Semisynthetic Pneumococcal Glycoconjugate Nanovaccine.

Pneumococcal conjugate vaccines offer an excellent safety profile and high protection against the serotypes comprised in the vaccine. However, inclusion of protein antigens fromStreptococcus pneumoniaecombined with potent adjuvants and a suitable delivery system are expected to both extend protection to serotype strains not represented in the formulation and stimulate a broader immune response, thus more effective in young children, elderly, and immunocompromised populations. Along this line, nanoparticle (NP) delivery systems can enhance the immunogenicity of antigens by protecting them from degradation and increasing their uptake by antigen-presenting cells, as well as offering co-delivery with adjuvants. We report herein the encapsulation of a semisynthetic glycoconjugate (GC) composed of a synthetic tetrasaccharide mimicking theS. pneumoniae serotype 14 capsular polysaccharide (CP14) linked to the Pneumococcal surface protein A (PsaA) using chitosan NPs (CNPs). These GC-loaded chitosan nanoparticles (GC-CNPs) were not toxic to human monocyte-derived dendritic cells (MoDCs), showed enhanced uptake, and displayed better immunostimulatory properties in comparison to the naked GC. A comparative study was carried out in mice to evaluate the immune response elicited by the glycoconjugate-administered subcutaneously (SC), where the GC-CNPs displayed 100-fold higher IgG response as compared with the group treated with nonencapsulated GC. Overall, the study demonstrates the potential of this chitosan-based nanovaccine for efficient delivery of glycoconjugate antigens.

Nanoscale Mapping of Recombinant Viral Proteins: From Cells to Virus-Like Particles.

Influenza recombinant proteins and virus-like particles (VLPs) play an important role in vaccine development (e.g., CadiFlu-S). However, their production from mammalian cells suffers from low yields and lack of control of the final VLPs. To improve these issues, characterization techniques able to visualize and quantify the different steps of the process are needed. Fluorescence microscopy represents a powerful tool able to image multiple protein targets; however, its limited resolution hinders the study of viral constructs. Here, we propose the use of super-resolution microscopy and in particular of DNA-point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy as a characterization method for recombinant viral proteins on both cells and VLPs. We were able to quantify the amount of the three main influenza proteins (hemagglutinin (HA), neuraminidase (NA), and ion channel matrix protein 2 (M2)) per cell and per VLP with nanometer resolution and single-molecule sensitivity, proving that DNA-PAINT is a powerful technique to characterize recombinant viral constructs.

Role of Survivin in Bladder Cancer: Issues to Be Overcome When Designing an Efficient Dual Nano-Therapy.

Bladder cancer is the 10th most diagnosed cancer, with almost 10 M cancer deaths last year worldwide. Currently, chemotherapy is widely used as adjuvant therapy after surgical transurethral resection. Paclitaxel (PTX) is one of the most promising drugs, but cancer cells acquire resistance, causing failure of this treatment and increasing the recurrence of the disease. This poor chemotherapeutic response has been associated with the overexpression of the protein survivin. In this work, we present a novel dual nano-treatment for bladder cancer based on the hypothesis that the inhibition of survivin in cancer cells, using a siRNA gene therapy strategy, could decrease their resistance to PTX. For this purpose, two different polymeric nanoparticles were developed to encapsulate PTX and survivin siRNA independently. PTX nanoparticles showed sizes around 150 nm, with a paclitaxel loading of around 1.5%, that produced sustained tumor cell death. In parallel, siRNA nanoparticles, with similar sizes and loading efficiency of around 100%, achieved the oligonucleotide transfection and knocking down of survivin expression that also resulted in tumor cell death. However, dual treatment did not show the synergistic effect expected. The root cause of this issue was found to be the cell cycle arrest produced by nuclear survivin silencing, which is incompatible with PTX action. Therefore, we concluded that although the vastly reported role of survivin in bladder cancer, its silencing does not sensitize cells to currently applied chemotherapies.

Towards a Quantitative Single Particle Characterization by Super Resolution Microscopy: From Virus Structures to Antivirals Design.

In the last year the COVID19 pandemic clearly illustrated the potential threat that viruses pose to our society. The characterization of viral structures and the identification of key proteins involved in each step of the cycle of infection are crucial to develop treatments. However, the small size of viruses, invisible under conventional fluorescence microscopy, make it difficult to study the organization of protein clusters within the viral particle. The applications of super-resolution microscopy have skyrocketed in the last years, converting this group into one of the leading techniques to characterize viruses and study the viral infection in cells, breaking the diffraction limit by achieving resolutions up to 10 nm using conventional probes such as fluorescent dyes and proteins. There are several super-resolution methods available and the selection of the right one it is crucial to study in detail all the steps involved in the viral infection, quantifying and creating models of infection for relevant viruses such as HIV-1, Influenza, herpesvirus or SARS-CoV-1. Here we review the use of super-resolution microscopy (SRM) to study all steps involved in the viral infection and antiviral design. In light of the threat of new viruses, these studies could inspire future assays to unveil the viral mechanism of emerging viruses and further develop successful antivirals against them.

