Therapeutic Effects of Zymomonas mobilis on Experimental DSS-Induced Colitis Mouse Model.

Zymomonas mobilis, a Gram-negative bacteria observed in some popular beverages, is considered safe and has been studied for its potential therapeutic benefits. In this study, we explored its effects on the inflammatory process, tissue integrity, differential gene expression, and microbiota composition in an experimental dextran sulfate sodium (DSS)-induced colitis model in mice. As a result, Z. mobilis alleviated the symptoms caused by DSS administration, as indicated by reduced weight loss, disease activity index, a significant reduction in the colon weight/length ratio, and histopathological improvement. Also, Z. mobilis could restore the mucosal barrier as well as increase the expression of Muc3 and Ocln genes. An analysis of 16S rRNA sequences showed that Z. mobilis alters gut microbiota, increasing Akkermansia muciniphila abundance and decreasing Escherichia coli. Furthermore, Z. mobilis seems to be involved in potentiating a regulatory phenotype by inducing immunomodulatory genes like Tgfb, Il5, Il10, and Foxp3 and reducing the relative mRNA expression of proinflammatory cytokines TNF, Il6, and Il17. Our data suggest that Z. mobilis could alleviate disease progression and be considered a possible probiotic adjuvant for pathologies of the bowel.

Progress on Phage Display Technology: Tailoring Antibodies for Cancer Immunotherapy.

The search for innovative anti-cancer drugs remains a challenge. Over the past three decades, antibodies have emerged as an essential asset in successful cancer therapy. The major obstacle in developing anti-cancer antibodies is the need for non-immunogenic antibodies against human antigens. This unique requirement highlights a disadvantage to using traditional hybridoma technology and thus demands alternative approaches, such as humanizing murine monoclonal antibodies. To overcome these hurdles, human monoclonal antibodies can be obtained directly from Phage Display libraries, a groundbreaking tool for antibody selection. These libraries consist of genetically engineered viruses, or phages, which can exhibit antibody fragments, such as scFv or Fab on their capsid. This innovation allows the in vitro selection of novel molecules directed towards cancer antigens. As foreseen when Phage Display was first described, nowadays, several Phage Display-derived antibodies have entered clinical settings or are undergoing clinical evaluation. This comprehensive review unveils the remarkable progress in this field and the possibilities of using clever strategies for phage selection and tailoring the refinement of antibodies aimed at increasingly specific targets. Moreover, the use of selected antibodies in cutting-edge formats is discussed, such as CAR (chimeric antigen receptor) in CAR T-cell therapy or ADC (antibody drug conjugate), amplifying the spectrum of potential therapeutic avenues.

New Anti-Flavivirus Fusion Loop Human Antibodies with Zika Virus-Neutralizing Potential.

Zika virus infections exhibit recurrent outbreaks and can be responsible for disease complications such as congenital Zika virus syndrome. Effective therapeutic interventions are still a challenge. Antibodies can provide significant protection, although the antibody response may fail due to antibody-dependent enhancement reactions. The choice of the target antigen is a crucial part of the process to generate effective neutralizing antibodies. Human anti-Zika virus antibodies were selected by phage display technology. The antibodies were selected against a mimetic peptide based on the fusion loop region in the protein E of Zika virus, which is highly conserved among different flaviviruses. Four rounds of selection were performed using the synthetic peptide in two strategies: the first was using the acidic elution of bound phages, and the second was by applying a competing procedure. After panning, the selected VH and VL domains were determined by combining NGS and bioinformatic approaches. Three different human monoclonal antibodies were expressed as scFvs and further characterized. All showed a binding capacity to Zika (ZIKV) and showed cross-recognition with yellow fever (YFV) and dengue (DENV) viruses. Two of these antibodies, AZ1p and AZ6m, could neutralize the ZIKV infection in vitro. Due to the conservation of the fusion loop region, these new antibodies can potentially be used in therapeutic intervention against Zika virus and other flavivirus illnesses.

In silico construction of a multiepitope Zika virus vaccine using immunoinformatics tools.

