The rhabdovirus Maraba has recently been characterized as a potent oncolytic virus. In the present study, we engineered an attenuated Maraba strain, defined as MG1, to express a melanoma-associated tumor antigen. Its ability to mount an antitumor immunity was evaluated in tumor-free and melanoma tumor-bearing mice. Alone, the MG1 vaccine appeared insufficient to prime detectable adaptive immunity against the tumor antigen. However, when used as a boosting vector in a heterologous prime-boost regimen, MG1 vaccine rapidly generated strong antigen-specific T-cell immune responses. Once applied for treating syngeneic murine melanoma tumors, our oncolytic prime-boost vaccination protocol involving Maraba MG1 dramatically extended median survival and allowed complete remission in more than 20% of the animals treated. This work describes Maraba virus MG1 as a potent vaccine vector for cancer immunotherapy displaying both oncolytic activity and a remarkable ability to boost adaptive antitumor immunity.

TLR ligands are critical activators of innate immunity and are being developed as vaccine adjuvants. However, their usefulness in conjunction with NOD-like receptor agonists remains poorly studied. In this study, we evaluated a new ligand that targets both TLR2 and NOD2 receptors. We assessed its ability to enhance dendritic cell maturation in vitro in addition to improving systemic and mucosal immune responses in mice. The chimeric NOD2/TLR2 ligand induced synergistic upregulation of dendritic cell maturation markers, costimulatory molecules, and secretion of proinflammatory cytokines compared with combinations of separate ligands. Furthermore, when coadministered with biodegradable nanoparticles carrying a model Ag, the ligand was able to induce high Ag-specific IgA and IgG titers at both systemic and mucosal sites after parenteral immunizations. These findings point out the potential utility of chimeric molecules TLR/NOD as adjuvants for vaccines to induce systemic and mucosal immune responses.

The zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real‐time observations of vascular flow of nanoparticles and their interactions with cells throughout the body. As a consequence, this system enables the acquisition of quantitative data that are difficult to obtain in rodents. Until now, a few studies using the zebrafish model have only described semiquantitative results on key nanoparticle parameters. Here, a MACRO dedicated to automated quantitative methods is described for analyzing important parameters of nanoparticle behavior, such as circulation time and interactions with key target cells, macrophages, and endothelial cells. Direct comparison of four nanoparticle (NP) formulations in zebrafish embryos and mice reveals that data obtained in zebrafish can be used to predict NPs' behavior in the mouse model. NPs having long or short blood circulation in rodents behave similarly in the zebrafish embryo, with low circulation times being a consequence of NP uptake into macrophages or endothelial cells. It is proposed that the zebrafish embryo has the potential to become an important intermediate screening system for nanoparticle research to bridge the gap between cell culture studies and preclinical rodent models such as the mouse.

The development of safe and effective vaccines for cancer and infectious diseases remains a major goal in public health. Over the last two decades, controlled release of vaccine antigens and immunostimulant molecules has been achieved using nanometer or micron-sized delivery vehicles synthesized using biodegradable polymers. In addition to achieving a depot effect, enhanced vaccine efficacy using such delivery vehicles has been attributed to efficient targeting of antigen presenting cells such as dendritic cells. Biodegradable and biocompatible poly(lactic acid) and poly(lactic-co-glycolic acid) polymers belong to one such family of polymers that have been a popular choice of material used in the design of these delivery vehicles. This review summarizes research findings from ourselves and others highlighting the promise of poly(lactic acid)- and poly(lactic-co-glycolic acid)-based vaccine carriers in enhancing immune responses.

The zebrafish is extensively used as an animal model for human and fish diseases. However, our understanding of the structural organization of its immune system remains incomplete, especially the mucosa-associated lymphoid tissues (MALTs). Teleost MALTs are commonly perceived as diffuse and scattered populations of immune cells throughout the mucosa. Yet, structured MALTs have been recently discovered in Atlantic salmon (Salmo salar L.), including the interbranchial lymphoid tissue (ILT) in the gills. The existence of the ILT was only recently identified in zebrafish and other fish species, highlighting the need for in-depth characterizations of the gill-associated lymphoid tissue (GIALT) in teleosts. Here, using 3-D high-resolution microscopy, we analyze the GIALT of adult zebrafish with an immuno-histology approach that reveals the organization of lymphoid tissues via the labeling of T/NK cells with an antibody directed to a highly conserved epitope on the kinase ZAP70. We show that the GIALT in zebrafish is distributed over at least five distinct sub-regions, an organization found in all pairs of gill arches. The GIALT is diffuse in the pharyngeal part of the gill arch, the interbranchial septum and the filaments/lamellae, and structured in two sub-regions: the ILT, and a newly discovered lymphoid structure located along each side of the gill arch, which we named the Amphibranchial Lymphoid Tissue (ALT). Based on RAG2 expression, neither the ILT nor the ALT constitute additional thymi. The ALT shares several features with the ILT such as presence of abundant lymphoid cells and myeloid cells embedded in a network of reticulated epithelial cells. Further, the ILT and the ALT are also a site for T/NK cell proliferation. Both ILT and ALT show structural changes after infection with Spring Viraemia of Carp Virus (SVCV). Together, these data suggest that ALT and ILT play an active role in immune responses. Comparative studies show that whereas the ILT seems absent in most neoteleosts (“Percomorphs”), the ALT is widely present in cyprinids, salmonids and neoteleosts, suggesting that it constitutes a conserved tissue involved in the protection of teleosts via the gills.

