Boosting antigen-specific T cell activation with lipid-stabilized protein nanoaggregates

Vaccines based on protein antigens have numerous advantages over inactivated pathogens, including easier manufacturing and improved safety. However, purified antigens are weakly immunogenic, as they lack the spatial organization and the associated 'danger signals' of the pathogen. Formulating vaccines as nanoparticles enhances the recognition by antigen presenting cells, boosting the cell-mediated immune response. This study describes a nano-precipitation method to obtain stable protein nanoaggregates with uniform size distribution without using covalent cross-linkers. Nanoaggregates were formed via microfluidic mixing of ovalbumin (OVA) and lipids in the presence of high methanol concentrations. A purification protocol was set up to separate the nanoaggregates from OVA and liposomes, obtained as byproducts of the mixing. The nanoaggregates were characterized in terms of morphology, zeta-potential and protein content, and their interaction with immune cells was assessed in vitro. Antigen-specific T cell activation was over 6-fold higher for nanoaggregates compared to OVA, due in part to the enhanced uptake by immune cells. Lastly, a two-dose immunization with nanoaggregates in mice induced a significant increase in OVA-specific CD8+ T splenocytes compared to soluble OVA. Overall, this work presents for the first time the microfluidic production of lipid-stabilized protein nanoaggregates and provides a proof-of-concept of their potential for vaccination

A preliminary investigation on hydrogel disks imbibed with enriched transfersomes as promising tool to promote the cutaneous deposition of vaccine antigens

The skin is an appealing and easily accessible route of administration for fast mass vaccination. However, it relies mainly on injection, meaning no immunization is achieved locally. In addition, pain, fear, and injuries strongly affect and reduce the effectiveness and adherence of patients to vaccination plans. Non-invasive routes of administration offer a safe and reliable solution. On these bases, three series of phospholipid vesicles were eco-friendly developed and adequately modified to pursue cutaneous immunization. Ovalbumin was selected as the model antigen, whereas transfersomes were chosen over liposomes because of their higher capability to cross the skin. Glycerol, sodium hyaluronate, or their combination were added to further improve their penetration, and this led to small (<60 nm), homogenous vesicles (polydispersity index <0.2), stable for up to 9 months. The colloidal formulations, alone or combined with occlusive strategies (achieved by cling film or hydrogel disks), were tested on tape-stripped skin to facilitate their application in a potential real-life context. While the enriched transfersomes alone provided better ovalbumin accumulation into the skin, the combination with cling film and especially with the hydrogel disks boosted their performance even further (using the hydrogel disks, in the epidermis, the ovalbumin accumulated ranged from ∼30 to ∼40 μg/cm2 and in the dermis from ∼4 to ∼10 μg/cm2). Despite the antigen-presenting assay do not show any immunogenicity for these vesicles (p > 0.05 with respect to the ovalbumin solution tested at the same dose), they are expected to perform better than the ovalbumin solution due to the protection and the enhanced skin deposition they provide. In addition, all the formulations can be safely administered as they are highly biocompatible in a wide range of doses (>100 % from 0.05 to 50 μg/mL of ovalbumin) and do not exert any inflammatory response. However, further studies are needed to confirm these promising preliminary results

Massive-Scale RNA-Seq Analysis of Non Ribosomal Transcriptome in Human Trisomy 21

Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high-throughput RNA sequencing have revealed the complexity of transcriptomes, most of them rely on polyA enrichment protocols, able to detect only a small fraction of total RNA content. On the opposite end, massive-scale RNA sequencing on rRNA-depleted samples allows the survey of the complete set of coding and non-coding RNA species, now emerging as novel contributors to pathogenic mechanisms. Hence, in this work we analysed for the first time the complete transcriptome of human trisomic endothelial progenitor cells to an unprecedented level of resolution and sensitivity by RNA-sequencing. Our analysis allowed us to detect differential expression of even low expressed genes crucial for the pathogenesis, to disclose novel regions of active transcription outside yet annotated loci, and to investigate a plethora of non-polyadenilated long as well as short non coding RNAs. Novel splice isoforms for a large subset of crucial genes, and novel extended untranslated regions for known genes—possibly novel miRNA targets or regulatory sites for gene transcription—were also identified in this study. Coupling the rRNA depletion of samples, followed by high-throughput RNA-sequencing, to the easy availability of these cells renders this approach very feasible for transcriptome studies, offering the possibility of investigating in-depth blood-related pathological features of Down syndrome, as well as other genetic disorders

Vectorized delivery of alpha-Galactosylceramide and tumor antigen on filamentous bacteriophage fd induces protective immunity by enhancing tumor-specific T cell response

