Recherche & Développement

R&D : Publications

La technologie de vaxinano repose sur un système unique et innovant d'administration d'antigènes exploitant des nanoparticules biocompatibles sans danger et sans utilisation d’adjuvant. Une sélection de publications scientifiques majeures est listée ci-dessous.

Starch-based NP act as antigen delivery systems without immunomodulating effect


Plos one

Auteur: François Fasquelle, Laurent Dubuquoy, Didier Betbeder

The nasal route of immunization has become a real alternative to injections. It is indeed
described as more efficient at inducing immune protection, since it initiates both mucosal
and systemic immunity, thus protecting against both the infection itself and the transmission
of pathogens by the host. However, the use of immunomodulators should be limited since
they induce inflammation. Here we investigated in vitro the mechanisms underlying the
enhancement of antigen immunogenicity by starch nanoparticles (NPL) delivery systems in
H292 epithelial cells, as well as the NPL’s immunomodulatory effect. We observed that NPL
had no intrinsic immunomodulatory effect but enhanced the immunogenicity of an E. coli
lysate (Ag) merely by increasing its intracellular delivery. Moreover, we demonstrated the
importance of the NPL density on their efficiency by comparing reticulated (NPL) and non-reticulated particles (NPL-NR). These results show that an efficient delivery system is sufficient to induce a mucosal immune response without the use of immunomodulators.

Voir la publication

Le vaccin nasal fabriqué à partir du parasite Leishmania donovani inactivé protège et induit une réponse immunitaire specifique contre la leishmaniose viscérale


Plos Neglected Tropical Diseases

Auteur: Doumet Georges Helou, Aurelie Mauras , François Fasquelle , Juliane Sousa Lanza , Philippe M. Loiseau, Didier Betbeder , Sandrine Cojean

Voir la publication

Effective nanoparticle-based nasal vaccine against latent and congenital toxoplasmosis in sheep.


Frontiers in Immunology

Auteur: Ducournau C., Moiré N., Carpentier R., Herkt C., Lantier I., Betbeder D. and Dimier-Poisson I.

Toxoplasma gondii is a parasitic protozoan of worldwide distribution, able to infect all warm-blooded animals, but particularly sheep. Primary infection in pregnant sheep leads to millions of abortions and significant economic losses for the livestock industry. Moreover, infected animals constitute the main parasitic reservoir for humans. Therefore, the development of a One-health vaccine seems the best prevention strategy. Following earlier work, a vaccine constituted of total extract of Toxoplasma gondii proteins (TE) associated with maltodextrin nanoparticles (DGNP) was developed in rodents. In this study we evaluated the ability of this vaccine candidate to protect against latent and congenital toxoplasmosis in sheep. After two immunizations by either intranasal or intradermal route this DGNP/TE vaccine generated specific Th1-cellular immune responses, mediated by APC-secretion of IFN-γ and IL-12. Secretion of IL-10 appeared to regulate this Th1 response for intradermally vaccinated sheep but was absent in intranasally-vaccinated animals. Finally, protection against latent toxoplasmosis and transplacental transmission were explored. Intranasal vaccination led to a marked decrease of brain cysts compared with the non-vaccinated group. This DGNP/TE vaccine administered intranasally conferred a high level of protection against latent toxoplasmosis and its transplacental transmission in sheep, highlighting the potential for development of such a vaccine for studies in other species.

Voir la publication

Importance of phospholipid core for mucin hydrogel penetration and mucosal cell uptake of maltodextrin nanoparticles


ACS Appl. Bio Mater.

Auteur: Francois Fasquelle, Rodolphe Carpentier, Bastien Demouveaux, Jean-Luc Desseyn, and Didier Betbeder

Nanoparticles (NP) used as intranasal antigen delivery systems are a promising way to vaccinate. However NP have to cross the mucus gel and penetrate into mucosal cells to deliver antigens and an antagonism exists between mucopenetrating NP rarely able to interact with cells and NP designed to deliver antigens often described as mucoadhesive. Here we compared the ability of cationic maltodextrin-based NP without (NP+) or with a lipid core (NPL) to interact with mucins and airway epithelial cells. We showed that the presence of the lipid core increased the NPL mobility in mucus by lowering interactions with mucins. Similarly, the uptake and the protein delivery by NPL in airway epithelial cells was not hindered by mucins. This highlights the importance of the lipids in the NPL, which are more efficient in crossing mucus while maintaining interaction with epithelial cells, an intermediate behavior between muco-adhesive and mucopenetrating NP.

