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.

Off the beaten path: Novel mRNA-nanoformulations for therapeutic vaccination against HIV.

Over the last few years, immunotherapy for HIV in general and therapeutic vaccination in particular, has received a tremendous boost, both in preclinical research and in clinical applications. This interest is based on the evidence that the immune system plays a crucial role in controlling HIV infection, as shown for long-term non-progressors and elite controllers, and that immune responses can be manipulated towards targeting conserved epitopes. So far, the most successful approach has been vaccination with autologous dendritic cells (DCs) loaded ex vivo with antigens and activation signals. Although this approach offers much promise, it also comes with significant drawbacks such as the requirement of a specialized infrastructure and expertise, as well as major challenges for logistics and storage, making it extremely time consuming and costly. Therefore, methods are being developed to avoid the use of ex vivo generated, autologous DCs. One of these methods is based on mRNA for therapeutic vaccination. mRNA has proven to be a very promising vaccine platform, as the coding information for any desired protein, including antigens and activation signals, can be generated in a very short period of time, showing promise both as an off-the-shelf therapy and as a personalized approach. However, an important drawback of this approach is the short half-life of native mRNA, due to the presence of ambient RNases. In addition, proper immunization requires that the antigens are expressed, processed and presented at the right immunological site (e.g. the lymphoid tissues). An ambivalent aspect of mRNA as a vaccine is its capacity to induce type I interferons, which can have beneficial adjuvant effects, but also deleterious effects on mRNA stability and translation. Thus, proper formulation of the mRNA is crucially important. Many approaches for RNA formulation have already been tested, with mixed success. In this review we discuss the state-of-the-art and future trends for mRNA-nanoparticle formulations for HIV vaccination, both in the prophylactic and in the therapeutic setting.

A Sacrificial PLA Block Mediated Route to Injectable and Degradable PNIPAAm-Based Hydrogels.

Thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-based injectable hydrogels represent highly attractive materials in tissue engineering and drug/vaccine delivery but face the problem of long-term bioaccumulation due to non-degradability. In this context, we developed an amphiphilic poly(D,L-lactide)-b-poly(NIPAAm-co-polyethylene glycol methacrylate) (PLA-b-P(NIPAAm-co-PEGMA)) copolymer architecture, through a combination of ring-opening and nitroxide-mediated polymerizations, undergoing gelation in aqueous solution near 30 °C. Complete hydrogel mass loss was observed under physiological conditions after few days upon PLA hydrolysis. This was due to the inability of the resulting P(NIPAAm-co-PEGMA) segment, that contains sufficiently high PEG content, to gel. The copolymer was shown to be non-toxic on dendritic cells. These results thus provide a new way to engineer safe PNIPAAm-based injectable hydrogels with PNIPAAm-reduced content and a degradable feature.

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.

Tailoring mRNA Vaccine to Balance Innate/Adaptive Immune Response.

mRNA vaccine platforms present numerous advantages, such as versatility, rapid production, and induction of cellular and humoral responses. Moreover, mRNAs have inherent adjuvant properties due to their complex interaction with pattern recognition receptors (PRRs). This recognition can be either beneficial in activating antigen-presenting cells (APCs) or detrimental by indirectly blocking mRNA translation. To decipher this Janus effect, we describe the different innate response mechanisms triggered by mRNA molecules and how each element from the 5' cap to the poly-A tail interferes with innate/adaptive immune responses. Then, we emphasize the importance of some critical steps such as production, purification, and formulation as key events to further improve the quality of immune responses and balance innate and adaptive immunity.

Self-assembled amphiphilic copolymers as dual delivery system for immunotherapy.

Subunit vaccines using recombinant antigens appear as the privileged vaccination technology for safety reasons but still require the development of carriers/adjuvants ensuring optimal immunogenicity and efficacy. Micelles from self-assembled amphiphilic copolymers have recently emerged as highly relevant and promising candidates owing to their ease of preparation, low size (entering in lymphatic capillaries for reaching lymph nodes), size/surface tunability and chemical versatility enabling introduction of stimuli (e.g. pH) responsive features and biofunctionalization with dedicated molecules. In particular, research efforts have increasingly focused on dendritic cells (DCs) targeting and activation by co-delivering (with antigen) ligands of pattern recognition receptors (PRRs, e.g. toll-like receptors). Such strategy has appeared as one of the most effective for eliciting CD 8+ T-cell response, which is crucial in the eradication of tumors and numerous infectious diseases. In this short review, we highlight the recent advances in such micelle-based carriers in subunit vaccination and how their precise engineering can be a strong asset for guiding and controlling immune responses.

