5th Assia Pacific Global Summit and Expo on VACCINES AND VACCINATION. Brisbane, Australia, 27-July, 2015 (12:05 -12: 30 pm)

A dendritic cell targeted vaccine loaded with A dendritic cell targeted vaccine loaded with a GAPDH peptide confers wide protection to a GAPDH peptide confers wide protection to listeriosis in susceptible and resistant mice.listeriosis in susceptible and resistant mice.

Carmen Álvarez DomínguezInstituto de Investigación Marqués de Valdecilla-IDIVAL.

Santander. Spain

Filum: FirmicutesType: BacilliOrder: BacillalesFamily: Listeriaceae Genus : ListeriaSpecie : Listeria monocytogenes

Listeria monocytogenes:

Features:•Bacterium gram-positive•Rod•Flagella (motility)•Intracellular facultative•13 serotypes (95% isolates in food-related outbreaks)

o 1/2ao 4b (80%)

Localization:•Ubiquitous: silage, vegetables, water, animal haeces & food•Saprophyte

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

Listeria contaminated

food

Bladder

Lymph nodes

blood

Liver

Spleen

Brain

Foetus

Placenta

Human listeriosis:

• Infectious diseases:

(meningitis, septicemia, encephalitis, abortions, pre-term deliveries)

• Transmission: (food, rare by contact, mother-fetus)

• Tropism: placenta-fetus, CNS

• Treatment: antibiotics (difficult access, doses, schedule)

• Cells: monocytes-macrophages, epithelial, endothelial, dendritic cells.

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

GAPDH

Cossart P. & Toledo-Arana A. Microbes Infect. (2008). 10(9):1041-50

Htp

∆LLO/∆ActA-attenuated∆GAPDH-lethal

Listeria cell cycle & virulence factors:

Immune system action Infection

① Cells Innate immunity:Phagocytes: MØ, DC, PMNNK

② Cells adaptive immunity:B cells: antibodiesT cells: CD4, CD8

- Natural anti-Listeria immunity (human microbiota)

1. Listeriosis cases has increased 10X in last 8 years 1-3 cases/year per hospital (low) 13-14 cases (medium)

o Similar incidence in pregnant women/neonateso Higher incidence in elderly/immuno-compromise (autoimmune, liver transplants,

oncologic)

Causes:o Change in diet prepared and long-storage food (Listeria grows -20ºC)o Change in bacteria/immunity 50X virulence strains (20 years ago)o New biological treatments anti-TNF/autoimmune patientso Number of liver transplanted patients increases 100X in last 5 years

2. No vaccine available for Listeria (~status than other intracellular bacteria)3. Attenuated bacteria do not confer protection4. Requirement of LLO to induce immunity & protection

• Listeria-based vaccines are available for cancer patients requirement vaccine

Vaccine status in listeriosis & epidemiology:(Advaxis Lm-LLO-E7)

1. Risk groupsa. Low risk healthy population (all ages)b. High risk-1 pregnant women/fetus, elderlyc. High risk-2 immunecompromise patients (cancer, liver transplants,

autoimmune)

2. Vectorsa. Cellular vectors (DC, MØs)b. Vectors targeted to DC

3. Antigens (epitopes)• LLO (listeriolysin O) (LLO91-99/LLO296-304/LLO189-201)• GAPDH (glyceraldehyde-3-phosphate-dehydrogenase)

(GAPDH1-22)

Development of a vaccine for listeriosis:

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

Non-toxic Without live pathogens Effective Ag-specific response Powerful innate immune response

Cellular vectors: Dendritic cells (*DC) and macrophages (MØ) Vectors targeted to DC (Nanoparticles/GNP)

2. Vectors Safe & induce cellular immune response

Main features:

Putative vectors:

CELL LYSATES

PREPARE VACCINE VECTOR WITH DENDRITIC CELLS

Listeria infect DCs & localize in MIIC compartments >>> expression of markers- DC maturationLLO responsable for survival & >>maturation markers in DC

DC = VACCINE VECTOR

Ag processing/LLO

CD11c+F4/80+MHC-II+CD11blow

*GAPDH

GAPDH

Saprophyte intracellular life

① Virulence factors in Ag-processing route: MHC-I & MHC-II LLO & GAPDH

Factor Stage Diana celular Inmune response

InlA 1 E-cadherina ?

InlB 1 gC1qR/MetR ?

p60 1 ? MHC-I/II

*ActA 1 HSPG-R ?

Profil. 3 Arp2/3 MHC-I

*GAPDH 2 Rab5a MHC-I/II

PI-PLC 2f enlaces PI/GPI ?

*LLO 2f Ctsd/MAPK MHC-I/II

PC-PLC 4 fosf.(PC,PE,PS) ?