We have exploited the properties of filamentous bacteriophage fd to deliver immunologically active lipids together with antigenic peptides. Filamentous bacteriophages resemble for size, capability to be permeable to blood vessels, and high density antigen expression, a nature-made nanoparticle. In addition, their major coat protein pVIII, which is arranged to form a tubular shield surrounding the phage genome, has a high content of hydrophobic residues promoting lipid association. We conjugated bacteriophages to alpha-GalactosylCeramide (α-GalCer), a lipid antigen-stimulating invariant natural killer T (iNKT) cells and capable of inducing their anti-tumoral activities. We found that bacteriophage fd/α-GalCer conjugates could repeatedly stimulate iNKT cells in vitro and in vivo, without inducing iNKT anergy. Moreover, co-delivery of α-GalCer and a MHC class I restricted tumor-associated antigenic determinant to antigen-presenting cells via bacteriophages strongly boosted adaptive CD8+ T cell response and efficiently delayed tumor progression. Co-delivery of a tumor antigen and iNKT-stimulatory lipid on the surface of filamentous bacteriophages is a novel approach to potentiate adaptive anti-cancer immune responses, overcoming the current limitations in the use of free α-GalCer and may represent an attractive alternative to existing delivery methods, opening the path to a potential translational usage of this safe, inexpensive, and versatile tool

Induction of human NK cell-mediated cytotoxicity by CD40 triggering on antigen presenting cells

Engagement of CD40 on antigen presenting cells (APC) is central to the initiation of cell-mediated immune response. Here, we investigated the ability of CD40 ligation on APC to induce NK cell-mediated cytotoxicity in the human system and the mechanism(s) underlying this process. We showed that APC (consisting in adherent peripheral blood mononuclear cells) (PBMC), prestimulated with anti-CD40 monoclonal antibodies and co-cultured with autologous non-adherent PBMC for 5-9 days, induced CD3-/CD56+ NK cell-mediated cytotoxicity as well as CD3+/CD56+ T cell-mediated unrestricted cytotoxic activity. The generation of NK cell-mediated cytotoxicity was independent on cell-to-cell contact between CD40-triggered APC and NK cells. Moreover. we found that IL-12 did not play a role in NK cells induction by anti-CD40 priming, while IL-2 and IL-15 did play a role. Our results provide an insight into the mechanism by which NK cells are activated in peripheral blood and useful informations for therapeutic application of anti-CD40 antibodies. (C) 2003 Elsevier Science (USA). All rights reserved

Triggering DTH and CTL Activity by fd Filamentous Bacteriophages: Role of CD4+ T Cells in Memory Responses

The ability of fd bacteriophage particles to trigger different arms of the immune system has been previously shown by us with particular emphasis on the ability of phages to raise CTL responses in vitro and in vivo. Here we show that fd virions in the absence of adjuvants are able to evoke a DTH reaction mediated by antigen specific CD8+ T cells. In addition, we analyzed the induction of CTL responses in mice depleted of CD4+ T cells, and we observed that short-term secondary CTL responses were induced in the absence of CD4+ T cells while induction of long-term memory CTLs required the presence of CD4+ T lymphocytes. These results examine the cellular mechanism at the basis of fd efficiency and provide new elements to further validate the use of fd particles for eliciting and monitoring antigen-specific CTLs

The use of filamentous bacteriophage fd to deliver MAGE-A10 or MAGE-A3 HLA-A2-restricted peptides and to induce strong antitumor CTL responses

Delivery of tumor-associated antigen (TAA)-derived peptides in a high immunogenic form represents one of the key issues for effective peptide-based cancer vaccine development. Here, we report the ability of non-pathogenic filamentous bacteriophage fd virions to deliver HLA-A2-restricted MAGE-A10254-262- or MAGE-A3271-279-derived peptides and elicit potent specific CTL responses in vitro and in vivo. Interestingly, human anti-MAGE-A3271-279-specific CTLs were able to kill human MAGE-A3+ tumor cells, even if these cells naturally express a low amount of MAGE-A3271-279 peptide-HLA epitope surface complexes and are usually not recognized by CTLs generated by conventional stimulation procedures. MAGE-A3271-279-specific/CD8+ CTL clones were isolated from in vitro cultures and their high avidity for antigen recognition was assessed. Moreover, in vivo tumor protection assay showed that vaccination of humanized HHD (HLA-A2.1+/H2-Db+) transgenic mice with phage particles expressing MAGE-A3271-279-derived peptides hampered tumor growth. Overall, these data indicate that engineered filamentous bacteriophage virions increase substantially the immunogenicity of delivered TAA-derived peptides, thus representing a novel powerful system for the development of effective peptide-based cancer vaccines.L'articolo è disponibile sul sito dell'editore http://www.jimmunol.or