Poly(lactic acid) nanoparticles and cell-penetrating peptide potentiate mRNA-based vaccine expression in dendritic cells triggering their activation.

Messenger RNA-based vaccines have the potential to trigger robust cytotoxic immune responses, which are essential for fighting cancer and infectious diseases like HIV. Dendritic Cells (DCs) are choice targets for mRNA-based vaccine strategies, as they link innate and adaptive immune responses and are major regulators of cytotoxic and humoral adaptive responses. However, efficient delivery of antigen-coding mRNAs into DC cytosol has been highly challenging. In this study, we developed an alternative to lipid-based mRNA delivery systems, using poly(lactic acid) nanoparticles (PLA-NPs) and cationic cell-penetrating peptides as mRNA condensing agent. The formulations are assembled in two steps: (1) formation of a polyplex between mRNAs and amphipathic cationic peptides (RALA, LAH4 or LAH4-L1), and (2) adsorption of polyplexes onto PLA-NPs. LAH4-L1/mRNA polyplexes and PLA-NP/LAH4-L1/mRNA nanocomplexes are taken up by DCs via phagocytosis and clathrin-dependent endocytosis, and induce strong protein expression in DCs in vitro. They modulate DC innate immune response by activating both endosome and cytosolic Pattern Recognition Receptors (PRRs), and induce markers of adaptive responses in primary human DCs in vitro, with prevalent Th1 signature. Thus, LAH4-L1/mRNA and PLA-NP/LAH4-L1/mRNA represent a promising platform for ex vivo treatment and mRNA vaccine development.

Nanoparticle-Based Dressing: The Future of Wound Treatment?

Reconstructing functional skin after a wound remains a challenge due to the complexity of healing. In this regard, biocompatible nanoparticles (NPs) carrying and releasing bioactive drugs in a controlled and sustained manner may significantly improve the efficacy of wound therapies compared with current treatments. Topical administration of drug-loaded NPs allows optimal delivery to the dermis and improves product efficacy. Furthermore, associating NPs with scaffolds represents a new concept of 'dressing'. Experimental in vivo, ex vivo, and in vitro models have been developed in preclinical assays to evaluate the beneficial effects of nanoparticulate dressings. Drug-loaded NPs are promising tools for innovative wound healing treatment, especially with regard to their multifunctional properties.

Identification of Novel Smoothened Ligands Using Structure-Based Docking.

The seven transmembrane protein Smoothened is required for Hedgehog signaling during embryonic development and adult tissue homeostasis. Inappropriate activation of the Hedgehog signalling pathway leads to cancers such as basal cell carcinoma and medulloblastoma, and Smoothened inhibitors are now available clinically to treat these diseases. However, resistance to these inhibitors rapidly develops thereby limiting their efficacy. The determination of Smoothened crystal structures enables structure-based discovery of new ligands with new chemotypes that will be critical to combat resistance. In this study, we docked 3.2 million available, lead-like molecules against Smoothened, looking for those with high physical complementarity to its structure; this represents the first such campaign against the class Frizzled G-protein coupled receptor family. Twenty-one high-ranking compounds were selected for experimental testing, and four, representing three different chemotypes, were identified to antagonize Smoothened with IC50 values better than 50 µM. A screen for analogs revealed another six molecules, with IC50 values in the low micromolar range. Importantly, one of the most active of the new antagonists continued to be efficacious at the D473H mutant of Smoothened, which confers clinical resistance to the antagonist vismodegib in cancer treatment.

Identification and Analysis of Antiviral Compounds Against Poliovirus.

The Global Polio Eradication Initiative, launched in 1988, had as its goal the eradication of polio worldwide by the year 2000 through large-scale vaccinations campaigns with the live attenuated oral PV vaccine (OPV) (Griffiths et al., Biologicals 34:73-74, 2006). Despite substantial progress, polio remains endemic in several countries and new imported cases are reported on a regular basis ( http://www.polioeradication.org/casecount.asp ).It was recognized by the poliovirus research community that developing antivirals against poliovirus would be invaluable in the post-OPV era. Here, we describe three methods essential for the identification of selective inhibitors of poliovirus replication and for determining their mode of action by time-of-drug-addition studies as well as by the isolation of compound-resistant poliovirus variants.