Voir la publication

Prevention of influenza virus infection and transmission by intranasal administration of a porous maltodextrin nanoparticle-formulated vaccine


International Journal of Pharmaceutics

Auteur: Le MQ., Ye L., Bernasconi V., Carpentier R., Fasquelle F., Lycke N., Staeheli P. and Betbeder D.

Influenza vaccines administered intramuscularly exhibit poor mucosal immune responses in the respiratory tract which is the prime site of the infection. Intranasal vaccination is a potential route for vaccine delivery which has been demonstrated effective in inducing protective immune responses in both systemic and mucosal compartments. For this purpose, nanoparticles have been used as antigen delivery systems to improve antigen capture by immune cells. In this paper we demonstrate efficient delivery of viral antigens to airway epithelial cells, macrophages and dendritic cells, using polysaccharide nanoparticles (NPL), leading to a strong protection against influenza virus infection. A formulation combining split Udorn virus antigens with NPL and the mucosal protein adjuvant CTA1-DD was administered intranasally and resulted in an enhanced specific humoral immune response. Furthermore, NPL carrying split Udorn, with or without CTA1-DD, inhibited virus transmission from infected to uninfected naive mice. These results demonstrate that an intranasal delivery system combining NPL, mucosal adjuvant CTA1-DD and split virus antigens confers robust protection against influenza infection and inhibits virus transmission.

Voir la publication

Porous Maltodextrin-Based Nanoparticles: A Safe Delivery System for Nasal Vaccines


Journal of Nanomaterials

Auteur: Carpentier R, Platel A, Salah N, Nesslany F and Betbeder D

Vaccination faces limitations and delivery systems additionally appear to have potential as tools to trigger protective immune responses against diseases. The nanoparticles studied are cationic maltodextrin-based nanoparticles with an anionic phospholipid core (NPL); they are a promising antigen delivery system and their efficacy as drug vectors against complex diseases such as toxoplasmosis has already been demonstrated. Cationic compounds are generally described as toxic, therefore it is of interest to evaluate the behavior of these NPL in vitro and in vivo. Here, we studied the in vitro toxicity (cytotoxicity and ROS induction in intestinal and airway epithelial cell lines) and the in vivo tolerability and genotoxicity of these nanoparticles administered by the nasal route to a rodent model. In vitro these NPL were not cytotoxic and did not induce any ROS production. In vivo, even at very large doses (1,000 times the expected human dose), no adverse effect and no genotoxicity were observed in lungs, stomach, colon or liver. This study shows that these NPL can be safely used.

Voir la publication

Porous Nanoparticles With Self-Adjuvanting M2e-Fusion Protein and Recombinant Hemagglutinin Provide Strong and Broadly Protective Immunity Against Influenza Virus Infections


Fontiers in Immunology

Auteur: Bernasconi V, Bernocchi B, Ye L, Lê MQ, Omokanye A, Carpentier R, Schön K, Saelens X, Staeheli P, Betbeder D, Lycke N.

Due to the high risk of an outbreak of pandemic influenza, the development of a broadly protective universal influenza vaccine is highly warranted. The design of such a vaccine has attracted attention and much focus has been given to nanoparticle-based influenza vaccines which can be administered intranasally. This is particularly interesting since, contrary to injectable vaccines, mucosal vaccines elicit local IgA and lung resident T cell immunity, which have been found to correlate with stronger protection in experimental models of influenza virus infections. Also, studies in human volunteers have indicated that pre-existing CD4+ T cells correlate well to increased resistance against infection. We have previously developed a fusion protein with 3 copies of the ectodomain of matrix protein 2 (M2e), which is one of the most explored conserved influenza A virus antigens for a broadly protective vaccine known today. To improve the protective ability of the self-adjuvanting fusion protein, CTA1-3M2e-DD, we incorporated it into porous maltodextrin nanoparticles (NPLs). This proof-of-principle study demonstrates that the combined vaccine vector given intranasally enhanced immune protection against a live challenge infection and reduced the risk of virus transmission between immunized and unimmunized individuals. Most importantly, immune responses to NPLs that also contained recombinant hemagglutinin (HA) were strongly enhanced in a CTA1-enzyme dependent manner and we achieved broadly protective immunity against a lethal infection with heterosubtypic influenza virus. Immune protection was mediated by enhanced levels of lung resident CD4+ T cells as well as anti-HA and -M2e serum IgG and local IgA antibodies.

