Engineering nanoparticle-based vaccines: Implications for the quality of humoral and cellular immunity

Vaccination is undoubtedly a major success in modern medicine. Yet, today there are pathogens for which no licensed or fully protective vaccines have been created. Development of vaccines based on subunit components has proven to be a safe and cost effective method for eliciting protection against pathogenic infections. It erases safety concern related to immunization with live attenuated viruses, and it offers the flexibility to target a specific antigen as well as to tune the quality of the response by selecting a precise immunostimulatory compound. Still, the ability of subunit vaccines to stimulate an immune response is usually weaker than traditional vaccine preparations. One of the approaches used to enhance immunogenicity of subunit vaccines is antigen loading or presentation by biomaterial-based carriers, especially in particulate shapes. For this purpose, our group has recently developed two nanocarriers, nanoparticles (NPs) and polymersomes (PSs) that differ considerably between each other in their physical and chemical properties. In this thesis, we evaluated the impact of antigen delivery by nanocarriers on the magnitude, and more importantly, on the quality of cellular and humoral responses. We assessed how NPs modified the CD8+ T cell response to a viral MHCI epitope and demonstrated that presentation of antigen by NPs significantly enhances the magnitude of the cellular responses. Moreover, we showed that the quality of the memory CD8+ T cells was considerably altered, with NPs promoting a robust pool of memory CD8+ T cells with an effector-like phenotype. Recent work by our group showed that T cell responses induced by NPs and PSs differ significantly depending on the delivery vehicle, with NPs enhancing cytotoxic T cell responses and PS augmenting CD4+ T cells. Herein, we demonstrated that the preferential T cell activation is the consequence of a distinct intracellular trafficking and a differential organ biodistribution of the antigen, which is promoted by the properties of the nanocarrier. Furthermore, we found that PSs are better at enhancing CD4+ T cell activation and inducing T follicular helper (Tfh) cells, which translated in an increase in germinal center B cells. Considering these results, we decided to evaluate the feasibility of a PS-based vaccine for the delivery of an antigen derived from Lassa virus, the Lassa Glycoprotein 1 (LASV-GP1). Our data showed that PSs promote an enhancement in the quality of the antibodies against LASV-GP1, which display an increased protein binding because of a broader epitope targeting. Finally, we demonstrated that, besides classical antigen-presenting cells, non-hematopoietic stromal cells also scavenge foreign antigens. We showed that lymph node lymphatic endothelial cell (LECs) internalize and process exogenous proteins in inflammatory and non-inflammatory conditions in vivo. Although with our approach we could not detect the implications of LECs presentation in the functional outcome of immune responses, we hypothesize that the elucidation of the roles of LECs on the tuning of immunity will bring novel modes of immunomodulation of vaccines. In summary, this thesis describes a comprehensive study of the adaptive immune responses generated after intradermal delivery of particulate-based subunit vaccines.

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

Therapeutic Immunomodulation of the Lymphoid-like Tumor Environment Using Nanoparticulate Formulations

