Characterization of Immune Modulating Lipid Nanoparticles

 

Characterization of Immune Modulating Lipid Nanoparticles

  Overview

•  Vaccination & Adjuvants

•  Immune response

•  Immunological Characterization of Versamune®

Vaccine  Development  

Vaccination •  Cost effective modality for various diseases •  Induce lasting immune response •  Useful for prophylactic and therapeutic intervention

Vaccine  Development  

Vaccine Antigens •  Derived form live/attenuated pathogen potential virulence hazardous to immunocompromised •  Recombinant DNA Technology considered safer poorly immunogenic: require adjuvant

Vaccine  Development  

Adjuvant •  Substance accelerating the immune response: non immunogenic •  Depot: Alum •  Immune Stimulators: Toll-like receptor agonists •  Delivery Vehicles: Liposomes/nanoparticles •  Clinically approved list of adjuvants limited •  Alum: Aluminum hydroxide/phosphate •  AS04: Cervarix® (GSK) Alum+MPL A •  AS03: H5N1 (GSK) o/w not commercially available •  MF59: Fluad® (Novartis) o/w emulsion (squalene) •  Montanide™: w/o emulsion

Vaccine  Development  

Adjuvant Properties • 

Safe & Easily administered

• 

Elicit desired immune response

• 

Maintain integrity of antigen

Liposome  based  Nanopar5cles  as  Adjuvants  

•  Used extensively for drug delivery

•  Proven clinical safety and biocompatibility

•  Ease of manufacture and scale-up

Versamune® Nanotechnology Vector

Sterile Injectable Solution Avg. Particle Size 100-200 nm IM or Sub-Q injections First pure enantiomer formulation In Phase I HPV related neoplasia

R-DOTAP: (R)-1,2-Dioleoyloxy-3-triethylammoniumpropane chloride

Immune Response: General Overview Infection/Vaccination Innate Immunity

Adaptive Immunity T cells

MΦ, PMNL Complement

B cells

Ag presentation

CD4+, Th

Dendritic Cells

Cytokines

Inflammation

CD8+, CTL

Plasma Cell

Lyse Cells

Antibodies

Interaction of Nanoparticle with Dendritic Cells: Antigen Presentation   Dendritic Cells Phagocytose Antigen Secrete Pro-Inflammatory Cyto/Chemokines Upregulate Costimulatory molecules Present Antigen to T cells CD4 via MHC II CD8 via MHC I (cross presentation)

