Bernard Verrier

New insights in mucosal vaccine development.

Mucosal surfaces are the major entrance for infectious pathogens and therefore mucosal immune responses serve as a first line of defence. Most current immunization procedures are obtained by parenteral injection and only few vaccines are administered by mucosal route, because of its low efficiency. However, targeting of mucosal compartments to induce protective immunity at both mucosal sites and systemic level represents a great challenge. Major efforts are made to develop new mucosal candidate vaccines by selecting appropriate antigens with high immunogenicity, designing new mucosal routes of administration and selecting immune-stimulatory adjuvant molecules. The aim of mucosal vaccines is to induce broad potent protective immunity by specific neutralizing antibodies at mucosal surfaces and by induction of cellular immunity. Moreover, an efficient mucosal vaccine would make immunization procedures easier and be better suited for mass administration. This review focuses on contemporary developments of mucosal vaccination approaches using different routes of administration.

Type I IFN Counteracts the Induction of Antigen-Specific Immune Responses by Lipid-Based Delivery of mRNA Vaccines

The use of DNA and viral vector-based vaccines for the induction of cellular immune responses is increasingly gaining interest. However, concerns have been raised regarding the safety of these immunization strategies. Due to the lack of their genome integration, mRNA-based vaccines have emerged as a promising alternative. In this study, we evaluated the potency of antigen-encoding mRNA complexed with the cationic lipid 1,2-dioleoyl-3trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE ) as a novel vaccination approach. We demonstrate that subcutaneous immunization of mice with mRNA encoding the HIV-1 antigen Gag complexed with DOTAP/DOPE elicits antigen-specific, functional T cell responses resulting in specific killing of Gag peptide-pulsed cells and the induction of humoral responses. In addition, we show that DOTAP/DOPE complexed antigen-encoding mRNA displays immune-activating properties characterized by secretion of type I interferon (IFN) and the recruitment of proinflammatory monocytes to the draining lymph nodes. Finally, we demonstrate that type I IFN inhibit the expression of DOTAP/DOPE complexed antigen-encoding mRNA and the subsequent induction of antigen-specific immune responses. These results are of high relevance as they will stimulate the design and development of improved mRNA-based vaccination approaches.

Traffic of poly(lactic acid) nanoparticulate vaccine vehicle from intestinal mucus to sub-epithelial immune competent cells

Mucosal immunization is designed to induce strong immune responses at portal of pathogen entry. Unfortunately, mechanisms underlying the fate of the vaccine vector co-administered with antigens are still partially uncovered and limit further development of mucosal vaccines. Hence, poly(lactic acid) (PLA) nanoparticles being a versatile vaccine vehicle, we have analyzed the fate of these PLA nanoparticles during their uptake at intestinal mucosal sites, both in vivo and ex vivo, to decipher the mechanisms involved during this process. We first designed specific fluorescent PLA nanoparticles exhibiting strong colloidal stability after encapsulation of either 6-coumarin or CellTrace BODIPY before monitoring their transport through mucosa in the mouse ligated ileal loop model. The journey of the particles appears to follow a three-step process. Most particles are first entrapped in the mucus. Then, crossing of the epithelial barrier takes place exclusively through M-cells, leading to an accumulation in Peyers patches (PP). Lastly, we noticed specific interaction of these PLA nanoparticles with underlying B cells and dendritic cells (DCs) of PP. Furthermore, we could document that DCs engulfing some nanoparticles could exhibit a TLR8+ specific expression. Specific targeting of these two cell types strongly supports the use of PLA nanoparticles as a vaccine delivery system for oral use. Indeed, following oral gavage of mice with PLA nanoparticles, we were able to observe the same biodistribution patterns, indicating that these nanoparticles specifically reach immune target required for oral immunization.

Dectin-1 Is Essential for Reverse Transcytosis of Glycosylated SIgA-Antigen Complexes by Intestinal M Cells

This work reports the long-awaited identification of Dectin-1 and Siglec-5 as the M cell co-receptors that mediate the reverse transcytosis of secretory IgA molecules to mount a gut immune response.

Encapsulation of Nod1 and Nod2 receptor ligands into poly(lactic acid) nanoparticles potentiates their immune properties

Most successful vaccines are able to induce persistent antibody responses that can last a lifetime. Emerging evidences indicate that activation of immune cells through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) or Nod-like receptors (NLRs) may be critical mechanisms. Among PRRs, the use of TLR ligands as adjuvants is already largely described whereas the use of NLRs ligands remains largely unexplored. As activation of intracytoplasmic NLRs is able to induce proinflammatory molecules, the added value of encapsulation of Nod1 and Nod2 receptor ligands into Poly(Lactic Acid) (PLA) biodegradable nanocarriers to modulate their immune properties on human dendritic cells (DCs) maturation has been evaluated. Their ability to induce systemic immune responses in mice was also measured and compared to free ligands and the Alum adjuvant. Nod ligands encapsulated into PLA NPs were efficiently taken up by DCs and subsequently induced a strong up-regulation of maturation markers and the enhancement of proinflammatory cytokine secretion by DCs. Furthermore, co-injection of encapsulated Nod-ligands with PLA particles carrying Gag p24 HIV-1 antigen allowed a 100 fold increase in antibody responses in comparison to Alum. These results suggest that encapsulation of Nod ligands into PLA-NPs could be an effective way to improve vaccine efficiency.

The biased nucleotide composition of HIV-1 triggers type I interferon response and correlates with subtype D increased pathogenicity.

