Evaluating the use of nanoparticles as an antigen delivery system

The delivery of antigens on degradable nanoparticles, directly to dendritic cells residing in lymph nodes, offers great opportunities for vaccine design. This master thesis investigates the issues related to the development of vaccines based on ultrasmall (25nm)nanoparticles, capable of draining to lymph nodes via lymphatic flow and to spontaneously activate complement factor C3 as an adjuvant. The first part investigates a Mycobacterium tuberculosis preventive vaccine in collaboration with Northwestern (Jamie Carter and Garrett Green) and EPFL students (Marie Ballester and Bastien Schyrr) in Cape Town. Research plan for the preclinical testing of potential vaccine formulations to be used as a tuberculosis vaccine was investigated as well as adjuvancy, antigen dosage determination, efficacy, safety testing, vaccine manufacture, regulatory issues, and transition to clinical trials. In the second part, a peptide derived from the Apical Membrane Antigen 1 (AMA1) of the malaria causing parasite Plasmodium falciparum, conjugated to 28nm nanoparticles was used to immunize mice and proved to create an immune response in 4 out of 11 mice after 4 injections. Antibodies produced by the responding mice were able to recognize the native antigen on the parasite in Indirect Immunofluorescence and Western blotting Assays. The nanoparticles were retrieved in mice lymph nodes by Immunohistochemistry and were located near antigen presenting cells

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

Nanoparticle Based Tuberculosis Vaccine

Marie Ballester, Sai Reddy

Tuberculosis (TB) is a growing world health problem, especially in developing countries. The only available vaccine is the hundred years old Bacille de Calmette et Guérin (BCG). It has been shown to reduce infant mortality, but it is not an effective method for preventing TB in adults. A new vaccine that can safely and effectively be administered in infancy and confers long-term protection against TB is urgently needed. Using a novel nanoparticle (NP) delivery platform developed in the laboratories of Prof. Swartz and Prof. Hubbell at the Swiss Federal Institute of Technology in Lausanne (EPFL), we are proposing a research plan for the development of a TB vaccine. The NP platform has been shown to be extremely stable, safe, cheap, and able to induce strong T-cell responses, and thus we expect it has potential to be an effective vaccine that holds advantages over the current candidates. We describe a preclinical testing plan that includes best formulation selection, dosage determination, efficacy, and safety testing. We also explored manufacturing process, regulatory issues, and the transition to clinical trials. This part of the project was done in Cape Town as part of a MS Honor Program in Bioengineering for Global Health 2008, and it reflects the work of Jamie Carter (Northwestern University), Garrett Green (Northwestern University), Bastien Schyrr (EPFL), Alexandre de Titta (EPFL) and myself. Before we can couple a TB antigen to the NPs, the basics of this vaccine platform behavior need to be explored. My research in Prof. Swartz's laboratory (EPFL) on the characterization of uptake and processing of NPs by dendritic cells (DCs) will be presented. This is key since the immune response relies on the way the antigen is uptaken and processed. Data suggesting caveolin dependent uptake, presence of NPs in the cytoplasm, and presentation via MHC class I and MHC class II molecules was collected by a set of immunofluorescence experiments on mice DCs.

2008

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