Iraklis Kourtis

Cancer is a leading cause of death worldwide. Understanding the underlying mechanisms that lead to tumor escape and evasion is pivotal for the design of new therapeutic applications. This thesis takes an immunobioengineering approach, and navigates through identifying the immunological parameters relevant in the tumor microenvironment, proposing and developing a strategy in response to address these parameters using nanosized drug and antigen carriers. It is widely accepted that changes in the immunological equilibriumof the tumor microenvironment are crucial for the progression, dissemination and metastasis of a developing tumor. Motivated by tumors' natural secretion of CCL21, a chemokine known for dendritic (DC) migration towards the lymphatics, and for attracting DC and T cells in the lymph node (LN), we perturbed the physiological secretion of CCL21 of B16-F10 melanomas, deriving both a knock-down (CCL21low) and an over-expressing (CCL21high) variant. Interestingly, in immunocompetent mice, growth in CCL21-secreting tumors had an advantage over the CCL21low sub-clones and was CCR7 dependent. CCL21low tumors harbored more antigen specific T cells, while CCL21-secreting, more T regulatory cells (Tregs), which also correlated with subsequent biochemical polarization. Moreover, CCL21-secreting tumors formed a reticular fibroblastic (FRC) network (ERTR7+gp38+CCL21+) reminiscent of the lymphoid tissue of the paracortex (T cell zone) within LNs. The lymphoidlike stroma was orchestrated by the recruitment of CCR7+ adult lymphoid tissue inducers (LTis), responsible for LN formation during embryogenesis, present only in the CCL21-secreting tumors. In mice lacking LTis (Rorc(γt)-deficient) control tumors displayed impaired growth, suggesting that tumor growth is stroma dependent. In addition, the stroma of control or CCL21high tumors was infiltrated by more myeloid suppressor cells (MDSCs) than in CCL21low variant. MDSCs are a heterogeneous population of immature myeloid precursor cells that repress T cell activation and antigen presentation in chronic inflammation and cancer, hampering the efficacy of anti-tumoral vaccinations. CCL21's ability to protect allografts was further demonstrated in non-syngeneic recipients and in ectopically CCL21 transduced βTC tumor models. By mimicking secondary lymphoid organs, tumors may hi-jack and modulate the immune response from immunogenic to tolerogenic to allow for growth and metastasis. Intrigued by the excessive stroma infiltration of MDSCs in CCL21-secreting tumors, we performed a detailed spatiotemporal biodistribution analysis of ultrasmall (sub 50 nm) nanoparticles (NPs) in naïve and tumor bearing mice. Intradermally administered NPs rapidly drained to the LNs and spleen and persistently targetedmajor phagocytic populations that play a key role in hosting an immune response in naïve and tumor bearing mice. Furthermore, tumor-induced myeloid compartments, mainlyMDSCs, were highly targeted naturally within the tumormicroenvironment and spleen (tumor macroenvironment) 12 h post administration, without the use of site specific ligand. Following the MDSCs' natural preference towards phagocytosing NPs, and taking advantage of their excessive reductive cytoplasm, we designed a macromolecular pro-drug formulation that bears a cytotoxic payload and can be released in highly reductive microenvironments. Specifically, we engineered a reducible delivery system of 6-tioguanine (TG) in various formulations. Remarkably, bothMDSC compartments were ablated, monocytic ∼20 fold (CD11b+Ly6c+), polymorphonuclear ∼100 fold (CD11b+Ly6g+), for at least 7 d after administration in tumor bearing mice. As the bioidstribution revealed that apart from MDSCs, other antigen presenting cells (APCs) have phagocytosed NPs, we observed only a ∼2 fold decrease in APCs, suggesting that the rest could be used for vaccination. Taking into account that cancer vaccines and cancer immunotherapy rely on the efficient antigen presentation and education of naïve T cells by professional APCs, we explored the ability of NPs to deliver antigens in the appropriate sub-cellular compartment for antigen presentation and activation of T cells. Using the model antigen ovalbumin, we illustrated that exogenous antigen coupled to NPs by reducible bonds were internalized by DCs in vivo and in vitro. The conjugated antigen was preferentially released from the NPs with a reducible formulation, and was successfully presented in the major histocompatibility complex (MHC) I and II, which lead to activation and proliferation of antigen specific CD8+ and CD4+ T cells in vivo, respectively. Taking together, the nanoparticle platformcould be used efficiently for both inducing cytotoxicity and invoking an immune response. From discovery of new suppressive niches to therapeutical applications, we envisioned a two prong strategy that combined I) a modulatory regime to upset the balance of theMDSC populations both within the tumor microenvoronent and systemically with II) a cancer vaccine with the potential to significantly improve anti-tumoral therapeutic applications. This thesis demonstrated that such an approach is both possible and probable.

EPFL2012