Ag & peptide MHC Sequence

LLO 91-99 I GYKDGNEYI

LLO 215-234 IISQLIAKFGTAFKAVNNSLNV

LLO 354-371 II DEVQIIDGLNGDLRDILK

GAPDH 1-22 I/II MTVKVGINGFGRIGRLAFRRIQ

p60 217-225 I KYGVSVQDI

P60 449-457 I IYVGNGQMI

p60 301-312 II EAAKPAPAPSTN

Mpl 84-92 I GYLTDNDEI

ActA I ?

(Endosomes/Phagosomes)

3. Antigens (epitopes) induce CD4 & CD8 immunity

LLO1-491

(Traffic 6: 252-265, 2005; Traffic 9(3): 325-337, 2008; Mol Microbiol 72:668-682, 2008;

GAPDH1-22

3. Antigens (epitopes) LLO: old virulence factor GAPDH: new virulence factor

MHC-II/GAPDHRab5a/GAPDH

- Ag processing compartments(phagosomes)- Induce CD4+ & CD8+

immunityPatent: P200602175(3)

BM-DC(+GM-CSF) BM-DC(+10µg/ml LLO91-99 orLLO189-201 or GAPDH1-22)

7 d(2X)

i.p

3 d/i.p

DC-GAPDH1-22 = protection but >>Th1 response than DC-LLO91-99 vaccine

CD4+ & CD8+ response; while DC-LLO91-99 only CD8+ MØ vaccines were toxic discarded their use

Design 1: Low risk fellows (all ages)- DC-LLO91-99 & DC-GAPDH1-22

(Front. Cell. Infect. Microbiol. 4, 22: 1-11. doi:10.3389/ fcimb.2014.00022. eCollection 2014. )

listeriosis

Design 2: Low risk fellows (all ages)- Nanovaccine/adjuvant (Advax) • Dr. S.Penadés (CIC-biomaGUNE,

Donosti)

• Dr. M. Marradi (CIC-biomaGUNE)

• Dr. N. Petrovosky (Flinders U. Adelaide, Australia) (ADVAXTM)

*GNP-LLO91-99PROTECTION

VAC: NV:

CF

U x

107 SPLEENS

97% 96% 83%

- splenomegalia- granulomatosis

Rodriguez-Del Rio et al., 2015. Vaccine . 33,12: 1465-73; Calderon-Gonzalez et al., 2015. Hum Vac Immunother (in press).

- elderly- *neonates

+ Adv

ax

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

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

Conclusions with vaccines for low risk group:

What about a vaccine for high risk group?

A. Binding predictions to MHC-I/MHC-II compare to LLO:• LLO91-99/Kb 67.5 (low risk group)-MHC-I• GAPDH5-15/Kb 1.6• LLO19O-201/IAb 75.1 (low risk group)-MHC-II• GAPDH8-22/IAb 74.95• GADPH4-18/IAb 69.15

B. GAPDH5-15 loopC. GADPH8-22 a-helix

Epitopes of strong binding to MHC-I & MHC-II:•LLO190-201 (only to MHC-II)•LLO91-99 (intermediate binder to MHC-I)•GAPDH1-15

•GAPDH1-22

BIOINFORMATIC PREDICTIONS OF LLO EPITOPES IN LOW RISK GROUP

(Front. Cell. Infect. Microbiol. 4, 22: 1-11. doi:10.3389/ fcimb.2014.00022. eCollection 2014. )

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

Design 3: High risk fellows (immunosuppressed patients)-DC vaccines

Method: Epitopes for DC-vaccines in listeriosis = Bioinformatics/T cell assay+Advax

DC-activation in vitro DC-inoculation/DTH assay

s.c DTH humans

1.- Protection: peptides binding to MHC-I: DC-GAPDH1-22/1-15>DC-LLO91-99>DC-LLO296-304

2.- Rule for DC-vaccines/listeriosis: - 4 intermediate binders to MHC-I+1 weak binder to MHC-II

3.- Vaccine for high risk group: DC-GAPDH1-15, DC-GAPDH1-22

- Cancer, autoimmne patients- Pregnant women fetus

Design 3: High risk groups (immunosuppressed patients)-DC-vaccines

Design 3: High risk groups (immunosuppressed patients)-DC-vaccines

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

T cell response B cell response

Spleenmarkers

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

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

Conclusions with vaccines for high risk group:

DC-GAPDH1-15 & DC-GAPDH1-22 high & low risk groups

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

FINAL CONCLUSIONS AND PERSPECTIVES:

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

-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)

Grants: SAF2012-34203, SAF2009-08695, SAF2006-08968 & API2010/03/SAF2009-08695, AIP2012-SAF2012-34203

- 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)

EXTERNAL COLLABORATORS:

MEMBERS OF THE GROUP: