieved with insertion of plasmids encoding the tumor antigen [126] , or by their integration in the bacterial
chromosome [220] . These antigens would be expressed as chimeric proteins along with Lm virulence
factors [221] such as LLO or actin assembly inducing-protein (ActA) [222] . Lm uses ActA for motility and
intercellular propagation and its immunogenic features increase the immune response towards tumor
antigens with poor immunogenicity [223] . These experimental studies were oriented to measure efficacy
in recently developed vaccines. Among these vaccines, Lm-LLO-E7 was studied for cervical cancer
models [224] , Lm-her2-neu for metastatic breast cancer [211] , Lm-LLO-PSA for prostate cancer [225] , Lm-MPFG
for hepatocellular carcinoma [226] and LM-Kras for pancreatic ductal adenocarcinoma [227] and others; all of
them reporting suppression in growth and even regression [228] .

Lm utilization as live vector could induce systemic disease in immunocompromised individuals limiting
its use for human vaccines [221] . Different strains have been cultured with specific gene deletionsto guarantee
their safety [229-232] . Among these new strains, only XFL-7 and LmΔactA/ΔplcB have been used in clinical
trials. The XFL-7 strain was created with chromosomal deletion in its Prfa gene. This gene codes for an
activating transcription factor needed for bacterial virulence factor expression. In order to increase its
expression, a complementation of a multicopy plasmid with a heterolog gene was introduced [231] . The
LmΔactA/ΔplcB strain was made with a deletion of its virulence genes ActA and inlB-used for surface
proteins codification that favors cell invasion-to prevent capture from non-phagocytic cells and reduce
hepatic damage [232] .

The first clinical trial to assess safety with Lm administration in cancer patients utilized attenuated
strains as vaccines, specially Lm-LLO-E7 [126] . The latter was made from XLF-7 strains to express E7
oncoantigen from human papilloma virus serotype 16 (HPV16). This vaccine was also designed to treat
[15]
cervical cancer , and other tumors induced by HPV16 such as oropharyngeal cancer [224] . In this open,
nonrandomized, uncontrolled study, Maciag et al. [185] assessed safety and viability of Lm-LLO-E7 via
9
10
9
intravenous administration with intervals of 21 days. Doses of 1 × 10 , 3.3 × 10 or 1 × 10 Colony-Forming
Units (CFU) were administered to 15 patients with invasive cervical carcinoma in advanced stages and
refractory to conventional therapy. Despite the fact that all the patients presented systemic adverse effects
in the study (fever, vomit, headache, muscle aches, tachycardia, hypotension, anemia) most of them were
[15]
alleviated during the first 12-h post dose, responding to symptomatic treatment whenever necessary .

Safety of Lm-LLO-E7 administration in humans is still under study with insertion of plasmid encoded
resistance to chloramphenicol required for bacterial survival in vivo [233] . Phase II clinical trials to assess
efficacy and safety in patients with oropharyngeal cancer (NCT01598792) were suspended after a patient
developed systemic listeriosis following vaccination [234] . This shows the need for a new attenuation,
especially for their use on immunocompromised patients.