Diapositiva 1

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Listeria monocytogenes

• Features
• Bacterium gram-positive
• Rod
• Flagella (motility)
• Intracellular facultative
• 13 serotypes (95 isolates in food-related
  outbreaks)
• 1/2a
• 4b (80)
• Localization
• Ubiquitous silage, vegetables, water, animal
  haeces food
• Saprophyte

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Human listeriosis
Risk groups elderly, neonates, pregnant women,
immunocompromise patients (cancer, liver
transplants, autoimmune diseases)

• Incidence
• Low 1-3 cases/year/100.000 inhab
• Non-symptomatic infections (flu)

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Listeria cell cycle virulence factors
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- Natural anti-Listeria immunity (human
microbiota)
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Vaccine status in listeriosis epidemiology

• Listeriosis cases has increased 10X in last 8
  years
• 1-3 cases/year per hospital (low) ? 13-14 cases
  (medium)
• Similar incidence in pregnant women/neonates
• Higher incidence in elderly/immuno-compromise
  (autoimmune, liver transplants, oncologic)
• Causes
• Change in diet ? prepared and long-storage food
  (Listeria grows -20ºC)
• Change in bacteria/immunity ?50X virulence
  strains (20 years ago)
• New biological treatments ? anti-TNF/autoimmune
  patients
• Number of liver transplanted patients increases
  100X in last 5 years
• No vaccine available for Listeria (status than
  other intracellular bacteria)
• Attenuated bacteria do not confer protection
• Requirement of LLO to induce immunity
  protection
• Listeria-based vaccines are available for cancer
  patients ? requirement vaccine

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Development of a vaccine for listeriosis

• Risk groups
• Low risk ? healthy population (all ages)
• High risk-1 ? pregnant women/fetus, elderly
• High risk-2? immunecompromise patients (cancer,
  liver transplants, autoimmune)
• Vectors
• Cellular vectors (DC, MØs)
• Vectors targeted to DC
• Antigens (epitopes)
• LLO (listeriolysin O) (LLO91-99/LLO296-304/LLO189-
  201)
• GAPDH (glyceraldehyde-3-phosphate-dehydrogenase)
  (GAPDH1-22)

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1. Risk groups ? Animal models

• C57BL/6 ? resistant to listeriosis
• model for low risk group
• healthy population of all ages
• Balb/c ? high sensitive to listeriosis
• model for high risk group
• Immuno-compromise patients

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• Vectors ? Safe induce cellular immune response
• Main features

Putative vectors
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1. Antigens (epitopes) ? induce CD4 CD8 immunity

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• Antigens (epitopes) ? LLO old virulence factor
• ? GAPDH new virulence factor

- Ag processing compartments (phagosomes) -
Induce CD4 CD8 immunity
MHC-II/GAPDH Rab5a/GAPDH
Patent P200602175(3)
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Design 1 Low risk fellows (all ages)-
DC-LLO91-99 DC-GAPDH1-22
DC-GAPDH1-22 ? protection but gtgtTh1 response
than DC-LLO91-99 vaccine ? CD4 CD8
response while DC-LLO91-99 only CD8 MØ
vaccines were toxic ? discarded their use
(Front. Cell. Infect. Microbiol. 4, 22 1-11.
doi10.3389/ fcimb.2014.00022. eCollection 2014. )
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Design 2 Low risk fellows (all ages)-
Nanovaccine/adjuvant (Advax)

• elderly
• neonates

• Dr. S.Penadés (CIC-biomaGUNE, Donosti)
• Dr. M. Marradi (CIC-biomaGUNE)
• Dr. N. Petrovosky (Flinders U. Adelaide,
  Australia) (ADVAXTM)

PROTECTION
GNP-LLO91-99
VAC
- splenomegalia - granulomatosis

• GNP-LLO91-99/Advax vaccine
• Protection in C57BL/6 (low risk)
• Medium protection in Balb/c (high risk)
• Good CD4 CD8 Listeria-specific immunity

