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

International audienceControlled 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 IFNγ and TNFα 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

Peripherally Administered Nanoparticles Target Monocytic Myeloid Cells, Secondary Lymphoid Organs and Tumors in Mice

Nanoparticles have been extensively developed for therapeutic and diagnostic applications. While the focus of nanoparticle trafficking in vivo has traditionally been on drug delivery and organ-level biodistribution and clearance, recent work in cancer biology and infectious disease suggests that targeting different cells within a given organ can substantially affect the quality of the immunological response. Here, we examine the cell-level biodistribution kinetics after administering ultrasmall Pluronic-stabilized poly(propylene sulfide) nanoparticles in the mouse. These nanoparticles depend on lymphatic drainage to reach the lymph nodes and blood, and then enter the spleen rather than the liver, where they interact with monocytes, macrophages and myeloid dendritic cells. They were more readily taken up into lymphatics after intradermal (i.d.) compared to intramuscular administration, leading to similar to 50% increased bioavailability in blood. When administered i.d., their distribution favored antigen-presenting cells, with especially strong targeting to myeloid cells. In tumor-bearing mice, the monocytic and the polymorphonuclear myeloid-derived suppressor cell compartments were efficiently and preferentially targeted, rendering this nanoparticulate formulation potentially useful for reversing the highly suppressive activity of these cells in the tumor stroma

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 cell

The TLR4 Agonist Fibronectin Extra Domain A is Cryptic, Exposed by Elastase-2; use in a fibrin matrix cancer vaccine

Fibronectin (FN) is an extracellular matrix (ECM) protein including numerous fibronectin type III (FNIII) repeats with different functions. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4). Here, by producing various FN fragments containing FNIII EDA, we found that FNIII EDA's immunological activity depends upon its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with its neighboring FNIII repeats (FNIII 9-10-11) enhanced its activity in agonizing TLR4, while C-terminal extension with the native FNIII 12-13-14 heparin-binding domain abrogated it. In addition, we reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Activity of the C-terminally extended FNIII EDA could be restored after cleavage of the FNIII 12-13-14 domain by elastase 2. FN being naturally bound to the ECM, we immobilized FNIII EDA-containing FN fragments within a fibrin matrix model along with antigenic peptides. Such matrices were shown to stimulate cytotoxic CD8(+) T cell responses in two murine cancer models

Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory recall at low dose

In subunit vaccines, strong CD8(+) T-cell responses are desired, yet they are elusive at reasonable adjuvant doses. We show that targeting adjuvant to the lymph node (LN) via ultrasmall polymeric nanoparticles (NPs), which rapidly drain to the LN after intradermal injection, greatly enhances adjuvant efficacy at low doses. Coupling CpG-B or CpG-C oligonucleotides to NPs led to better dual-targeting of adjuvant and antigen (codelivered on separate NPs) in cross-presenting dendritic cells compared with free adjuvant. This led to enhanced dendritic cell maturation and T helper 1 (Th1)-cytokine secretion, in turn driving stronger effector C8(+) T-cell activation with enhanced cytolytic profiles and, importantly, more powerful memory recall. With only 4 mu g CpG, NP-CpG-B could substantially protect mice from syngeneic tumor challenge, even after 4 mo of vaccination, compared with free CpG-B. Together, these results show that nanocarriers can enhance vaccine efficacy at a low adjuvant dose for inducing potent and long-lived cellular immunity

Nanoparticle conjugation and pulmonary delivery enhance the protective efficacy of Ag85B and CpG against tuberculosis

Vaccines that drive robust T-cell immunity against Mycobacterium tuberculosis (Mtb) are needed both for prophylactic and therapeutic purposes. We have recently developed a synthetic vaccine delivery platform with Pluronic-stabilized polypropylene sulfide nanoparticles (NPs), which target lymphoid tissues by their small size (∼ 30 nm) and which activate the complement cascade by their surface chemistry. Here we conjugated the tuberculosis antigen Ag85B to the NPs (NP-Ag85B) and compared their efficacy in eliciting relevant immune responses in mice after intradermal or pulmonary administration. Pulmonary administration of NP-Ag85B with the adjuvant CpG led to enhanced induction of antigen-specific polyfunctional Th1 responses in the spleen, the lung and lung-draining lymph nodes as compared to soluble Ag85B with CpG and to the intradermally-delivered formulations. Mucosal and systemic Th17 responses were also observed with this adjuvanted NP formulation and vaccination route, especially in the lung. We then evaluated protection induced by the adjuvanted NP formulation following a Mtb aerosol challenge and found that vaccination with NP-Ag85B and CpG via the pulmonary route displayed a substantial reduction of the lung bacterial burden, both compared to soluble Ag85B with CpG and to the corresponding intradermally delivered formulations. These findings highlight the potential of administrating NP-based formulations by the pulmonary route for TB vaccination

Nanoparticles target lymph node dendritic cells better after i.d. vs. i.m. delivery.

<p>(<b>a</b>) Blood concentrations of Dy649-labeled NPs after i.v., i.m. and i.d. administration. (<b>b</b>) Heat maps representing the median percentage of NP⁺ cells for indicated cell populations. Note that maxima vary from 10% in total leukocytes to 100% in monocytes. <i>P</i> values were computed by comparing the adjusted means of each organ between i.d. and i.m. for each cell type with a two-tailed Student's t-test. (<b>c</b>) Importance of route of administration for each cellular subtype. The log-likelihood ratio represents the likelihood of the alternate model, i.e. the model without taking account the route of administration, over the likelihood of the full factorial model. <i>P</i> values were computed using the <i>Chi</i> Square test between the alternate model and the full model for each population. For 144 h, <i>n</i> = 2, for all else, <i>n</i>≥4. Leukocytes: CD45⁺, mature myeloid DCs: CD11c⁺CD11b⁺I/A^b+, cross-presenting DCs: CD11c⁺CD8α⁺ I/A^b+, immature myeloid DCs: CD11c⁺CD11b⁺I/A^b−, immature lymphoid DCs: CD11c⁺CD11b⁻I/A^b−, medullary/red pulp (RP) macrophages (MØ): CD11b⁺F4/80⁺, monocytes: CD11b⁺GR1^midSSC^lowF4/80⁺, granulocytes: CD11b⁺GR1^highSSC^high, T cells: CD3ε⁺, B cells: B220⁺. Draining lymph nodes are indicated by Ax: axillary, Br: brachial, In: inguinal, Po: popliteal; Sp: spleen. *<i>p</i>≤0.05, **<i>p</i><0.01, ***<i>p</i><0.005.</p