endosomes

ERK

Antigen:MHC complex Co-stimulatory molecules

Cytokines

 Versamune®  Effects  on  Dendri5c  Cells  

maximum was 2 days post injection (Fig. 5A). general phenomenon by liposomes. Using BMDC, theaccumulation results These data were confirmed by immunohistochemistry staining showed that only cationic liposomes (DOTAP and DOEPC) (Fig. 5B). The same data also demonstrated that more lymphocould induce the MCP-1/CCL2 release from cytes BMDC. Neutral migrated to the draining lymph nodes upon DOTAP/E7 treatment, in the 44 enlargement of the nodes. (DOPC and DOG) and negativelyW.charged (DOPS and resulting DOPG) Yan et al. / Molecular Immunology (2007) 3672–3681 3677 Fig. 3. DOTAP induces activation of ERK and p38 in BMDC. (A) Time The inhibitors of ERK and p38 pathways were also liposomes could not (Fig. 2A). course study. At day 6, BMDC were seeded in 12-well plateAlso in the shown density is a tertiary amine formulated in the DOTAP/E7 liposomes. DOTAP/E7/Uof 106 /mL/well. They were withDODAP, 75 !M DOTAP liposomes the analog ofincubated DOTAP, could not for induce the activity either,release was also attenuated in the cells treated with siRNA, 0126 and DOTAP/E7/SB203580 formed clear suspension but indicated time. The cells were harvested and subjected to Western blotting analsuggesting that the activity requires quaternary amino ysis using antibodies indicated in the figure. The same membrane wasaprobed which blocked ERK1 gene (Fig. 4B). However, even PD98059 washeaddifficult to formulate in DOTAP/E7. The expression stable with ERK2 antibody to serve as a loading DOTAP-induced activa- in DOTAP/E7/drug group in the lipid.control. LPS(B)was also included the experiments formulations had similar zeta potentials, parthough ERK1 was down-regulated, LPS-induced CCL2 release tion of ERK was negatively regulated by p38. As described in Fig. 2C, BMDC ticleCCL2 sizes and antigen loading capacities as the DOTAP/E7, as a positive control. Fig. 2A also shows that the inducwere pre-incubated with indicated inhibitors for 20 min, followed by 75 !M could not be attenuated, suggesting that LPS utilizes different DOTAP liposome for 30 min.liposomes (C) DOTAP-induced ERKaactivation tiontreatment by cationic reaches maximumindicating amount that in 24drug h, incorporation did not significantly alter the physical characteristics of thepathway vaccine. DOTAP/E7/U-0126 was mainly through PI-3 kinase and negatively regulated by p38. As described to induce CCL2 release (Fig. 4B). This data because 48 h incubation did not result in any more chemokines.signaling 3678were subcutaneously injected to the W. Yan et al. / Molecular Immunology 44 (2007) 3672–3681 in B, BMDC were pre-incubated with indicated inhibitors for 20 min, followed and DOTAP/E7/SB203580 also demonstrated that ERK1 siRNA treatment did not affect Ourliposome data treatment also show that DOTAP-induced CCL2 by 75 !M DOTAP for 30 min. mice.expression Consistentlyiswith in vitro data, DOTAP/E7 formulation there is significant ver dose-dependent (Fig. 2B). Five micromoles of DOTAP couldotherofsignaling could induce accumulation CCL2 in the pathways. draining lymph nodes larity, charge and meth and U-0126 SB-203580 CCL2 inducinduce significant amount of CCL2 from BMDC and and maxiSo reciprocally far, our regulated data demonstrated DOTAP- induced for ERK actispecific application tion (Fig. 6A). In order to determine whether ERK and p38 Time course of BMDC Treated with mum induction was reached at 45–75 !M. To identify whichvation and the downstream induction of CCL2 in antigen vitro.ratio, Wethe lipos 75uM DOTAP pathway was involved in the CCL2 induction by DOTAP inhave investigated if the above signaling mechanism plays antigenaloading role in the DOTAP Dose dependent release CCL2 from BMDC tion. From the safety p BMDC, several inhibitors specific to distinct signaling pathin the adjuvant activity of DOTAP in vivo. DOTAP/E7 lipo- and wi biocompatible ways were used. PD98059 and U-0126, specific inhibitors for 2005; O’Hagan and Si immunize the ERK pathway, almost completely abolished CCL2 pro-some vaccine formulation was used to subcutaneouslyalso been used to intr assayed duction, but surprisingly, SB203586, an inhibitor of the p38mice and the draining lymph nodes were collected and MHC class I pathway accumupathway, synergistically increased CCL2 production inducedby ELISA for CCL2. The data shows that CCL2(Chikh and Schutze-R Since by DOTAP (Fig. 2C). Our data also clearly showed that PKClated in the draining lymph nodes after immunization and cationic the lipo charged cell membran pathway and Src kinase are not involved in the CCL2 produc-maximum accumulation was 2 days post injection (Fig. 5A). and vaccine delivery ( tion induced by DOTAP, because GF109203X (PKC inhibitor)These data were confirmed by immunohistochemistry Traditionally, stainingcationic and PP2 (Src kinase inhibitor) had no inhibition effect, respecterms of activating inn (Fig. 5B). The same data also demonstrated