A Cationic Micelle Complex Improves CD8+T Cell Responses in Vaccination Against Unmodified Protein Antigen

Nanoscale carrier platforms promote immune responses to vaccination by facilitating delivery of vaccine components to immunologically relevant sites. The technique is particularly valuable for subunit vaccination, in which coadministration of immunostimulatory adjuvant is known to enhance immune responses to protein antigen. The fabrication of polymer-based nanoparticle vaccines commonly requires covalent attachment of vaccine components to the carrier surface. In contrast, we here describe a cationic micelle vaccination platform in which antigen and adjuvant loading is mediated by noncovalent molecular encapsulation and electrostatic complexation. Cationic micelles were generated through self-assembly of a polyarginine-conjugated poly(ethylene glycol)-b-poly(propylene sulfide) (PEG PPS) diblock copolymer amphiphile, with or without encapsulation of monophosphoryl lipid A (MPLA), an amphiphilic experimental vaccine adjuvant. Micelle complexes were subsequently formed by complexation of ovalbumin (OVA) and CpG-B oligodeoxynucleotide (a second experimental adjuvant) to the cationic micelles. In a 35-day study in mouse, micelle mediated codelivery of OVA antigen and CpG-B enhanced cellular and humoral responses to vaccination. These outcomes were highlighted in spleen and lymph node CD8(+) T cells, with significantly increased populations of IFN gamma(+), TNF alpha(+), and polyfunctional IFN gamma(+) TNF alpha(+) cells. Elevated cytokine production is a hallmark of robust cytotoxic T lymphocyte (CTL) responses sought in next-generation vaccine technologies. Increased production of OVA-specific IgGI, IgG2c, and IgG3 also confirmed micelle enhancement of humoral responses. In a subsequent 35-day study, we explored micelle-mediated vaccination against OVA antigen coadministered with MPLA and CpG-B adjuvants. A synergistic effect of adjuvant coadministration was observed in micelle-free vaccination but not in groups immunized with micelle complexes. This outcome underlines the advantage of the micelle carrier: we achieved optimal cellular and humoral responses to vaccination by use of this nanoparticle platform with a single adjuvant. In particular, enhanced CTL responses support future development of the cationic micelle platform in experimental cancer vaccines and for vaccination against reticent viral pathogens.

Exploiting immunofunctional nanoparticles as vaccines and synthetic pathogens

Sai Reddy

The emergence of recombinant DNA technology has shifted research towards developing subunit vaccines, which use protein or peptide antigens instead of live/attenuated pathogens. Recent research has also shown that the best way to get sustained immunity is to deliver an antigen directly to antigen-presenting cells (APCs), specifically dendritic cells (DCs). However there are two difficulties to overcome in targeting DCs for use in subunit vaccines: (1) DCs are present in a low concentration in peripheral tissue (where typical vaccines are delivered) compared to secondary lymphoid organs such as lymph nodes. Thus delivering antigen to DCs in lymph nodes offers a powerful alternative for vaccine technology. (2) Activating DCs requires co-delivering an adjuvant also sometimes known as a molecular "danger signal" which matures DCs and initiates the subsequent adaptive immune response. Adjuvants are needed since most protein and peptide antigens are insufficiently immunogenic. Current danger signals being explored in experimental vaccine formulations mimic bacterial components (e.g., lipopolysaccharide (LPS), unmethylated CpG DNA) and often signal through a class of pattern recognition receptors knows as Toll-like receptors (TLRs), however limitations exist in that these often cause toxic side effects by inducing high levels pro-inflammatory cytokines. To address the need to target DCs, we used a nanoparticle delivery approach. We investigated the delivery of 20, 45, and 100 nm diameter poly(ethylene glycol)-stabilized poly(propylene sulfide) (PPS) nanoparticles to DCs in the lymph nodes. These nanoparticles consisted of a cross-linked rubbery core of PPS surrounded by a hydrophilic corona of poly(ethylene glycol). The PPS domain is capable of carrying hydrophobic drugs and degrades within oxidative environments. 20 nm particles were most readily taken up into lymphatics following interstitial injection, while both 20 and 45 nm nanoparticles showed significant retention in lymph nodes, displaying a consistent and strong presence at 24, 72, 96 and 120 h post-injection. Nanoparticles were internalized by up to 40-50% of lymph node DCs (and APCs) without the use of a targeting ligand, and the site of internalization was in the lymph nodes rather than at the injection site. Finally, an increase in nanoparticle-containing DCs (and other APCs) was seen at 96 h vs. 24 h, suggesting an infiltration of these cells to lymph nodes. Thus, PPS nanoparticles of 20-45 nm showed potential for vaccine delivery specifically targeting DCs in lymph nodes. Next we exploited our nanoparticle technology as a vaccine platform. We engineered antigen-bearing nanoparticle vaccines with two novel features: lymph node-targeting and in situ complement activation. Following intradermal injection, ultra-small nanoparticles (25 nm) were readily transported by interstitial flows into lymphatic capillaries, which leads to their accumulation in lymph nodes resident DCs. We further designed the nanoparticle surface chemistry to activate the complement cascade, thus spontaneously generating a danger signal in situ for DC activation. With ovalbumin as a model antigen conjugated to the nanoparticles, we demonstrated humoral and cellular immunity in mice. Finally we exploited nanoparticles as "synthetic pathogens" to isolate the effect of free surface thiols on complement activation and the subsequent immunological response induced. We found that surfaces with both hydroxyl and thiol groups activated complement to significantly higher levels than hydroxl groups alone. We then demonstrated that nanoparticles that induced thiol-associated complement activation were potent danger signals that were able to mature dendritic cells to express high levels of costimulatory molecules and the Th1 promoting cytokine interuleukin-12. Moreover, this dendritic cell maturation was dependent on a Toll-like receptor 4 (TLR4) signaling pathway and independent of the adaptor protein myeloid differentiation protein 88 (MyD88). Thus we uncover a potential direct link between complement and TLR4 signaling that is dependent on the specific activation of complement by thiol-expressing surfaces. In conclusion, this thesis develops a novel vaccine methodology, which is at the interface of nanotechnology and biotechnology, by harnessing lymphatic transport to target DCs and complement activation to activate adaptive immunity; we demonstrated an elegant vaccine technology platform for applications that include global health. Finally we exploit our nanoparticles as synthetic pathogens to discover fundamental mechanisms connecting the complement system and TLR signaling.