Toward antiviral therapy/prophylaxis for rhinovirus-induced exacerbations of chronic obstructive pulmonary disease: challenges, opportunities, and strategies.

Chronic obstructive pulmonary disease (COPD) is a life-threatening lung illness characterized by persistent and progressive airflow limitation. Exacerbations of COPD contribute to the severity of this pathology and accelerate disease progression. To date, pharmacological treatment of both stable COPD patients and patients experiencing exacerbations is mainly symptomatic with bronchodilators and steroids as the mainstay of therapy. Bacteria trigger such exacerbations in a number of cases; hence, antibiotics might be included in the treatment as well. Several respiratory viruses are frequently detected in sputum from patients during COPD exacerbations. These include influenza viruses, respiratory syncytial virus, and, most often, rhinoviruses. In this review, we discuss the potential use of an anti-rhinovirus drug for the treatment and prophylaxis of rhinovirus-induced COPD exacerbations and the path forward toward the development and use of such a drug. Copyright © 2015 John Wiley & Sons, Ltd.

In vitro characterisation of a pleconaril/pirodavir-like compound with potent activity against rhinoviruses.

BACKGROUND: Rhinovirus infections do not only cause common colds, but may also trigger severe exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Even though rhinoviruses have been the focus of extensive drug development efforts in the past, an anti-rhinoviral drug still has to make it to the market. In the past, the viral capsid protein VP1 has been shown to be an important target for the development of antiviral molecules. Furthermore, many different chemical scaffolds appear to possess the properties that are required to inhibit virus replication by this mechanism of action. I-6602, an analogue of the rhinovirus inhibitor pirodavir, was previously identified as a potent inhibitor of rhinovirus infection. Here, we describe the antiviral activity of its analogue ca603, a molecule with a modified linker structure, and corroborate its mechanism of action as a capsid binder. FINDINGS: The molecule ca603 shows antiviral activity against a panel of rhino-and enteroviruses. Cross-resistance is observed against viruses with mutations that render them resistant to the inhibitory effect of the capsid binder pleconaril and thermostability assays demonstrate that the compound binds and stabilizes the viral capsid. Binding of the molecule to the VP1 protein is corroborated by in silico modeling. CONCLUSIONS: It is confirmed that ca603 inhibits rhinovirus replication by interaction with the VP1 protein and, by this, allows to further expand the chemical diversity of capsid-binding molecules.

The enterovirus 3C protease inhibitor SG85 efficiently blocks rhinovirus replication and is not cross-resistant with rupintrivir.

The novel enterovirus protease inhibitor (PI) SG85 effectively inhibits the in vitro replication of 14 rhinoviruses representative of species A and B (median 50% effective concentration, 0.04 µM). A low-level SG85-resistant variant was selected that carried amino acid substitutions S127G and T143A in the 3C protease. Both substitutions are required for low-level resistance to SG85, as demonstrated by reverse genetics. Interestingly, there is no cross-resistance to SG85 and rupintrivir (another PI); a structural explanation is provided for this observation.

Exploration of the anti-enterovirus activity of a series of pleconaril/pirodavir-like compounds.

BACKGROUND: The Enterovirus genus of the Picornaviridae is represented by several viral pathogens that are associated with human disease, namely Poliovirus 1, Enterovirus 71 and Rhinoviruses. Enterovirus 71 has been associated with encephalitis, while Rhinoviruses are a major cause of asthma exacerbations and chronic obstructive pulmonary disease. Based on the structure of both pleconaril and pirodavir, we previously synthesized some original compounds as potential inhibitors of Rhinovirus replication. METHODS: These compounds were explored for in vitro antiviral potential on other human pathogenic Enteroviruses, namely Enterovirus 71 on rhabdo-myosarcoma cells, Coxsackievirus B3 on Vero cells, Poliovirus 1 and Echovirus 11 on BGM cells. RESULTS: Activity was confirmed for compound against Rhinovirus 14. Furthermore, few compounds showed a cell-protective effect on Enterovirus 71, presented a marked improvement as compared to the reference drug pleconaril for inhibitory activity on both Enterovirus 71 and Poliovirus 1. The most striking observation was the clear cell protective effect for the set of analogues in a virus-cell-based assay for Echovirus 11 with an effective concentration (EC50) as low as 0.3 µM (Selectivity index or SI = 483), and selectivity indexes greater than 857 (EC50 = 0.6 µM) and 1524 (EC50 = 0.33 µM). CONCLUSION: Some of the evaluated compounds showed potent and selective antiviral activity against several enterovirus species, such as Enterovirus 71 (EV-A), Echovirus 11 (EV-B), and Poliovirus 1 (EV-C). This could be used as a starting point for the development of other pleconaril/pirodavir-like enterovirus inhibitors with broad-spectrum activity and improved effects as compared to the reference drugs.

