Sublingual Vaccination by polysaccharide-based patch – BuccaVac
Sublingual vaccine against flu
Mucosal administration, and in particular sublingual administration, of vaccines allows the induction of both systemic and mucosal immunity. However, liquid administration leads to a dilution of the vaccine in the saliva. BuccaVac proposes the development of a sublingual patch for the induction of a strong immunity against flu virus by induction of a local response in the upper and lower respiratory tract, providing a strong protection directly at the portal of entry of the virus.
Original design of a buccal patch for human sublingual vaccination oriented towards a FLU vaccine by induction of a neutralizing mucosal immune response.
We propose a pioneering approach that has never been addressed to date and will go well beyond the current state-of-the-art strategies for sublingual vaccination: association of a mucoadhesive delivery system with a particulate antigen formulation containing an immunostimulant. Such new technology involves the development of a biocompatible, biodegradable, biomimetic and mucoadhesive patch that will create a novel vaccination mode. <br />The buccal patches will be made of chitosan (CHI) which presents excellent mucoadhesive properties and hyaluronic acid (HyA) which is widely present in the human saliva.<br />The two polysaccharides CHI and HyA will be assembled by the Layer-by-Layer (LbL) assembly technique that is a simple, inexpensive, automatized, highly versatile and bottom-up processing technology. The free-standing membranes (patches) obtained have few centimetres of diameter and tens of microns thick (possibility of miniaturisation for animal experimentation).<br />Afterwards, the patches will be loaded by two proteins moieties, one antigen from Influenza A and one mucosal adjuvant targeting APCs. The addition of an adjuvant is envisaged to overcome the highly tolerogenic buccal environment. The antigen (either as a free protein or presented at the surface of a biodegradable nanoparticle (NP) to mimic viral presentation) is the hemagglutinin (HA) protein from pandemic Influenza A H1N1 (A/California/07/2009) which is one of the most common circulating type. In order to specifically target the APCs and reduce the migration time from the submucosa to the surface, the patch will be adjuvanted with a chemoattractive cytokine that will be loaded into the patch and released during the initial steps of patch dissolution (by salivary enzymes and pH). <br />Indeed, the dissolution process will have to be controlled to specifically deliver the loaded molecules to the mucosa, i.e. at the contact site. It is our ambition to have a biomedical device validated in in vivo preclinical studies (mouse and NHP) that may contribute to increase the scientific knowledge in mucosal immunity and provide efficient and innovative solutions to mucosal vaccination.
BuccaVac aims to develop a novel patch for influenza prevention in order to circumvent the limitations of the vaccines that are used nowadays. The project is organised in 3 WPs .
WP1 was devoted to the development of the patch loaded with 1/ NPs decorated with the flu antigen and 2/ a biological adjuvant. The flu antigen (HA protein) was combined with NPs (size around 120 nm) ensuring a strong entrapping efficacy in the patch, without dispersing HA immunogen in the saliva. To reverse tolerance of mucosal dendritic cells, a chemoattractive cytokine was added as a mucosal adjuvant at the surface of the patch and will be delivered during initial steps of patch dissolution.
In the WP2, biocompatibility and bioactivity was evaluated on models of human buccal cell lines and after sublingual administration in mouse. MHC2+ immune cell recruitment and the local secretion of pro-inflammatory cytokines were assessed. The adjuvant properties of the different vaccine formulations was assessed in vitro and in vivo by measurement of cytokine production by Luminex and by testing the activation of reporter cell lines.
Finally, in WP3 (ongoing), the best formulation will be tested in vivo in mice in order to assess eventual irritation and compare the immunogenicity of the vaccine formulations as well as the ability to protect against a Flu lethal challenge. Anti-HA antibody titres will be determined from serum, saliva and bronchio-alveolar lavages to track the kinetics of the systemic humoral immune response as well as the mucosal one, and their neutralizing capacities against influenza virus. A special focus will be made on the kinetics of activation of the germinal centers in draining lymph nodes that are clue actors in the induction of the immune response. The finality of the project is to select a sublingual formulation to determine its immunogenic potential on non-human primates.