Zika virus (ZIKV) is an arbovirus from the Flaviviridae family and Flavivirus genus. Neurological events have been associated with ZIKV-infected individuals, such as Guillain-Barré syndrome, an autoimmune acute neuropathy that causes nerve demyelination and can induce paralysis. With the increase of ZIKV infection incidence in 2015, malformation and microcephaly cases in newborns have grown considerably, which suggested congenital transmission. Therefore, the development of an effective vaccine against ZIKV became an urgent need. Live attenuated vaccines present some theoretical risks for administration in pregnant women. Thus, we developed an in silico multiepitope vaccine against ZIKV. All structural and non-structural proteins were investigated using immunoinformatics tools designed for the prediction of CD4 + and CD8 + T cell epitopes. We selected 13 CD8 + and 12 CD4 + T cell epitopes considering parameters such as binding affinity to HLA class I and II molecules, promiscuity based on the number of different HLA alleles that bind to the epitopes, and immunogenicity. ZIKV Envelope protein domain III (EDIII) was added to the vaccine construct, creating a hybrid protein domain-multiepitope vaccine. Three high scoring continuous and two discontinuous B cell epitopes were found in EDIII. Aiming to increase the candidate vaccine antigenicity even further, we tested secondary and tertiary structures and physicochemical parameters of the vaccine conjugated to four different protein adjuvants: flagellin, 50S ribosomal protein L7/L12, heparin-binding hemagglutinin, or RS09 synthetic peptide. The addition of the flagellin adjuvant increased the vaccine's predicted antigenicity. In silico predictions revealed that the protein is a probable antigen, non-allergenic and predicted to be stable. The vaccine's average population coverage is estimated to be 87.86%, which indicates it can be administered worldwide. Peripheral Blood Mononuclear Cells (PBMC) of individuals with previous ZIKV infection were tested for cytokine production in response to the pool of CD4 and CD8 ZIKV peptide selected. CD4 + and CD8 + T cells showed significant production of IFN-γ upon stimulation and IL-2 production was also detected by CD8 + T cells, which indicated the potential of our peptides to be recognized by specific T cells and induce immune response. In conclusion, we developed an in silico universal vaccine predicted to induce broad and high-coverage cellular and humoral immune responses against ZIKV, which can be a good candidate for posterior in vivo validation.

A Germline-Encoded Structural Arginine Trap Underlies the Anti-DNA Reactivity of a Murine V Gene Segment.

Autoimmunity may have its origins of early repertoire selection in developmental B cells. Such a primary repertoire is probably shaped by selecting B cells that can efficiently perform productive signaling, stimulated by self-antigens in the bone marrow, such as DNA. In support of that idea, we previously found a V segment from VH10 family that can form antibodies that bind to DNA independent of CDR3 usage. In this paper we designed four antibody fragments in a novel single-chain pre-BCR (scpre-BCR) format containing germinal V gene segments from families known to bind DNA (VH10) or not (VH4) connected to a murine surrogate light chain (SLC), lacking the highly charged unique region (UR), by a hydrophilic peptide linker. We also tested the influence of CDR2 on DNA reactivity by shuffling the CDR2 loop. The scpre-BCRs were expressed in bacteria. VH10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the VH4 segment did not. The CDR2 loop shuffling hampered VH10 reactivity while displaying a gain-of-function in the nonbinding VH4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the VH10 CDR2 as the possible cross-reactive structural element. We presented evidence of DNA reactivity hardwired in a V gene, suggesting a structural mechanism for innate autoreactivity. Therefore, while autoreactivity to DNA can lead to autoimmunity, efficiently signaling for B cell development is likely a trade-off mechanism leading to the selection of potentially autoreactive repertoires.

Discovering Selected Antibodies From Deep-Sequenced Phage-Display Antibody Library Using ATTILA.

Phage display is a powerful technique to select high-affinity antibodies for different purposes, including biopharmaceuticals. Next-generation sequencing (NGS) presented itself as a robust solution, making it possible to assess billions of sequences of the variable domains from selected sublibraries. Handling this process, a central difficulty is to find the selected clones. Here, we present the AutomaTed Tool For Immunoglobulin Analysis (ATTILA), a new tool to analyze and find the enriched variable domains throughout a biopanning experiment. The ATTILA is a workflow that combines publicly available tools and in-house programs and scripts to find the fold-change frequency of deeply sequenced amplicons generated from selected VH and VL domains. We analyzed the same human Fab library NGS data using ATTILA in 5 different experiments, as well as on 2 biopanning experiments regarding performance, accuracy, and output. These analyses proved to be suitable to assess library variability and to list the more enriched variable domains, as ATTILA provides a report with the amino acid sequence of each identified domain, along with its complementarity-determining regions (CDRs), germline classification, and fold change. Finally, the methods employed here demonstrated a suitable manner to combine amplicon generation and NGS data analysis to discover new monoclonal antibodies (mAbs).

