Hepatitis C virus (HCV) infects 3% of world population being responsible for nearly half a million deaths annually urging the need for a prophylactic vaccine. Retrovirus like particles are commonly used scaffolds for antigens presentation being the core of diverse vaccine candidates. The immunogenicity of host proteins naturally incorporated in retrovirus was hypothesized to impact the performance of retrovirus based vaccines. In this work, the capacity of engineered retrovirus like particles devoided of host protein CD81 to display HCV envelope antigens was compared to non-engineered particles. A persistent inability of CD81 negative VLPs to incorporate HCV E2 protein as a result from the inefficient transport of HCV E2 to the plasma membrane, was observed. This work enabled the identification of a CD81-mediated transport of HCV E2 while stressing the importance of host proteins for the development of recombinant vaccines.

To generate a panel of antibodies binding human breast cancers, a human single chain Fv phage display library was selected for rapid internalization into the SK-BR-3 breast cancer cell line. Thirteen unique antibodies were identified within the 55 cell binding antibodies studied, all of them showing specific staining of tumor cells compare to normal epithelial cells. Two of the antibodies bound the ErbB2 oncogene while 6 bound the tumor marker transferrin receptor (TfR). By developing a scFv immunoprecipitation method, we were able to use LC-MS/MS to identify the antigen bound by one of the antibodies (3GA5) as FPRP (prostaglandin F2alpha receptor-regulatory protein)/EWI-F/CD9P-1 (CD9 partner 1) an Ig superfamily member that has been described to interact directly with CD9 and CD81 tetraspanins and to be overexpressed in adherent cancer cell lines. Although the 3GA5 scFv had no direct anti-proliferative effect, intracellular expression of the scFv was able to knockdown CD9P-1 expression and could be used to further define the role of the tetraspanin system in proliferation and metastasis. Moreover, the 3GA5 scFv was rapidly internalized into breast tumor cells and could have potential for the targeted delivery of cytotoxic agents to breast cancers. This study is the proof of principle that the direct selection of phage antibody libraries on tumor cells can effectively lead to the identification and functional characterization of relevant tumor markers.

Several studies have reported the roles of Trichinella spiralis extracellular vesicles in immune regulation and pathogen diagnosis. Currently, the T. spiralis muscle larvae excretory/secretory product (Ts-ML-ES) is the antigen recommended by the International Commission on Trichinellosis (ICT) for serological diagnosis of trichinellosis. However, it can only be used to detect middle and late stages of infections, and cross-reactions with other parasite detections occur. Therefore, there is a need to identify antigens for specific detection of early stage trichinellosis.

Immunotherapies with antibody-drug-conjugates (ADC) and CAR-T cells, targeted at tumor surface antigens (surfaceome), currently revolutionize clinical oncology. However, target identification warrants a better understanding of the surfaceome and how it is modulated by the tumor microenvironment. Here, we decode the surfaceome and endocytome and its remodeling by hypoxic stress in glioblastoma (GBM), the most common and aggressive brain tumor in adults. We employed a comprehensive approach for global and dynamic profiling of the surfaceome and endocytosed (endocytome) proteins and their regulation by hypoxia in patient-derived GBM cultures. We found a heterogeneous surface-endocytome profile and a divergent response to hypoxia across GBM cultures. We provide a quantitative ranking of more than 600 surface resident and endocytosed proteins, and their regulation by hypoxia, serving as a resource to the cancer research community. As proof-of-concept, the established target antigen CD44 was identified as a commonly and abundantly expressed surface protein with high endocytic activity. Among hypoxia induced proteins, we reveal CXADR, CD47, CD81, BSG, and FXYD6 as potential targets of the stressed GBM niche. We could validate these findings by immunofluorescence analyses in patient tumors and by increased expression in the hypoxic core of GBM spheroids. Selected candidates were finally confronted by treatment studies, showing their high capacity for internalization and ADC delivery. Importantly, we highlight the limited correlation between transcriptomics and proteomics, emphasizing the critical role of membrane protein enrichment strategies and quantitative mass spectrometry. Our findings provide a comprehensive understanding of the surface-endocytome and its remodeling by hypoxia in GBM as a resource for exploration of targets for immunotherapeutic approaches in GBM.