Voir la publication

Residence time and uptake of porous and cationic maltodextrin-based nanoparticles in the nasal mucosa: Comparison with anionic and cationic nanoparticles.


International Journal of Pharmaceutics

Auteur: Le MQ, Carpentier R, Lantier I, Ducournau C, Dimier-Poisson I, Betbeder D.

Different types of biodegradable nanoparticles (NP) have been studied as nasal mucosa cell delivery systems. These nanoparticles need to strongly interact with mucosa cells to deliver their payload. However, only a few simultaneous comparisons have been made and it is therefore difficult to determine the best candidate. Here we compared 5 types of nanoparticles with different surface charge (anionic or cationic) and various inner compositions as potential vectors: cationic and anionic liposomes, cationic and anionic PLGA (Poly Lactic co-Glycolic Acid) NP and porous and cationic maltodextrin NP (cationic surface with an anionic lipid core: NPL). We first quantified their nasal residence time after nasal administration in mice using in vivo live imaging and NPL showed the longest residence time. In vitro endocytosis on mucosal cells (airway epithelial cells, macrophages and dendritic cells) using labeled nanoparticles were performed by flow cytometry and confocal microscopy. Among the 5 nanoparticles, NPL were taken up to the greatest extent by the 3 different cell lines and the endocytosis mechanisms were characterized. Taken together, we observed that the nanoparticles' cationic surface charge is insufficient to improve mucosal residence time and cellular uptake and that the NPL are the best candidates to interact with airway mucosal cells.

Voir la publication

Nasal Nanovaccines


International Journal of Pharmaceutics - 2017 Jul 8;530(1-2):128-138

Auteur: Bernocchi B., Carpentier, R., Betbeder D.

Nasal administration of vaccines is convenient for the potential stimulation of mucosal and systemic immune protection. Moreover the easy accessibility of the intranasal route renders it optimal for pandemic vaccination. Nanoparticles have been identified as ideal delivery systems and adjuvants for vaccine application. Heterogeneous protocols have been used for animal studies. This complicates the understanding of the formulation influence on the immune response and the comparison of the different nanoparticles approaches developed. Moreover anatomical and immunological differences between rodents and humans provide an additional hurdle in the rational development of nasal nanovaccines. This review will give a comprehensive expertise of the state of the art in nasal nanovaccines in animals and humans focusing on the nanomaterial used.

Voir la publication

Synthetic parasites: a successful mucosal nanoparticle vaccine against Toxoplasma congenital infection in mice.


FUTURE MICROBIOLOGY Volume: 12 Pages 393-405

Auteur: Ducournau, C.; Nguyen, T.; Carpentier, R.; […] Betbeder, D.; Dimier-Poisson, I.

Aims: Development of protein vaccine to prevent congenital infection is a major public health priority. Our goal is the design of mucosal synthetic pathogen inducing protective immune responses against congenital toxoplasmosis.
Materials & Methods: Mice were immunized intranasally, establishing pregnancy and challenging orally. Placental immune response, congenital infection, pup growth, parasitic load rates were studied.
Results: Pups born to vaccinated infected dams had significantly fewer brain cysts, no intraocular inflammation and normal growth. Protection was associated with a placental cellular Th1 response down-regulated by IL-6 and correlated with persistence of vaccine for few hours in the nose before being totally eliminated.
Conclusion: Our vaccine conferred high protection against congenital toxoplasmosis. These results provide support for future studies of other congenital vaccine.

Voir la publication

Mechanisms allowing protein delivery in nasal mucosa using NPL nanoparticles.



