In vivo intranasal delivery of coagulation factor IX: a proof-of-concept study.

BACKGROUND: Hemophilia B (HB) is a bleeding disorder characterized by coagulation factor (F) IX (FIX) deficiency. The current standard-of-care for severe HB is prophylaxis with long-term repetitive intravenous (i.v.) infusions of recombinant FIX (rFIX) with standard half-life or extended half-life. Unmet needs remain regarding the development of non-invasive administration routes for coagulation factors. The aim of this study was to evaluate the effectiveness of intranasal delivery (IND) of rFIX and rFIX fused to Fc fragment (rFIX-Fc) in mice. METHODS: Drops of rFIX and rFIX-Fc were deposited in the nostrils of wild-type, FcRn knock-out, FcRn humanized, and FIX knock-out mice. rFIX mucosal uptake was evaluated by measuring plasma FIX antigen and FIX activity (FIX:C) levels, and by performing histologic analysis of the nasal mucosa following IND. RESULTS: After IND, both rFIX and rFIX-Fc were equally delivered to the blood compartment, irrespective of the mouse strain studied, mostly through a passive mechanism of transportation across the mucosal barrier, independent of FcRn receptor. Both plasma FIX antigen and FIX:C activity levels increased following IND in FIX knock-out mice. CONCLUSION: This proof-of-concept study describes evidence supporting the nasal route as an alternative to FIX i.v. infusion for the treatment of HB.

Poloxamers Have Vaccine-Adjuvant Properties by Increasing Dissemination of Particulate Antigen at Distant Lymph Nodes.

Vaccine technology is still facing challenges regarding some infectious diseases, which can be addressed by innovative drug delivery systems. In particular, nanoparticle-based vaccines combined with new types of adjuvants are actively explored as a platform for improving the efficacy and durability of immune protection. Here, biodegradable nanoparticles carrying an antigenic model of HIV were formulated with two combinations of poloxamers, 188/407, presenting or not presenting gelling properties, respectively. The study aimed to determine the influence of poloxamers (as a thermosensitive hydrogel or a liquid solution) on the adaptive immune response in mice. The results showed that poloxamer-based formulations were physically stable and did not induce any toxicity using a mouse dendritic cell line. Then, whole-body biodistribution studies using a fluorescent formulation highlighted that the presence of poloxamers influenced positively the dissemination profile by dragging nanoparticles through the lymphatic system until the draining and distant lymph nodes. The strong induction of specific IgG and germinal centers in distant lymph nodes in presence of poloxamers suggested that such adjuvants are promising components in vaccine development.

DLin-MC3-Containing mRNA Lipid Nanoparticles Induce an Antibody Th2-Biased Immune Response Polarization in a Delivery Route-Dependent Manner in Mice.

mRNA-based vaccines have made a leap forward since the SARS-CoV-2 pandemic and are currently used to develop anti-infectious therapies. If the selection of a delivery system and an optimized mRNA sequence are two key factors to reach in vivo efficacy, the optimal administration route for those vaccines remains unclear. We investigated the influence of lipid components and immunization route regarding the intensity and quality of humoral immune responses in mice. The immunogenicity of HIV-p55Gag encoded mRNA encapsulated into D-Lin-MC3-DMA or GenVoy-ionizable lipid-based LNPs was compared after intramuscular or subcutaneous routes. Three sequential mRNA vaccines were administrated followed by a heterologous boost composed of p24-HIV protein antigen. Despite equivalent IgG kinetic profiles of general humoral responses, IgG1/IgG2a ratio analysis showed a Th2/Th1 balance toward a Th1-biased cellular immune response when both LNPs were administrated via the intramuscular route. Surprisingly, a Th2-biased antibody immunity was observed when DLin-containing vaccine was injected subcutaneously. A protein-based vaccine boost appeared to reverse this balance to a cellular-biased response correlated to an increase in antibody avidity. Our finding suggests that the intrinsic adjuvant effect of ionizable lipids appears to be dependent on the delivery route used, which could be relevant to reach potent and long-lasting immunity after mRNA-based immunization.

Induction of a strong and long-lasting neutralizing immune response by dPreS1-TLR2 agonist nanovaccine against hepatitis B virus.