EPFL2008

Polymer micelles with pyridyl disulfide-coupled antigen travel through lymphatics and show enhanced cellular responses following immunization

Karen Dane, Jackson Eby, Sachiko Hirosue, Jeffrey Alan Hubbell, Melody Swartz

Poly(ethylene glycol)-stabilized poly(propylene sulfide) core (PEG-PPS) nanoparticles (NPs) smaller than 50 nm efficiently travel to draining lymph nodes and interact with antigen-presenting cells (APCs) to induce potent immune responses following intradermal immunization. To determine if a similar system could be developed that could be more easily and reproducibly prepared and eliminated faster in vivo, we created block copolymers of PEG-bl-PPS capable of self-assembling into 25-35 nm micelles (MCs). Biodistribution studies showed that these MCs were able to travel to draining lymph nodes, where they preferentially interacted with APCs. To couple cysteine-containing antigens to the surface of the MCs, a new polymer was synthesized with a terminal pyridyl disulfide (PDS), forming PDS-PEG-bl-PPS-benzyl. When mice were immunized in conjunction with free CpG as an adjuvant, ovalbumin-conjugated MCs (MC-Ova) generated more (2.4-fold) Ova-specific CD8(+) T cells in the blood and higher (1.7-fold) interferon-gamma levels from splenocytes upon restimulation than in mice immunized with free Ova and CpG. When comparing this MC platform to our PEG-PPS NPs with disulfide-linked Ova, no significant differences were found in the measured responses. These results indicate that PDS-functionalized MCs are efficient antigen delivery vehicles that enhance immune responses compared to immunization with free protein. (C) 2012 Acts Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Elsevier Sci Ltd2012

Nano-sized drug-loaded micelles deliver payload to lymph node immune cells and prolong allograft survival

Karen Dane, Jackson Eby, Jeffrey Alan Hubbell, Melody Swartz, Alice Anna Tomei

By delivering immunomodulatory drugs in vivo directly to lymph nodes draining an injection site, an opportunity exists to increase drug bioavailability to local immune cells. Importantly, particles smaller than 100 nm are efficiently transported through lymphatic vessels to draining lymph nodes. To investigate whether this approach could be used for local delivery of immunomodulatory drugs, amphiphilic poly (ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) block copolymers forming 50 nm micelles were used to encapsulate hydrophobic drugs. Micelle drainage was determined using fluorescent micelles and showed effective targeting of multiple immune cell subsets in lymph nodes. For functional studies of our formulations, two approaches were considered. To evaluate the efficacy of anti-inflammatory drug delivery, dendritic cell activation was shown to be prevented when mice were pretreated with micelles loaded with the glucocorticoid mometasone and then challenged with the TLR9 ligand, CpG. To evaluate whether immunosuppressive drug-loaded micelles were effective in prolonging MHC-mismatched allograft survival, BALB/c mice were treated for 14 consecutive days with drug-loaded micelles following transplantation of allogenic C57BL/6 tail skin. Micelles loaded with a mixture of rapamycin and tacrolimus prolonged allograft survival by 2-fold. Our results indicate that the drug-loaded micelle approach effectively targets the draining lymph nodes and exhibits proper immune regulation. (C) 2011 Elsevier B. V. All rights reserved.

Elsevier2011

Exploiting immunofunctional nanoparticles as vaccines and synthetic pathogens

Sai Reddy

EPFL2008