In bony fish, the gill filaments are essential for gas exchanges, but also are vulnerable to infection by water-borne microorganisms. Omnipresent across fish, gill-associated lymphoid tissues (GIALT) regulate interactions with local microbiota and halt infection by pathogens. A special GIALT structure has recently been found in Salmonids, the interbranchial lymphoid tissue (ILT). However, the structural variation of GIALT across bony fish remains largely unknown. Here, we show how this critical zone of interaction evolved across fishes. By labeling a conserved T-cell epitope on tissue sections, we find that several basal groups of teleosts possess typical ILT, while modern teleosts have lymphoepithelium of different shape and size at the base of primary gill filaments. Within Cypriniformes, neither body size variation between two related species, zebrafish and common carp, nor morphotype variation, did have a drastic effect on the structure of ILT. Thereby this study is the first to describe the presence of ILT in zebrafish. The ILT variability across fish orders seems to represent different evolutionary solutions to balancing trade-offs between multiple adaptations of jaws and pharyngeal region, and immune responses. Our data point to a wide structural variation in gill immunity between basal groups and modern teleosts.

Activation of mucosal immunity is a key milestone for next-generation vaccine development. Biocompatible polymer-based nanoparticles (NPs) are promising vectors and adjuvants for mucosal vaccination. However, their in vivo uptake by mucosae and their biodistribution in antigen-presenting cells (APCs) need to be better understood to optimize mucosal nanovaccine designs. Here, we assessed if APCs are efficiently targeted in a spontaneous manner by surfactant-free poly(lactic acid) nanoparticles (PLA-NPs) after mucosal administration. Combining histology and flow imaging approaches, we describe and quantify the mucosal uptake of 200 nm PLA-NPs in adult zebrafish. Following bath administration, PLA-NPs penetrated and crossed epithelial barriers from all exposed mucosae. In mucosae, PLA-NPs accumulated in APCs, which were identified as dendritic cells (DCs), macrophages, and IgZ+ B cells in gills and skin. PLA-NP uptake by phagocytes was specific to these cell types, as PLA-NPs were not detected in neutrophils. Importantly, quantitative analyses in gills revealed that DCs take up PLA-NPs with specifically high efficiency. This study shows that surfactant-free PLA-NPs, which display optimal biocompatibility, can spontaneously target DCs with high efficiency in vivo following mucosal administration, and highlights PLA-NPs as powerful platforms for mucosal vaccine delivery in the medical and veterinary fields, and particularly in aquaculture.

Background The developing zebrafish is an emerging tool in nanomedicine, allowing non-invasive live imaging of the whole animal at higher resolution than is possible in the more commonly used mouse models. In addition, several transgenic fish lines are available endowed with selected cell types expressing fluorescent proteins; this allows nanoparticles to be visualized together with host cells. Methods Here, we introduce the zebrafish neural tube as a robust injection site for cancer cells, excellently suited for high resolution imaging. We use light and electron microscopy to evaluate cancer growth and to follow the fate of intravenously injected nanoparticles. Findings Fluorescently labelled mouse melanoma B16 cells, when injected into this structure proliferated rapidly and stimulated angiogenesis of new vessels. In addition, macrophages, but not neutrophils, selectively accumulated in the tumour region. When injected intravenously, nanoparticles made of Cy5-labelled poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate) (PEG-PDPA) selectively accumulated in the neural tube cancer region and were seen in individual cancer cells and tumour associated macrophages. Moreover, when doxorubicin was released from PEG-PDPA, in a pH dependant manner, these nanoparticles could strongly reduce toxicity and improve the treatment outcome compared to the free drug in zebrafish xenotransplanted with mouse melanoma B16 or human derived melanoma cells. Interpretation The zebrafish has the potential of becoming an important intermediate step, before the mouse model, for testing nanomedicines against patient-derived cancer cells. Funding We received funding from the Norwegian research council and the Norwegian cancer society