that more lympho2001). However, we ( tively. Fig. 2C also indicates that PI-3 kinase and Gi-dependent tions) have recently sh G-protein-coupled-receptor (GPCR) were likely involved in thecytes migrated to the draining lymph nodes upon DOTAP/E7 CCL2 release upon DOTAP treatment, because wortmannin andtreatment, resulting in the enlargement of the nodes. protein of the Human 3. DOTAP induces activation of ERK and p38 in BMDC. (A) Time sulated in the liposom The inhibitors of ERK and p38 pathways were also pertussis toxin (PTx) had some inhibition effect, respectively. Fig. 2. CCL2 is released from BMDC upon treatment with DOTAP liposomes. (DOTAP/E7 or DOEP se study. At day 6, BMDC were seeded in 12-well plate in the density Fig. 5. DOTAP/E7 formulation induces the accumulation of CCL2 in the mice (A) Only cationic induce the CCL2liposomes. release from the BMDC. At day 6, injection, it It iswere known that some regulated by theformulated in liposomes the DOTAP/E7 DOTAP/E7/Ucutaneous 06 /mL/well. They incubated with 75chemokines !M DOTAP are liposomes the drainingfor lymph nodes. At day 0, the mice (n = 3) were s.c. immunized by BMDC were treated with 75 !M liposomes made from by structurally16different E7-positive ERK pathway (Yoo et al., 2005), and verified in our system. DOTAP/E7analformulation 0126 (100 nmoland of DOTAP and 10 !g of E7 peptide). At DOTAP/E7/SB203580 formed clear suspension but tumor cated time. The cells were harvested and subjected to Western blotting lipids for the indicated time. The supernant was assayed by ELISA as mentioned also induce expression Fig. 4. Down-regulation of ERK gene expression by siRNA approach attenuates indicated days, the mice sacrificed and the draining lymph nodes were colUsingindicated BMDC to activation of the ERK pathway, our datawere using antibodies in study the The same membrane was probed of figure. ERK attenuates PD98059 was difficult to!Lformulate DOTAP/E7. The stable in were the text. (B) DOTAPininduces CCL2 releasein from BMDC in a dose depenthe DOTAP-inducedKnockdown CCL2 release from BMDC. (A) ERK1 gene expression was lected. The draining lymph nodes either homogenized 100 ELISA and CD86 and activat showed DOTAP induced ERK phosphorylation inFBS10in PBS) minand then ERK2byantibody serve as a BMDC. loading (B) DOTAP-induced activablocked siRNA afterto 24 hthat treatment in (B)control. ERK1BMDC blockage by siRNA buffer (10% analyzed by ELISA (A) or6, immunochemically CCL2 release form Fig. 6. Inhibition ofAt ERK both attenuates accumulation in the draining dent manner. day BMDC the were treated with 75 !M DOTAP liposomes 2006). These DOTAP/E7/drug formulations had similar zeta potentials, par-findings Attenuation ofCCL2 Anti-tumor specifically attenuated DOTAP-induced CCL2 release from BMDC. DOTAP: stained by CCL2 antibody (B) lymph asfor mentioned in the text. the Original magnification: nodes and blocks the anti-tumor activity of DOTAP/E7 formulation. (A)Inhibitors and phospho-ERK (pERK) stayed at a high level for 40 min of ERK was negatively regulated by p38. As described in Fig. 2C, BMDC 24 h, and then supernant was assayed by ELISA. (C) were adjuvant, leading to th responses inhibition 75 !M, LPS: 100 ng/mL. * p < 0.05 compared with control siRNA, n = 3. ×400. sizes and antigen loading the DOTAP/E7, CCL2 the drainingwith lymphERK nodescapacities was reciprocally as regulated usedaccumulation to study thein signaling pathways involved in the DOTAP-induced CCL2 (Fig.with 3A).indicated In addition, p38 was slightly activated by!M DOTAP.ticle pre-incubated inhibitors foralso 20 min, followed by 75 In the current repo by ERK and p38 pathways. Tumor free mice (n = 5) were s.c. injected with indicating that drug incorporation did not significantly alter release from BMDC. At day 6, BMDC were pre-incubated with inhibitors for the adjuvant activity o However, of I"B and ERK subsequent TAP liposome treatmentnoforphosphorylation 30 min. (C) DOTAP-induced activationdegraDOTAP/E7 co-formulated with or without 20 !g drug (SB-203580 or U-0126). Molecular Immunology, 2007,44(15):3672-81 20 min, followed by 75 !M DOTAP liposomes treatment for 24 h. The concenwas explored as an a At day 2, the draining lymph nodes were collected, and homogenized in 100 !L mainly through PI-3 kinase and negatively regulated by p38. As described dation were detected following DOTAP incubation (data notthe physical characteristics of the vaccine. DOTAP/E7/U-0126

Induc5on  of  Cell  Signaling  and  Cytokine  Release

   

 

Versamune® a Prototypical Nanoparticle Vector

Target Dendritic Cells for Antigen Presentation Increased Co-stimulatory molecules Enhanced antigen cross presentation Activates ERK leading to Enhanced Chemokine Secretion

Enhanced T cell Activation Increased antigen specific T cells Prophylactic and Therapeutic efficacy Potentiates Humoral Responses: B cells Increased NAb titers to H1N1

  Acknowledgements

University of Kentucky Jerold Woodward, PhD Martin Ward UNC Leaf Huang, PhD