Multifaceted antibodies development against synthetic α-dystroglycan mucin glycopeptide as promising tools for dystroglycanopathies diagnostic.

Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient's quality of life. They are associated with altered glycosylation of the dystrophin-glycoprotein (DGC) complex components, such as α-dystroglycan (α-DG), fundamental in the structural and functional stability of the muscle fiber. The diagnosis of dystroglycanopathies is currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic hypoglycosylated muscle condition. Thus, modified α-DG mucins have been considered potential targets for the development of new diagnostic strategies toward these diseases. In this context, this work describes the synthesis of the hypoglycosylated α-DG mimetic glycopeptide NHAc-Gly-Pro-Thr-Val-Thr[αMan]-Ile-Arg-Gly-BSA (1) as a potential tool for the development of novel antibodies applicable to dystroglycanopathies diagnosis. Glycopeptide 1 was used for the development of polyclonal antibodies and recombinant monoclonal antibodies by Phage Display technology. Accordingly, polyclonal antibodies were reactive to glycopeptide 1, which enables the application of anti-glycopeptide 1 antibodies in immune reactive assays targeting hypoglycosylated α-DG. Regarding monoclonal antibodies, for the first time variable heavy (VH) and variable light (VL) immunoglobulin domains were selected by Phage Display, identified by NGS and described by in silico analysis. The best-characterized VH and VL domains were cloned, expressed in E. coli Shuffle T7 cells, and used to construct a single chain fragment variable that recognized the Glycopeptide 1 (GpαDG1 scFv). Molecular modelling of glycopeptide 1 and GpαDG1 scFv suggested that their interaction occurs through hydrogen bonds and hydrophobic contacts involving amino acids from scFv (I51, Y33, S229, Y235, and P233) and R8 and α-mannose from Glycopeptide 1.

Gene expression profile of human T cells following a single stimulation of peripheral blood mononuclear cells with anti-CD3 antibodies.

BACKGROUND: Anti-CD3 immunotherapy was initially approved for clinical use for renal transplantation rejection prevention. Subsequently, new generations of anti-CD3 antibodies have entered clinical trials for a broader spectrum of therapeutic applications, including cancer and autoimmune diseases. Despite their extensive use, little is known about the exact mechanism of these molecules, except that they are able to activate T cells, inducing an overall immunoregulatory and tolerogenic behavior. To better understand the effects of anti-CD3 antibodies on human T cells, PBMCs were stimulated, and then, we performed RNA-seq assays of enriched T cells to assess changes in their gene expression profiles. In this study, three different anti-CD3 antibodies were used for the stimulation: two recombinant antibody fragments, namely, a humanized and a chimeric FvFc molecule, and the prototype mouse mAb OKT3. RESULTS: Gene Ontology categories and individual immunoregulatory markers were compared, suggesting a similarity in modulated gene sets, mainly those for immunoregulatory and inflammatory terms. Upregulation of interleukin receptors, such as IL2RA, IL1R, IL12RB2, IL18R1, IL21R and IL23R, and of inhibitory molecules, such as FOXP3, CTLA4, TNFRSF18, LAG3 and PDCD1, were also observed, suggesting an inhibitory and exhausted phenotype. CONCLUSIONS: We used a deep transcriptome sequencing method for comparing three anti-CD3 antibodies in terms of Gene Ontology enrichment and immunological marker expression. The present data showed that both recombinant antibodies induced a compatible expression profile, suggesting that they might be candidates for a closer evaluation with respect to their therapeutic value. Moreover, the proposed methodology is amenable to be more generally applied for molecular comparison of cell receptor dependent antibody therapy.

Mucosal delivery of Lactococcus lactis carrying an anti-TNF scFv expression vector ameliorates experimental colitis in mice.

