Exploiting immunofunctional nanoparticles as vaccines and synthetic pathogens

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.

Exploiting lymphatic transport and complement activation in nanoparticle vaccines

Jeffrey Alan Hubbell, Sai Reddy, Melody Swartz

Antigen targeting and adjuvancy schemes that respectively facilitate delivery of antigen to dendritic cells and elicit their activation have been explored in vaccine development. Here we investigate whether nanoparticles can be used as a vaccine platform by targeting lymph node-residing dendritic cells via interstitial flow and activating these cells by in situ complement activation. After intradermal injection, interstitial flow transported ultra-small nanoparticles (25 nm) highly efficiently into lymphatic capillaries and their draining lymph nodes, targeting half of the lymph node-residing dendritic cells, whereas 100-nm nanoparticles were only 10% as efficient. The surface chemistry of these nanoparticles activated the complement cascade, generating a danger signal in situ and potently activating dendritic cells. Using nanoparticles conjugated to the model antigen ovalbumin, we demonstrate generation of humoral and cellular immunity in mice in a size- and complement-dependent manner.

Nature Publishing Group2007

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

Marcela Rincón-Restrepo

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.

EPFL2016

Development of a nano-adjuvant cancer vaccine and its evaluation for melanoma in combination with radiotherapy

Sylvie Hauert

Cancer is the second leading cause of death in the world and melanoma is the deadliest type of skin cancer. Although surgery, radiotherapy and chemotherapy are still standard treatments, the discovery of the role played by the immune system in cancer has allowed the development of new treatments, called immunotherapies. The goal of these treatments is to help the immune system eradicate cancerous cells, for example by increasing the anti-tumor response of the patient. In this particular case, the treatment has to activate cytotoxic CD8+ and CD4+ helper T cells against the cancer cells. This can be done by using subunit vaccines composed of tumor antigens, adjuvants and a delivery system, which will be internalized by dendritic cells (DCs) and induce its activation followed by the migration to the lymph nodes where it will educate T cells against specific antigens. Our laboratory has developed two different delivery systems, nanoparticles (NPs) and polymersomes (PSs) that can enhance the vaccineâs immune response by efficiently entering the lymphatic system, due to their size, and drain to the lymph node. Once they reach that location, they will be internalized by DCs and induce an anti-tumor response. In this thesis, we used these two nanocarriers to create a vaccine composed of different nano-adjuvants capable of activating several toll-like receptors simultaneously and test their efficacy at inducing an anti-tumor response in melanoma. We have developed and characterized the different nano-adjuvants, PS-MPLA, PS-CL075 and NP-CpG-B, and demonstrated, in vitro, synergistic effects on DC activation when PS-MPLA + PS-CL075 and PS-MPLA + NP-CpG-B were combined. In vivo, we showed the enhanced immune response with the different nano-adjuvant combinations, but only PS-MPLA + NP-CpG-B was able to delay tumor growth in melanoma. We then tested this vaccine in combination with radiotherapy, mimicking a clinical situation. We first demonstrated an enhanced immune response when combining a high irradiation dose, 15 Gy, with the nano-adjuvant vaccine draining the tumor-draining lymph node. Moreover, the vaccination of NP-CpG-B + PS-MPLA and radiotherapy increased mice survival by 17 days compared to mice only receiving radiotherapy. â Overall, we have demonstrated that combining different nano-adjuvants, targeting the tumor-draining lymph node, with radiotherapy enhanced significantly the survival in a melanoma model. This approach is particularly promising since ionizing radiation is a standard treatment in cancer and our vaccine can be applied to a multitude of cancer since it does not contain any antigen and its composition can easily be modified.