A: Deveopment of a patch made of CHI and HyA for the delivery of vaccine formulations by buccal route (results available in the publication resulting from the ANR BuccaVAc grant: doi: 10.1016/j.actbio.2021.04.024)
Sublingual vaccine patches were produces by the Layer by Layer (LbL) technology with a dipping robot. Mucoadhesive patches were conceived by green chemistry: natural origin of polysaccharides, no solvents, no crosslinking agents. The results obtained on the patch characterization perfectly answered the project expectations:
- Controlled biodegradability (degradation tests in artificial saliva, mass loss)
- Validated mucoadhseion in mouse (histology, whole body imaging)
- Biocompatibility: no toxicity was observed at 24h on human sublingual epithelial cells (Ho-1u-1, collaboration with GIMAP, St Etienne). No inflammation 6 or 24h after administration in the mouse (immunohistochemistry of MHCII+ cells, quantification of pro-inflammatory cytokines by Luminex).
- Efficient incorporation and delivery of an adjuvant protein (CCL20 cytokine), persistence of bioactivity after release
- Transport of model proteins in the sublingual mucosa in 10 min (histology, confocal microscopy)
- Adsorption and release of NPs : the adsorbed NPs at the surface of the patch are efficiently released during its degradation by salivary enzymes, and then uptaken (internalized) by APCs (dendritic cells) in culture.
B: Validation of a mucosal adjuvant
The use of the CCL20 chemokine as a mucosal adjuvant was assessed on a small immunization protocol (5 mice) with a modèle antigène adsorbed on NPs. This preliminary study, although encouraging, showed a limited mucosal response in the respiratory tract. This is why we decided to change the adjuvantation strategy and follow the project by the development of another mucosal adjuvant directly loaded inside the NPs. Two candidates were selected: a Nod2 agonist and an activator of the TLR7/8 pathway. The studies performed on these 2 particulate adjuvnats led to :
- the validation of the persistence of adjuvant abilities of the 2 molecules after loading onto the NP s (use of reporter cells lines HEK-Blue-hTLR7 et HEK-Blue-hNod2).
- the identification of blood cytokinic signature of the 2 candidates, 6h and 24h after sublingual administration as liquid formulation or formulated on the mucoadhesive patch (Luminex™ Cytokine Mouse Magnetic 20-Plex Panel).
The first 18 months of the BuccaVac project were very fruitful as they led to 1/ the validation of the LbL patch as an efficient delivery system for subunit vaccine delivery, and 2/ the identification of promising sublingual adjuvants.
The perspectives of BuccaVac are the in vivo validation of one of the two selected candidates. Immunisations on mouse model are ongoing to validate the induction of mucosal antibodies (anti-HA IgA) after sublinguale administration of formulations Patch-adjuvanted NPs-HA. Once the efficacy of the formulation confirmed, the WP3 will be condust as mentioned above.
PUBLICATIONS
1. Paris AL, Caridade S, Colomb E, Bellina M, Boucard E, Verrier B, Monge C. Sublingual protein delivery by a mucoadhesive patch made of natural polymers. Acta Biomater. 2021 Jul 1;128:222-235. doi: 10.1016/j.actbio.2021.04.024. Epub 2021 Apr 18. PMID: 33878475.
2. Paris AL, Colomb E, Verrier B, Anjuère F, Monge C. Sublingual vaccination and delivery systems. J Control Release. 2021 Apr 10;332:553-562. doi: 10.1016/j.jconrel.2021.03.017. Epub 2021 Mar 15. PMID: 33737202.
PATENT APPLICATION
« Système de libération de protéines par voie buccale » Submitted at INPI,12/11/2020, under the n° FR2011592
Mucosal vaccination mimics natural infection and induces a systemic and a local antibody secretion. Yet, there are still technological barriers to overcome for the development of this route of administration: increase the residence time of the antigen at the mucosal site, protect the antigen from degradation and induce an immune response at distant sites.
Herein we propose to develop a natural patch as an innovative carrier for buccal vaccine based on promising preliminary data. Such patch is obtained through the use of biodegradable polysaccharides as building blocks. The patch will contain a Flu antigen (model) and a specific mucosal adjuvant. This biomimetic patch will permit to deliver subunit vaccine components with a spatial and temporal control to optimize antigen uptake at the buccal mucosa/patch interface ensuring efficient immune responses. Patch efficacy will be assessed by sublingual administration on rodent and non-human primate.
Project coordination
Claire Monge (BIOLOGIE TISSULAIRE ET INGENIERIE THERAPEUTIQUE)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partner
LBTI - CNRS BIOLOGIE TISSULAIRE ET INGENIERIE THERAPEUTIQUE
Help of the ANR 316,312 euros
Beginning and duration of the scientific project:
January 2020
- 36 Months