Despite extensive study of extracellular vesicles (EVs), specifically exosomes (EXs) as biomarkers, important modulators of physiological or pathological processes, or therapeutic agents, relatively little is known about nonconventional sources of EXs, such as invertebrate or plant EXs, and their uses. Likewise, there is no clear information on the overview of storage conditions and currently used isolation methods, including new ones, such as microfluidics, which fundamentally affect the characterization of EXs and their other biomedical applications. The purpose of this review is to briefly summarize conventional and nonconventional sources of EXs, storage conditions and typical isolation methods, widely used kits and new "smart" technologies with emphasis on the influence of isolation techniques on EX content, protein detection, RNA, mRNA and others. At the same time, attention is paid to a brief overview of the direction of biomedical application of EXs, especially in diagnostics, therapy, senescence and aging and, with regard to the current situation, in issues related to Covid-19.

Extracellular vesicles (EVs) are secreted from various types of cells and have specific functions related to their origin. EVs are observed in the small intestinal lamina propria (lpEVs), but their function remains unclear. This study aimed to investigate the role of lpEVs.

An effective prophylactic vaccine would be beneficial for controlling and eradicating hepatitis C virus (HCV) infections. However, the high diversity across HCV genotypes is a major challenge for vaccine development. Selection of the appropriate immunogen is critical to elicit broad HCV neutralizing antibodies (NAbs). To increase the antigenic coverage of heterodimer glycoproteins, we designed and produced recombinant E1E2 antigens for genotypes 1a/1b/2a/3a/6a from an IgG Fc-tagged precursor protein in FreeStyle 293-F cells. The recombinant E1 and E2 antigens were localized and associated with the endoplasmic reticulum and co-purified from membrane extracts. By examining the interactions with HCV entry co-receptors and the blockade of HCV infection, we found that these purified Fc-E1E2 proteins displayed correct folding and function. Mouse immunization results showed that each recombinant E1E2 antigen could elicit a pangenotypic antibody response to itself and other genotypes. We also found that the pentavalent formula triggered a relatively higher and more uniform NAb titer and T cell response than monovalent antigens. Taken together, our findings may provide a useful strategy for the vaccine development of HCV and other viruses with highly heterogeneous surface glycoproteins.

Proteomic profiling of human embryonic stem cells (hESC) can identify cell fate determination and self-renewal biomarkers. Employing Fourier transform LC-ESI-MS/MS and MS(3) mass spectrometry, we obtained a membrane proteomic signature overlapping between hESC cultured on mouse embryonic fibroblast (MEF) feeders and those grown under MEF-free culture conditions. We identified 444 transmembrane or membrane-associated proteins, of which 157 were common between both culture conditions. Functional annotation revealed CD antigens (10%), adhesion proteins (4%), proliferation-associated proteins (4%), receptors (41%), transport proteins (21%), structural proteins (5%), and proteins with miscellaneous functions (15%). In addition, 15 CD antigens and a number of surface marker molecules not previously observed in hESC at a proteome level, e.g., Nodal modulator 1, CD222, transgelin-2, and CD81, were identified. In conclusion, we describe the first membrane proteome profile of hESC that is independent of culture conditions. These data can be used to define the phenotype of hESC.

Exosomes secreted from Mycobacterium avium-infected macrophages contain numerous antigens of both M. avium and the host cell and are involved in the induction and expression of the inflammatory responses in macrophages. The interaction between exosomes secreted from M. avium-infected macrophages and macrophage phagocytosis, cytokine secretion, immunostimulation, and apoptosis was analyzed. Upon stimulation with exosomes secreted from M. avium-infected macrophages, the phagocytosis of dextran by treated macrophages was increased. Furthermore, the expression of CD40, CD80, CD81, CD86, HLA-DR, and most notably CD195 was enhanced. Additionally, the secretion of IL-6, IL-8, IL-10, IFN-γ, and TNF-α was increased by stimulated macrophages. Exosome stimulation did not induce macrophage apoptosis when compared with macrophages infected with M. avium. Caspase expression, including that of caspases 3, 6, and 8, was also not altered in exosome stimulated macrophages. Thus exosomes trigger the inflammatory response in macrophages owing to the presence of bacterial antigens but have no effect on macrophage viability.

The hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a non-covalently linked heterodimer on the viral surface that mediates viral entry. E1, E2 and the heterodimer complex E1E2 are candidate vaccine antigens, but are technically challenging to study because of difficulties in producing natively folded proteins by standard protein expression and purification methods. To better comprehend the antigenicity of these proteins, a library of alanine scanning mutants comprising the entirety of E1E2 (555 residues) was created for evaluating the role of each residue in the glycoproteins. The mutant library was probed, by a high-throughput flow cytometry-based assay, for binding with the co-receptor CD81, and a panel of 13 human and mouse monoclonal antibodies (mAbs) that target continuous and discontinuous epitopes of E1, E2, and the E1E2 complex. Together with the recently determined crystal structure of E2 core domain (E2c), we found that several residues in the E2 back layer region indirectly impact binding of CD81 and mAbs that target the conserved neutralizing face of E2. These findings highlight an unexpected role for the E2 back layer in interacting with the E2 front layer for its biological function. We also identified regions of E1 and E2 that likely located at or near the interface of the E1E2 complex, and determined that the E2 back layer also plays an important role in E1E2 complex formation. The conformation-dependent reactivity of CD81 and the antibody panel to the E1E2 mutant library provides a global view of the influence of each amino acid (aa) on E1E2 expression and folding. This information is valuable for guiding protein engineering efforts to enhance the antigenic properties and stability of E1E2 for vaccine antigen development and structural studies.

Coronavirus-2 (SARS-CoV-2) infection causes an acute respiratory syndrome accompanied by multi-organ damage that implicates a prothrombotic state leading to widespread microvascular clots. The causes of such coagulation abnormalities are unknown. The receptor tissue factor, also known as CD142, is often associated with cell-released extracellular vesicles (EV). In this study, we aimed to characterize surface antigens profile of circulating EV in COVID-19 patients and their potential implication as procoagulant agents.

MicroRNAs (miRNAs) in extracellular vesicles (EVs) play essential roles in cancer initiation and progression. Quantitative measurements of EV miRNAs are critical for cancer diagnosis and longitudinal monitoring. Traditional PCR-based methods, however, require multi-step procedures and remain as bulk analysis. Here, the authors introduce an amplification-free and extraction-free EV miRNA detection method using a CRISPR/Cas13a sensing system. CRISPR/Cas13a sensing components are encapsulated in liposomes and delivered them into EVs through liposome-EV fusion. This allows for accurately quantify specific miRNA-positive EV counts using 1 × 108  EVs. The authors show that miR-21-5p-positive EV counts are in the range of 2%-10% in ovarian cancer EVs, which is significantly higher than the positive EV counts from the benign cells (<0.65%). The result show an excellent correlation between bulk analysis with the gold-standard method, RT-qPCR. The authors also demonstrate multiplexed protein-miRNA analysis in tumor-derived EVs by capturing EpCAM-positive EVs and quantifying miR-21-5p-positive ones in the subpopulation, which show significantly higher counts in the plasma of cancer patients than healthy controls. The developed EV miRNA sensing system provides the specific miRNA detection method in intact EVs without RNA extraction and opens up the possibility of multiplexed single EV analysis for protein and RNA markers.