Hepatitis B virus remains a major medical burden with more than 250 million chronically infected patients worldwide and 900,000 deaths each year, due to the disease progression towards severe complications (cirrhosis, hepatocellular carcinoma). Despite the availability of a prophylactic vaccine, this infection is still pandemic in Western Pacific and African regions, where around 6% of the adult population is infected. Among novel anti-HBV strategies, innovative drug delivery systems, such as nanoparticle platforms to deliver vaccine antigens or therapeutic molecules have been investigated. Here, we developed polylactic acid-based biodegradable nanoparticles as an innovative and efficient vaccine. They are twice functionalized by (i) the entrapment of Pam3CSK4, an immunomodulator and ligand to Toll-Like-Receptor 1/2, and by (ii) the adsorption/coating of myristoylated (2-48) derived PreS1 from the HBV surface antigen, identified as the major viral attachment site on hepatocytes. We demonstrate that such formulations mimic HBV virion with an efficient peptide recognition by the immune system, and elicit potent and durable antibody responses in naive mice during at least one year. We also show that the most efficient in vitro viral neutralization was observed with NP-Pam3CSK4-dPreS1 sera. The immunogenicity of the derived HBV antigen is modulated by the likely synergistic action of both the dPreS1 coated nanovector and the adjuvant moiety. This formulation represents a promising vaccine alternative to fight HBV infection.

A Polylactide-Based Micellar Adjuvant Improves the Intensity and Quality of Immune Response.

Micelles from amphiphilic polylactide-block-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (PLA-b-P(NAS-co-NVP)) block copolymers of 105 nm in size were characterized and evaluated in a vaccine context. The micelles were non-toxic in vitro (both in dendritic cells and HeLa cells). In vitro fluorescence experiments combined with in vivo fluorescence tomography imaging, through micelle loading with the DiR near infrared probe, suggested an efficient uptake of the micelles by the immune cells. The antigenic protein p24 of the HIV-1 was successfully coupled on the micelles using the reactive N-succinimidyl ester groups on the micelle corona, as shown by SDS-PAGE analyses. The antigenicity of the coupled antigen was preserved and even improved, as assessed by the immuno-enzymatic (ELISA) test. Then, the performances of the micelles in immunization were investigated and compared to different p24-coated PLA nanoparticles, as well as Alum and MF59 gold standards, following a standardized HIV-1 immunization protocol in mice. The humoral response intensity (IgG titers) was substantially similar between the PLA micelles and all other adjuvants over an extended time range (one year). More interestingly, this immune response induced by PLA micelles was qualitatively higher than the gold standards and PLA nanoparticles analogs, expressed through an increasing avidity index over time (>60% at day 365). Taken together, these results demonstrate the potential of such small-sized micellar systems for vaccine delivery.

Polylactide-Based Reactive Micelles as a Robust Platform for mRNA Delivery.

PURPOSE: mRNA has recently emerged as a potent therapeutics and requires safe and effective delivery carriers, particularly prone to address its issues of poor stability and escape from endosomes. In this context, we designed poly(D,L-lactide) (PLA)-based micelles with N-succinimidyl (NS) ester decorated hydrophilic hairy corona to trap/couple a cationic fusogenic peptide and further complex mRNA. METHODS: Two strategies were investigated, namely (i) sequential immobilization of peptide and mRNA onto the micelles (layer-by-layer, LbL) or (ii) direct immobilization of peptide-mRNA pre-complex (PC) on the micelles. After characterization by means of size, surface charge, peptide/mRNA coupling/complexation and mRNA serum stability, carrier cytotoxicity and transfection capacity were evaluated with dendritic cells (DCs) using both GFP and luciferase mRNAs. RESULTS: Whatever the approach used, the micellar assemblies afforded full protection of mRNA in serum while the peptide-mRNA complex yielded complete mRNA degradation. In addition, the micellar assemblies allowed to significantly reduce the toxicity observed with the peptide-mRNA complex. They successfully transfected hard-to transfect DCs, with a superior efficiency for the LbL made ones (whatever mRNAs studied) showing the impact of the elaboration process on the carrier properties. CONCLUSIONS: These results show the relevance and potential of this new PLA/peptide based micelle platform to improve mRNA stability and delivery, while offering the possibility of further multifunctionality through PLA core encapsulation.

Recombinant Haemagglutinin Derived From the Ciliated Protozoan Tetrahymena thermophila Is Protective Against Influenza Infection.

Current influenza vaccines manufactured using eggs have considerable limitations, both in terms of scale up production and the potential impact passaging through eggs can have on the antigenicity of the vaccine virus strains. Alternative methods of manufacture are required, particularly in the context of an emerging pandemic strain. Here we explore the production of recombinant influenza haemagglutinin using the ciliated protozoan Tetrahymena thermophila. For the first time we were able to produce haemagglutinin from both seasonal influenza A and B strains. This ciliate derived material was immunogenic, inducing an antibody response in both mice and non-human primates. Mice immunized with ciliate derived haemagglutinin were protected against challenge with homologous influenza A or B viruses. The antigen could also be combined with submicron particles containing a Nod2 ligand, significantly boosting the immune response and reducing the dose of antigen required. Thus, we show that Tetrahymena can be used as a manufacturing platform for viral vaccine antigens.