The genome of human immunodeficiency virus (HIV) has an average nucleotide composition strongly biased as compared to the human genome. The consequence of such nucleotide composition on HIV pathogenicity has not been investigated yet. To address this question, we analyzed the role of nucleotide bias of HIV-derived nucleic acids in stimulating type-I interferon response in vitro. We found that the biased nucleotide composition of HIV is detected in human cells as compared to humanized sequences, and triggers a strong innate immune response, suggesting the existence of cellular immune mechanisms able to discriminate RNA sequences according to their nucleotide composition or to detect specific secondary structures or linear motifs within biased RNA sequences. We then extended our analysis to the entire genome scale by testing more than 1300 HIV-1 complete genomes to look for an association between nucleotide composition of HIV-1 group M subtypes and their pathogenicity. We found that subtype D, which has an increased pathogenicity compared to the other subtypes, has the most divergent nucleotide composition relative to the human genome. These data support the hypothesis that the biased nucleotide composition of HIV-1 may be related to its pathogenicity.

UV and X-ray structural studies of a 101-residue long Tat protein from a HIV-1 primary isolate and of its mutated, detoxified, vaccine candidate

The 101‐residue long Tat protein of primary isolate 133 of the human immunodeficiency virus type 1 (HIV‐1), wt‐Tat133 displays a high transactivation activity in vitro, whereas the mutant thereof, STLA‐Tat133, a vaccine candidate for HIV‐1, has none. These two proteins were chemically synthesized and their biological activity was validated. Their structural properties were characterized using circular dichroism (CD), fluorescence emission, gel filtration, dynamic light scattering, and small angle X‐ray scattering (SAXS) techniques. SAXS studies revealed that both proteins were extended and belong to the family of intrinsically unstructured proteins. CD measurements showed that wt‐Tat133 or STLA‐Tat133 underwent limited structural rearrangements when complexed with specific fragments of antibodies. Crystallization trials have been performed on the two forms, assuming that the Tat133 proteins might have a better propensity to fold in supersaturated conditions, and small crystals have been obtained. These results suggest that biologically active Tat protein is natively unfolded and requires only a limited gain of structure for its function. Proteins 2010.

Particle-based transcutaneous administration of HIV-1 p24 protein to human skin explants and targeting of epidermal antigen presenting cells

Transcutaneous immunization is a promising vaccination strategy for the treatment of infectious diseases and cancer. In this study, we investigate the combination of cyanoacrylate skin surface stripping (CSSS) and particle-based antigen delivery to target the HIV-1 p24 protein to skin antigen presenting cells (APC). The CSSS treatment pre-activates skin APC and opens hair follicles, where protein-loaded particles accumulate and allow for sustained delivery of the loaded antigen to perifollicular APC. We found that poly-lactic acid (PLA) and polystyrene (PS) particles targeted the adsorbed HIV-1 p24 protein to the hair follicles. Small amounts of PS and PLA particles were found to translocate to the epidermis and be internalized by skin cells, whereas most of the particles aggregated in the hair follicle canal, where they released the loaded antigen. The p24 protein diffused to the epidermis and dermis and was detected in skin cells, especially in Langerhans cells and dermal dendritic cells. Furthermore, the combination of CSSS and particle-based delivery resulted in activation and maturation of Langerhans cells (HLA-DR, CD80 and CD83). We conclude that particle-based antigen delivery across partially disrupted skin barrier is a feasible and effective approach to needle-free transcutaneous vaccination.

Directing vaccine immune responses to mucosa by nanosized particulate carriers encapsulating NOD ligands

Mucosal surfaces are a major portal of entry for many pathogens that are the cause of infectious diseases. Therefore, effective vaccines that induce a protective immune response at these sites are much needed. However, despite early success with the live attenuated oral polio vaccine over 50 years ago, only a few new mucosal vaccines have been subsequently licensed. Development of new adjuvants, comprising antigen delivery platforms and immunostimulatory molecules, are critical for the successful development of new mucosal vaccines. Among them, biodegradable nanoparticle delivery systems are promising and NOD-like receptors are considered as potential new targets for immunostimulatory molecules. In this work, different NOD1 and NOD2 ligands were encapsulated in polylactic acid (PLA) nanoparticles, coated with HIV-1 gag p24 antigen. We showed that these new formulations are able to induce proliferation of HIV-specific T cells from HIV(+) individuals as well as autophagy. In vivo, these formulations highly enhanced p24-specific systemic and mucosal immune responses in mice not only after mucosal administration but also after immunization via the parenteral route. Our results provide a rational approach for combining nanosized particulate carriers and encapsulated NOD receptor ligands as potent synergistic tools for induction of specific mucosal immunity.

Elaboration of glycopolymer-functionalized micelles from an N-vinylpyrrolidone/lactide-based reactive copolymer platform.

Glycopolymer-corona-based micelles are obtained in a one-pot procedure, through reaction of D-mannosamine or D-glucosamine with the N-succinimidyl (NS) esters of a poly(D,L-lactide)-block-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (PLA-b-P(NAS-co-NVP)) amphiphilic copolymer (presenting quasi-alternating NAS/NVP units) in dimethyl sulfoxide (DMSO), followed by nanoprecipitation and dialysis against water. The glycopolymer micelles exhibit a higher CMC and size than those obtained from unmodified copolymer, due to increased hydrophilicity of the external block as a result of sugar derivatization, and the availability of the sugars at the micelle surface is evidenced through interactions with Concanavalin A (Con A) lectin, as attested by turbidimetric measurements and enzyme-linked lectin assay (ELLA). Interestingly, the glycopolymer micelles can be further used for hydrophobic molecule encapsulation and release, as shown with imiquimod, while keeping their interactions with con A intact. It is concluded that the PLA-based amphiphilic/reactive copolymer represents a versatile platform for glycopolymer-based micelle constructs for drug/vaccine delivery.