An inactivated whole-virus vaccine is currently used to prevent foot-and-mouth disease (FMD). Although this vaccine is effective, it offers short-term immunity that requires regular booster immunizations and has several side effects, including local reactions at the vaccination site. To address these limitations, herein, we evaluated the efficacy of bestatin as a novel small molecule adjuvant for inactivated FMD vaccines. Our findings showed that the FMD vaccine formulated with bestatin enhanced early, intermediate-, and particularly long-term immunity in experimental animals (mice) and target animals (pigs). Furthermore, cytokines (interferon (IFN)α, IFNβ, IFNγ, and interleukin (IL)-29), retinoic acid-inducible gene (RIG)-I, and T-cell and B-cell core receptors (cluster of differentiation (CD)28, CD19, CD21, and CD81) markedly increased in the group that received the FMD vaccine adjuvanted with bestatin in pigs compared with the control. These results indicate the significant potential of bestatin to improve the efficacy of inactivated FMD vaccines in terms of immunomodulatory function for the simultaneous induction of potent cellular and humoral immune response and a long-lasting memory response.

Human Gingival Mesenchymal Stem Cells (hGMSCs) are multipotential cells that can expand and differentiate in culture under specific and standardized conditions. In the present study, we have investigated whether in vitro pre-treatment of hGMSCs with Cannabidiol (CBD) can influence their expression profile, improving the therapeutic potential of this cell culture. Following CBD treatment (5 μM) for 24 h, gene expression analysis through Next Generation Sequencing (NGS) has revealed several genes differentially expressed between CBD-treated hGMSCs (CBD-hGMSCs) and control cells (CTR-hGMSCs) that were linked to inflammation and apoptosis. In particular, we have demonstrated that CBD treatment in hGMSCs prevented the activation of the NALP3-inflammasome pathway by suppressing the levels of NALP3, CASP1, and IL18, and in parallel, inhibited apoptosis, as demonstrated by the suppression of Bax. CBD treatment was also able to modulate the expression of the well-known mesenchymal stem cell markers (CD13, CD29, CD73, CD44, CD90, and CD166), and other surface antigens. Specifically, CBD led to the downregulation of genes codifying for antigens involved in the activation of the immune system (CD109, CD151, CD40, CD46, CD59, CD68, CD81, CD82, CD99), while it led to the upregulation of those implicated in the inhibition of the immune responses (CD47, CD55, CD276). In conclusion, the present study will provide a new simple and reproducible method for preconditioning hGMSCs with CBD, before transplantation, as an interesting strategy for improving the hGMSCs molecular phenotype, reducing the risk of immune or inflammatory reactions in the host, and in parallel, for increasing their survival and thus, their long-term therapeutic efficacy.

The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu3+-chelate or Eu3+-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2-10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145- and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa- cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs.

PRRSV is the etiological agent of one of the most important swine diseases with a significant economic burden worldwide and limitations in vaccinology. Exosomes are 30-100 nm vesicles of endocytic origin. Remarkably, immunizations with exosomes containing antigens from tumors or pathogens are capable of eliciting protective immune responses, albeit variably, in cancer and infectious diseases. Here we describe the isolation, molecular composition and immunogenicity of serum-derived exosomes from naïve animals, from PRRSV viremic animals and from animals previously PRRSV infected but already free of viruses (non viremic). Exosomes were isolated through size exclusion chromatography and characterized by different methodologies. Exosome-enriched fractions from naïve and natural infected animals contained classical tetraspanin exosomal markers (CD63 and CD81) and high concentrations of particles in the size-range of exosomes as detected by nanoparticle tracking analysis and cryo-TEM. NanoLC-MS/MS was used to identify viral antigens associated to exosomes. PRRSV-proteins were detected in serum samples from only viremic animals and from animals previously infected already free of viruses (non-viremic), but not in controls. Moreover, immune sera from pigs previously exposed to PRRSV specifically reacted against exosomes purified from non-viremic pig sera in a dose-dependent manner, a reactivity not detected when naïve sera was used in the assay. To facilitate future studies, a scaling-up process was implemented. To the best of our knowledge, this is the first molecular characterization of serum-derived exosomes from naïve pigs and pigs actively or previously infected with PRRSV. The presence of antigenic viral proteins in serum-derived exosomes free of virus, suggest their use as a novel vaccine approach against PRRSV.