Advax
Rodriguez-Del Rio et al., 2015. Vaccine . 33,12
1465-73 Calderon-Gonzalez et al., 2015. Hum Vac
Immunother (in press).
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Conclusions with vaccines for low risk group
1.- DC vaccines non-toxic, while MØ-vaccines
toxic 2.- DC-GAPDH1-22 DC-LLO91-99 were
efficient in low risk group - disadvantage
DC-LLO91-99 show lower Th1 response, only CD8
3.- GNP-LLO91-99 vaccines are only
efficient with Adjuvant (Advax) - disadvantage
do not work in high risk group
What about a vaccine for high risk group?
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(Front. Cell. Infect. Microbiol. 4, 22 1-11.
doi10.3389/ fcimb.2014.00022. eCollection 2014. )
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BIOINFORMATIC PREDICTIONS OF GAPDH EPITOPES IN
LOW HIGH RISK GROUPS

• Epitopes binding to MHC-I or MHC-II in low and
  high risk groups
• GAPDH1-15 ? 4 epitopes to MHC-I, 1 epitopes to
  MHC-II
• GAPDH1-22 ? 6 epitopes to MHC-I, 6 epitopes to
  MHC-II
• LLO91-99 ? 1 epitope to MHC-I (low high risk)
• LLO296-304 ? 1 epitope to MHC-I (low high risk)
• LLO189-201 ? 1 epitope to MHC-II (low risk)

GAPDH1-15 GAPDH1-22
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Design 3 High risk fellows (immunosuppressed
patients)-DC vaccines
Method Epitopes for DC-vaccines in listeriosis
Bioinformatics/T cell assayAdvax
s.c DTH humans
DC-activation in vitro
DC-inoculation/DTH assay
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Design 3 High risk groups (immunosuppressed
patients)-DC-vaccines
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Design 3 High risk groups (immunosuppressed
patients)-DC-vaccines
T cell response B cell response
Anti-IgM Ab LLO91-99 GAPDH1-22
control 0.156 0.005 0.163 0.04
NV 0.517 0.02 1.021 0.02
DC-GAPDH1-22 0.712 0.02 2.011 0.05
DC-LLO91-99 0.583 0.03 1.276 0.02
Spleen markers CD19 CD4 CD8 CD11c CD86
control 19 0.5 7 0.3 8 0.2 0.5 0.02
NV 15 0.5 18 0.2 17 0.2 26 0.8
DC-GAPDH1-22 15 0.3 26 0.4 28 0.5 70 0.7
DC-LLO91-99 15 0.2 7 0.2 27 0.3 70 0.6
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Conclusions with vaccines for high risk group
1.- DC-GAPDH1-22 DC-GAPDH1-15 were efficient
in high risk group - DC-GAPDH1-22
DC-GAPDH1-15 were efficient in low risk
group 2.- DC-LLO91-99 show some efficiency in
high risk group - DC-LLO91-99 was efficient in
low risk group 3.- DC-LLO296-304 show NO
efficiency in high risk group - DC-LLO296-304
was efficient in low risk group 4.-
DC-GAPDH1-22 DC-GAPDH1-15 induce Th1 B cell
responses (IgM) - DC-LLO91-99/DC-LLO296-304 do
not induce Th1 or B cell responses
DC-GAPDH1-15 DC-GAPDH1-22 high low risk groups
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FINAL CONCLUSIONS AND PERSPECTIVES
1.- Design to protect all populations against
listeriosis - GNP-LLO91-99/Advax (Nanovaccine)
- Safe for vaccination of infants/elderly 2.-
Design for patients at high risk of
listeriosis - DC-GAPDH1-15 DC-GAPDH1-15
(DC-vaccine) - Safe for vaccination of cancer
patients - Safe for vaccination of pregnant
women/fetus (induce Th1 B cell
responses) 3.- Preparing Nanovaccine design
with GAPDH1-22 - Expectations to cover ALL
fellows
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MEMBERS OF THE GROUP

• - Participants in this study
• Elisabet Frande-Cabanes (PhD student-IDICVAL)
• Ricardo Calderon-Gonzalez (PhD student-UC/IDIVAL)
• Lidia Alaez-Alvarez (Technician-IDIVAL)
• Dra. Carmen Alvarez-Dominguez (Director-IDIVAL)

• Other members of the group
• Dra. Susana Gomez-Salces (Associate Prof-UC)
• Dra. Sonsoles Yañez-Diaz (Physician
  Dermatol-HUMV)
• Montserrat Grifat (Undergraduate Student-UL)
• Lorena Vazquez-Rioja (Technician-IDIVAL)

EXTERNAL COLLABORATORS

• E. Pareja, R.Tobes (Era7 Bioinformatics, Granada)
• N. Petrovsky (Flinders Univ/Vaxine,Adelaide,Austra
  lia)