Auteur: Bernocchi, B.; Carpentier, R.; Lantier, I.; Ducournau, C.; Dimier-Poisson, I.; Betbeder, D.

The intranasal administration of proteins using nanoparticles is a promising approach for several applications, especially for mucosal vaccines. Delivery of protein within the epithelial barrier is a key point to elicit an immune response and nano-carrier has to show no toxicity. The aim of this work was to elucidate the interactions of cationic porous nanoparticles loaded with protein delivery for antigen delivery in the nose. We investigated the loading, the cellular delivery and the epithelial transcytosis of proteins associated to these nanoparticles containing an anionic lipid in their core (NPL). NPL were highly endocytosed by airway epithelial cells and significantly improved the protein delivery into the cell. In vitro transcytosis studies showed that NPL did not modify the in vitro epithelial permeability suggesting no toxicity of these carriers. Moreover protein and NPL did not translocate the epithelial barrier. In vivo studies demonstrated that NPL prolonged the nasal residence time of the protein and no NPL were found beyond the epithelial barrier in vivo, precluding a negative side effect. All together these results establish the NPL as a bio-eliminable and optimal vaccine carrier.

Voir la publication

Porous nanoparticles as delivery system of complex antigens for an effective vaccine


BIOMATERIALS Volume: 50 Pages: 164-175

Auteur: Dimier-Poisson, I.; Carpentier, R.; N'Guyen, T.; Dahmani, F.; Ducounau, C.; Betbeder, D.

Development of sub-unit mucosal vaccines requires the use of specific delivery systems or immune-modulators such as adjuvants to improve antigen immunogenicity. Nasal route for vaccine delivery by nanoparticles has attracted much interest but mechanisms triggering effective mucosal and systemic immune response are still poorly understood. Here we study the loading of porous nanoparticles (DGNP) with a total extract of Toxoplasma gondii antigens (TE), the delivery of TE by DGNP into airway epithelial, macrophage and dendritic cells, and the subsequent cellular activation. In vitro, DGNP are able to load complex antigens in a stable and quantitative manner. The outstanding amount of antigen association by DGNP is used to deliver TE in airway mucosa cells to induce a cellular maturation with an increased secretion of pro-inflammatory cytokines. Evaluation of nasal vaccine efficiency is performed in vivo on acute and chronic toxoplasmosis mouse models. A specific Th1/Th17 response is observed in vivo after vaccination with DGNP/TE. This is associated with high protection against toxoplasmosis regarding survival and parasite burden, correlated with an increased delivery of antigens by DGNP in airway mucosa cells. This study provides evidence of the potential of DGNP for the development of new vaccines against a range of pathogens.

Voir la publication

Airway delivery of peptides and proteins using nanoparticles


BIOMATERIALS Volume: 34 Issue: 2 Pages: 516-525

Auteur: Dombu, C.; Betbeder, D.

Delivery of peptides and proteins via the airways is one of the most exciting potential applications of nanomedicine. These macromolecules could be used for many therapeutic applications, however due to their poor stability in physiological medium and difficulties in delivering them across biological barriers, they are very difficult to use in therapy. Nanoparticulate drug delivery systems have emerged as one of the most promising technologies to overcome these limitations, owing mainly to their proven capacity to cross biological barriers and to enter cells in high yields, thus improving delivery of macromolecules. In this review, we summarize the current advances in nanoparticle designed for transmucosal delivery of peptides and proteins. Challenges that must be overcome in order to derive clinical benefits are also discussed.

Voir la publication

Influence of surface charge and inner composition of nanoparticles on intracellular delivery of proteins in airway epithelial cells


BIOMATERIALS Volume: 33 Issue: 35 Pages: 9117-9126

Auteur: Dombu, C.; Carpentier, R.; Betbeder, D.

