Adjuvant-free immunization with infective filarial larvae as lymphatic homing antigen carriers

Controlled infection with intestinal nematodes has therapeutic potential for preventing the symptoms of allergic and autoimmune diseases. Here, we engineered larvae of the filarial nematode Litomosoides sigmodontis as a vaccine strategy to induce adaptive immunity against a foreign, crosslinked protein, chicken egg ovalbumin (OVA), in the absence of an external adjuvant. The acylation of filarial proteins with fluorescent probes or biotin was not immediately detrimental to larval movement and survival, which died 3 to 5 days later. At least some of the labeled and skin-inoculated filariae migrated through lymphatic vessels to draining lymph nodes. The immunization potential of OVA-biotin-filariae was compared to that of an OVA-bound nanoparticulate carrier co-delivered with a CpG adjuvant in a typical vaccination scheme. Production of IFN gamma and TNF alpha by restimulated CD4+ cells but not CD8+ confirmed the specific ability of filariae to stimulate CD4(+) T cells. This alternative method of immunization exploits the intrinsic adjuvancy of the attenuated nematode carrier and has the potential to shift the vaccination immune response towards cellular immunity.

New immunomodulatory roles of lymphatic endothelium and implications for immunotherapy

Efthymia Vokali

The lymphatic system serves a critical role in fluid homeostasis, lipid metabolism and immune surveillance. The growing appreciation of its implication in various diseases challenges the conventional view of lymphatics as a passive transport system. Traditionally, the lymphatic endothelium has been perceived as a structural scaffold with certain immunological functions but no active involvement in immunomodulation. In the lymph nodes (LNs), the main sites of immune regulation in the periphery, lymphatic endothelial cells (LECs) come to close contact with immune cells, suggesting potential interactions. Indeed, LECs have been recently shown to suppress dendritic cell maturation and present peripheral tissue and tumor antigen for CD8+ T cell deletion. While LECs have only begun to be acknowledged as active regulators of immunity, their function and relative contribution in shaping immune responses is as yet poorly understood. This thesis aimed to elucidate the direct role of LECs in the induction of CD8+ T cell immunity and tolerance. First, we demonstrated that murine LECs can actively scavenge and cross-present exogenous antigen to cognate CD8+ T cells under non-inflamed conditions. By utilizing an in vitro coculture system and the model antigen ovalbumin, we investigated the antigen-specific interactions between LECs and CD8+ T cells. LEC-educated CD8+ T cells proliferated, exhibiting an activated phenotype, however, they displayed early-generation apoptosis and failed to produce effector cytokines. Our findings establish LECs as antigen-presenting cells and suggest that they may assist in the maintenance of peripheral tolerance during homeostasis. The particular differentiation state of LEC-educated CD8+ T cells prompted us to investigate whether they are terminally tolerized or they could escape the dysfunctional state. We demonstrated that LEC-educated CD8+ T cells adopted a distinct phenotype with central memory-like characteristics and shared multiple functional properties with memory cells. Upon antigen re-encounter, LEC-educated CD8+ T cells mounted proliferative responses and generated cytotoxic effector cells. More importantly, they participated in anti-infectious immunity while preserving a secondary-memory persistent population. Our findings reveal a unique differentiation state of antigen-experienced CD8+ T cells, generated under steady-state conditions, which remain inactive but can be functionally reactivated upon antigenic inflammatory challenge. This previously unanticipated feature of LECs triggered questions for their antigen-presenting function in an inflammatory setting. We asked whether the previously observed extensive proliferation of LECs in the LN during inflammation might directly influence the induction of immunity. We employed an anti-VEGFR3 blocking antibody to inhibit LEC proliferation and thus, reduce the number of LECs following vaccine immunization. Alternatively, we generated the Prox1-Cre-DTR mouse model, allowing for specific ablation of LECs following administration of diphtheria toxin in vivo. Our findings advance our perception of the relative contribution of LECs in the establishment of adaptive immunity. This thesis elucidates the multifaceted immunological role of LECs and strongly suggests the importance of harnessing their immunomodulatory function to enhance current vaccines and immunotherapeutic strategies. Looking forward, our work will contribute to future advances in the clinic.

EPFL2016

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

Impaired Humoral Immunity and Tolerance in K14-VEGFR-3-Ig Mice That Lack Dermal Lymphatic Drainage

Miriella Carmela Pasquier, Joseph Rutkowski, Melody Swartz, Susan Thomas

Lymphatic vessels transport interstitial fluid, soluble Ag, and immune cells from peripheral tissues to lymph nodes (LNs), yet the contribution of peripheral lymphatic drainage to adaptive immunity remains poorly understood. We examined immune responses to dermal vaccination and contact hypersensitivity (CHS) challenge in K14-VEGFR-3-Ig mice, which lack dermal lymphatic capillaries and experience markedly depressed transport of solutes and dendritic cells from the skin to draining LNs. In response to dermal immunization, K14-VEGFR-3-Ig mice produced lower Ab titers. In contrast, although delayed, T cell responses were robust after 21 d, including high levels of Ag-specific CD8(+) T cells and production of IFN-gamma, IL-4, and IL-10 upon restimulation. T cell-mediated CHS responses were strong in K14-VEGFR-3-Ig mice, but importantly, their ability to induce CHS tolerance in the skin was impaired. In addition, 1-y-old mice displayed multiple signs of autoimmunity. These data suggest that lymphatic drainage plays more important roles in regulating humoral immunity and peripheral tolerance than in effector T cell immunity. The Journal of Immunology, 2012, 189: 2181-2190.