The HIV-1 glycoprotein spike (gp120) is typically the first viral antigen that cells encounter before initiating immune responses, and is often the sole target in vaccine designs. Thus, characterizing the presence of cellular antigens on the surfaces of HIV particles may help identify new antiviral targets or impact targeting of gp120. Despite the importance of characterizing proteins on the virion surface, current techniques available for this purpose do not support high-throughput analysis of viruses, and typically only offer a semi-quantitative assessment of virus-associated proteins. Traditional bulk techniques often assess averages of viral preparations, which may mask subtle but important differences in viral subsets. On the other hand, microscopy techniques, which provide detail on individual virions, are difficult to use in a high-throughput manner and have low levels of sensitivity for antigen detection. Flow cytometry is a technique that traditionally has been used for rapid, high-sensitivity characterization of single cells, with limited use in detecting viruses, since the small size of viral particles hinders their detection. Herein, we report the detection and surface antigen characterization of HIV-1 pseudovirus particles by light scattering and fluorescence with flow cytometry, termed flow virometry for its specific application to viruses. We quantified three cellular proteins (integrin α4β7, CD14, and CD162/PSGL-1) in the viral envelope by directly staining virion-containing cell supernatants without the requirement of additional processing steps to distinguish virus particles or specific virus purification techniques. We also show that two antigens can be simultaneously detected on the surface of individual HIV virions, probing for the tetraspanin marker, CD81, in addition to α4β7, CD14, and CD162/PSGL-1. This study demonstrates new advances in calibrated flow virometry as a tool to provide sensitive, high-throughput characterization of the viral envelope in a more efficient, quantitative manner than previously reported techniques.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

Vaccine therapies based on virus-like particles (VLPs) are currently in the spotlight due to their potential for generating high immunogenic responses while presenting fewer side effects than conventional vaccines. These self-assembled nanostructures resemble the native conformation of the virus but lack genetic material. They are becoming a promising platform for vaccine candidates against several diseases due to the ability of modifying their membrane with antigens from different viruses. The coproduction of extracellular vesicles (EVs) when producing VLPs is a key phenomenon currently still under study. In order to characterize this extracellular environment, a quantitative proteomics approach has been carried out. Three conditions were studied: non-transfected, transfected with an empty plasmid as control, and transfected with a plasmid coding for HIV-1 Gag polyprotein. A shift in EV biogenesis has been detected upon transfection, changing the production from large to small EVs. Another remarkable trait found was the presence of DNA being secreted within vesicles smaller than 200 nm. Studying the protein profile of these biological nanocarriers, it was observed that EVs were reflecting an overall energy homeostasis disruption via mitochondrial protein deregulation. Also, immunomodulatory proteins like ITGB1, ENO3, and PRDX5 were identified and quantified in VLP and EV fractions. These findings provide insight on the nature of the VLP extracellular environment defining the characteristics and protein profile of EVs, with potential to develop new downstream separation strategies or using them as adjuvants in viral therapies.

Chronic infection with hepatitis C virus (HCV) remains a leading cause of liver-related pathologies and a global health problem, currently affecting more than 71 million people worldwide. The development of a prophylactic vaccine is much needed to complement the effective antiviral treatment available and achieve HCV eradication. Current strategies focus on increasing the immunogenicity of the HCV envelope glycoprotein E2, the major target of virus-neutralizing antibodies, by testing various expression systems or manipulating the protein conformation and the N-glycosylation pattern. Here we report the first evidence of successful production of the full-length HCV E2 glycoprotein in Nicotiana benthamiana, by using the Agrobacterium-mediated transient expression technology. Molecular and functional analysis showed that the viral protein was correctly processed in plant cells and achieved the native folding required for binding to CD81, one of the HCV receptors. N-glycan analysis of HCV-E2 produced in N. benthamiana and mammalian cells indicated host-specific trimming of mannose residues and possibly, protein trafficking. Notably, the plant-derived viral antigen triggered a significant immune response in vaccinated mice, characterized by the presence of antibodies with HCV-neutralizing activity. Together, our study demonstrates that N. benthamiana is a viable alternative to costly mammalian cell cultures for the expression of complex viral antigens and supports the use of plants as cost-effective production platforms for the development of HCV vaccines.