The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.

ZIPCO, a putative metal ion transporter, is crucial for Plasmodium liver-stage development.

The malaria parasite, Plasmodium, requires iron for growth, but how it imports iron remains unknown. We characterize here a protein that belongs to the ZIP (Zrt-, Irt-like Protein) family of metal ion transport proteins and have named ZIP domain-containing protein (ZIPCO). Inactivation of the ZIPCO-encoding gene in Plasmodium berghei, while not affecting the parasite's ability to multiply in mouse blood and to infect mosquitoes, greatly impairs its capacity to develop inside hepatocytes. Iron/zinc supplementation and depletion experiments suggest that ZIPCO is required for parasite utilization of iron and possibly zinc, consistent with its predicted function as a metal transporter. This is the first report of a ZIP protein having a crucial role in Plasmodium liver-stage development, as well as the first metal ion transporter identified in Plasmodium pre-erythrocytic stages. Because of the drastic dependence on iron of Plasmodium growth, ZIPCO and related proteins might constitute attractive drug targets to fight against malaria.

A cysteine protease inhibitor of plasmodium berghei is essential for exo-erythrocytic development.

Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.

A novel benzonitrile analogue inhibits rhinovirus replication.

OBJECTIVES: To study the characteristics and the mode of action of the anti-rhinovirus compound 4-[1-hydroxy-2-(4,5-dimethoxy-2-nitrophenyl)ethyl]benzonitrile (LPCRW_0005). METHODS: The antiviral activity of LPCRW_0005 was evaluated in a cytopathic effect reduction assay against a panel of human rhinovirus (HRV) strains. To unravel its precise molecular mechanism of action, a time-of-drug-addition study, resistance selection and thermostability assays were performed. The crystal structure of the HRV14/LPCRW_0005 complex was elucidated as well. RESULTS: LPCRW_0005 proved to be a selective inhibitor of the replication of HRV14 (EC(50) of 2 ±â€Š1 µM). Time-of-drug-addition studies revealed that LPCRW_0005 interferes with the earliest stages of virus replication. Phenotypic drug-resistant virus variants were obtained (≥30-fold decrease in susceptibility to the inhibitory effect of LPCRW_0005), which carried either an A150T or A150V amino acid substitution in the VP1 capsid protein. The link between the mutant genotype and drug-resistant phenotype was confirmed by reverse genetics. Cross-resistance studies and thermostability assays revealed that LPCRW_0005 has a similar mechanism of action to the capsid binder pleconaril. Elucidation of the crystal structure of the HRV14/LPCRW_0005 complex revealed the existence of multiple hydrophobic and polar interactions between the VP1 pocket and LPCRW_0005. CONCLUSIONS: LPCRW_0005 is a novel inhibitor of HRV14 replication that acts as a capsid binder. The compound has a chemical structure that is markedly smaller than that of other capsid binders. Structural studies show that LPCRW_0005, in contrast to pleconaril, leaves the toe end of the pocket in VP1 empty. This suggests that extended analogues of LPCRW_0005 that fill the full cavity could be more potent inhibitors of rhinovirus replication.

Synthesis, biological activity and structure-activity relationship of 4,5-dimethoxybenzene derivatives inhibitor of rhinovirus 14 infection.

Human rhinoviruses are a common cause of respiratory infections, and thus constitute an important target for medicinal chemistry. Still, no drug has been approved for clinical use. We report herein the discovery of dibenzenic derivatives with potent and specific in vitro anti-rhinoviral 14 activity. A total of 99 structural analogues were synthesized by an original synthesis method, i.e. through one organic agent Tetrakis(DimethylAmino)Ethylene (TDAE) and a structure-activity relationship was established. It was shown that 4,5-dimethoxy scaffold and the presence of a C-4 substituted aromatic moiety were necessary to the in vitro activity of these original agents. However, modifications on liker were not convincing. The benzonitrile derivative 23 was identified as the most potent and selective inhibitor of rhinovirus replication in these series (EC50 of 2 ± 0.5 µM, CC50 of 184 µM, selectivity index of 92).