Synthetic Rhamnose Glycopolymer Cell-Surface Receptor for Endogenous Antibody Recruitment.

Synthetic materials capable of engineering the immune system are of great relevance in the fight against cancer to replace or complement the current monoclonal antibody and cell therapy-based immunotherapeutics. Here, we report on antibody recruiting glycopolymers (ARGPs). ARGPs consist of polymeric copies of a rhamnose motif, which can bind endogenous antirhamnose antibodies present in human serum. As a proof-of-concept, we have designed ARGPs with a lipophilic end group that efficiently inserts into cell-surface membranes. We validate the specificity of rhamnose to attract antibodies from human serum to the target cell surface and demonstrate that ARGPs outperform an analogous small-molecule compound containing only one single rhamnose motif. The ARGP concept opens new avenues for the design of potent immunotherapeutics that mark target cells for destruction by the immune system through antibody-mediated effector functions.

Super-resolution microscopy reveals significant impact of M2e-specific monoclonal antibodies on influenza A virus filament formation at the host cell surface.

Influenza A virions are highly pleomorphic, exhibiting either spherical or filamentous morphology. The influenza A virus strain A/Udorn/72 (H3N2) produces copious amounts of long filaments on the surface of infected cells where matrix protein 1 (M1) and 2 (M2) play a key role in virus filament formation. Previously, it was shown that an anti-M2 ectodomain (M2e) antibody could inhibit A/Udorn/72 virus filament formation. However, the study of these structures is limited by their small size and complex structure. Here, we show that M2e-specific IgG1 and IgG2a mouse monoclonal antibodies can reduce influenza A/Udorn/72 virus plaque growth and infectivity in vitro. Using Immuno-staining combined with super-resolution microscopy that allows us to study structures beyond the diffraction limit, we report that M2 is localized at the base of viral filaments that emerge from the membrane of infected cells. Filament formation was inhibited by treatment of A/Udorn/72 infected cells with M2e-specific IgG2a and IgG1 monoclonal antibodies and resulted in fragmentation of pre-existing filaments. We conclude that M2e-specific IgGs can reduce filamentous influenza A virus replication in vitro and suggest that in vitro inhibition of A/Udorn/72 virus replication by M2e-specific antibodies correlates with the inhibition of filament formation on the surface of infected cells.

Nanoscale Mapping Functional Sites on Nanoparticles by Points Accumulation for Imaging in Nanoscale Topography (PAINT).

The ability of nanoparticles to selectively recognize a molecular target constitutes the key toward nanomedicine applications such as drug delivery and diagnostics. The activity of such devices is mediated by the presence of multiple copies of functional molecules on the nanostructure surface. Therefore, understanding the number and the distribution of nanoparticle functional groups is of utmost importance for the rational design of effective materials. Analytical methods are available, but to obtain quantitative information at the level of single particles and single functional sites, i. e., going beyond the ensemble, remains highly challenging. Here we introduce the use of an optical nanoscopy technique, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT), to address this issue. Combining subdiffraction spatial resolution with molecular selectivity and sensitivity, DNA-PAINT provides both geometrical and functional information at the level of a single nanostructure. We show how DNA-PAINT can be used to image and quantify relevant functional proteins such as antibodies and streptavidin on nanoparticles and microparticles with nanometric accuracy in 3D and multiple colors. The generality and the applicability of our method without the need for fluorescent labeling hold great promise for the robust quantitative nanocharacterization of functional nanomaterials.

Balanced production of ribosome components is required for proper G1/S transition in Saccharomyces cerevisiae.

Cell cycle regulation is a very accurate process that ensures cell viability and the genomic integrity of daughter cells. A fundamental part of this regulation consists in the arrest of the cycle at particular points to ensure the completion of a previous event, to repair cellular damage, or to avoid progression in potentially risky situations. In this work, we demonstrate that a reduction in nucleotide levels or the depletion of RNA polymerase I or III subunits generates a cell cycle delay at the G1/S transition in Saccharomyces cerevisiae. This delay is concomitant with an imbalance between ribosomal RNAs and proteins which, among others, provokes an accumulation of free ribosomal protein L5. Consistently with a direct impact of free L5 on the G1/S transition, rrs1 mutants, which weaken the assembly of L5 and L11 on pre-60S ribosomal particles, enhance both the G1/S delay and the accumulation of free ribosomal protein L5. We propose the existence of a surveillance mechanism that couples the balanced production of yeast ribosomal components and cell cycle progression through the accumulation of free ribosomal proteins. This regulatory pathway resembles the p53-dependent nucleolar-stress checkpoint response described in human cells, which indicates that this is a general control strategy extended throughout eukaryotes.