The delivery of protein in the airway using nanoparticles (NP) is an emerging strategy that shows encouraging results in vivo for several applications. However, the mechanisms by which NP deliver proteins to the inside of cells remain poorly understood. In this study, we investigated the intracellular delivery of ovalbumin (OVA) in human airway cells by two porous cationic polysaccharides nanoparticles. These NP have the same surface charge density but differ in that their inner core contains either cationic or anionic charges (respectively: NP+ and DGNP+). Confocal microscopy showed a rapid uptake of both NP by human airway cells, followed by a significant accumulation in clathrin vesicles and early endosomes. Both NP were found to associate OVA in a quantitative manner, and this association was stable even in presence of serum proteins. We observed that the two NP greatly increased OVA uptake by human airway cells, meanwhile FRET studies using FITC-labelled NP and TRITC-labelled OVA showed a gradual release of OVA from NP within cells, and this was much faster with DGNP+ than NP+. These results were confirmed using OVA–DQ to follow OVA degradation fragments within cells. Both NP increased intracellular proteolysis of OVA, however DGNP+ facilitated OVA escape from endosomes. Studies with trypsin and pepsin at different pH strongly suggested that both NP can protect (in the extracellular medium) or promote (in acidic endosomes) protein proteolysis, depending on the environment. Interestingly, the mechanisms involved could be explained as a function of protein global charge at different pH. All these results confirm the importance of not only the surface charge but also the inner composition of NP in determining their efficacy as tools for the delivery of proteins to different cellular compartments.

Voir la publication

Study of serum interaction with a cationic nanoparticle: Implications for in vitro endocytosis, cytotoxicity and genotoxicity


INTERNATIONAL JOURNAL OF PHARMACEUTICS Volume: 423 Issue: 1 Special Issue: SI Pages: 37-44

Auteur: Merhi, M.; Dombu, C.; Brient, A.; Chang, J.; Platel, A.; Le Curieux, F.; Marzin, D.; Nesslany, F.; Betbeder, D.

We used well-characterized and positively charged nanoparticles (NP+) to investigate the importance of cell culture conditions, specifically the presence of serum and proteins, on NP+ physicochemical characteristics, and the consequences for their endocytosis and genotoxicity in bronchial epithelial cells (16HBE14o-). NP+ surface charge was significantly reduced, proportionally to NP+/serum and NP+/BSA ratios, while NP+ size was not modified. Microscopy studies showed high endocytosis of NP+ in 16HBE14o-, and serum/proteins impaired this internalization in a dose-dependent manner. Toxicity studies showed no cytotoxicity, even for very high doses of NP+. No genotoxicity was observed with classic comet assay while primary oxidative DNA damage was observed when using the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG). The micronucleus test showed NP+ genotoxicity only for very high doses that cannot be attained in vivo. The low toxicity of these NP+ might be explained by their high exocytosis from 16HBE14o- cells. Our results confirm the importance of serum and proteins on nanoparticles endocytosis and genotoxicity.

Voir la publication

Characterization of endocytosis and exocytosis of cationic nanoparticles in airway epithelium cells.


NANOTECHNOLOGY Volume: 21 Issue: 35

Auteur: Dombu, C.; Kroubi, M.; Ribouche, R.; Matran, R.; Betbeder, D.

A major challenge of drug delivery using colloids via the airway is to understand the mechanism implied in their interactions with epithelial cells. The purpose of this work was to characterize the process of endocytosis and exocytosis of cationic nanoparticles (NPs) made of maltodextrin which were developed as a delivery system for antigens in vaccine applications. Confocal microscopy demonstrated that these NP are rapidly endocytosed after as little as 3 min incubation, and that the endocytosis was also faster than NP binding since most of the NPs were found in the middle of the cells around the nuclei. A saturation limit was observed after a 40 min incubation, probably due to an equilibrium becoming established between endocytosis and exocytosis. Endocytosis was dramatically reduced at 4 °C compared with 37 °C, or by NaN3 treatment, both results suggesting an energy dependent process. Protamine pretreatment of the cells inhibited NPs uptake and we found that clathrin pathway is implied in their endocytosis. Cholesterol depletion increased NP uptake by 300% and this phenomenon was explained by the fact that cholesterol depletion totally blocked NP exocytosis. These results suggest that these cationic NPs interact with anionic sites, are quickly endocytosed via the clathrin pathway and that their exocytosis is cholesterol dependent, and are similar to those obtained in other studies with viruses such as influenza.

Voir la publication