BACKGROUND: Anti-Tumor Necrosis Factor-alpha therapy has become clinically important for treating inflammatory bowel disease. However, the use of conventional immunotherapy requires a systemic exposure of patients and collateral side effects. Lactic acid bacteria have been shown to be effective as mucosal delivering system for cytokine and single domain antibodies, and it is amenable to clinical purposes. Therefore, lactic acid bacteria may function as vehicles for delivery of therapeutic antibodies molecules to the gastrointestinal tract restricting the pharmacological effect towards the gut. Here, we use the mucosal delivery of Lactococcus lactis carrying an anti-TNFα scFv expression plasmid on a DSS-induced colitis model in mice. RESULTS: Experimental colitis was induced with DSS administered in drinking water. L. lactis carrying the scFv expression vector was introduced by gavage. After four days of treatment, animals showed a significant improvement in histological score and disease activity index compared to those of untreated animals. Moreover, treated mice display IL-6, IL17A, IL1ß, IL10 and FOXP3 mRNA levels similar to health control mice. Therefore, morphological and molecular markers suggest amelioration of the experimentally induced colitis. CONCLUSION: These results provide evidence for the use of this alternative system for delivering therapeutic biopharmaceuticals in loco for treating inflammatory bowel disease, paving the way for a novel low-cost and site-specific biotechnological route for the treatment of inflammatory disorders.

MicroRNA expression profiles in human CD3+ T cells following stimulation with anti-human CD3 antibodies.

BACKGROUND: Anti-CD3 therapy can induce immunosuppression by several non mutually exclusive mechanisms that have been proposed to explain the therapeutic effect the administration anti-CD3 mAb, but its immunoregulatory mechanism is still not completely clear. In T cells, microRNAs (miRNAs) regulate several pathways, including those associated with immune tolerance. Here, we report changes in miRNA expression in T cells following treatment with anti-human CD3 antibodies. Peripheral blood mononuclear cells were cultured in the presence of the monoclonal antibody OKT3 or a recombinant fragment of humanized anti-CD3. Following these treatments, the expression profiles of 31 miRNA species were assessed in T cells using TaqMan arrays. RESULTS: Eight of the tested miRNAs (miR-155, miR-21, miR-146a, miR-210, miR-17, miR-590-5p, miR-106b and miR-301a) were statistically significantly up- or down-regulated relative to untreated cells. CONCLUSIONS: Stimulation of T cells with anti-human CD3 antibodies alters miRNA expression patterns, including of miRNA species associated with immune regulatory pathways.

Characterization of an In Vivo Z-DNA Detection Probe Based on a Cell Nucleus Accumulating Intrabody.

Left-handed Z-DNA is a physiologically unstable DNA conformation, and its existence in vivo can be attributed to localized torsional distress. Despite evidence for the existence of Z-DNA in vivo, its precise role in the control of gene expression is not fully understood. Here, an in vivo probe based on an anti-Z-DNA intrabody is proposed for native Z-DNA detection. The probe was used for chromatin immunoprecipitation of potential Z-DNA-forming sequences in the human genome. One of the isolated putative Z-DNA-forming sequences was cloned upstream of a reporter gene expression cassette under control of the CMV promoter. The reporter gene encoded an antibody fragment fused to GFP. Transient co-transfection of this vector along with the Z-probe coding vector improved reporter gene expression. This improvement was demonstrated by measuring reporter gene mRNA and protein levels and the amount of fluorescence in co-transfected CHO-K1 cells. These results suggest that the presence of the anti-Z-DNA intrabody can interfere with a Z-DNA-containing reporter gene expression. Therefore, this in vivo probe for the detection of Z-DNA could be used for global correlation of Z-DNA-forming sequences and gene expression regulation.

Identification and characterization of expressed retrotransposons in the genome of the Paracoccidioides species complex.

BACKGROUND: Species from the Paracoccidioides complex are thermally dimorphic fungi and the causative agents of paracoccidioidomycosis, a deep fungal infection that is the most prevalent systemic mycosis in Latin America and represents the most important cause of death in immunocompetent individuals with systemic mycosis in Brazil. We previously described the identification of eight new families of DNA transposons in Paracoccidioides genomes. In this work, we aimed to identify potentially active retrotransposons in Paracoccidioides genomes. RESULTS: We identified five different retrotransposon families (four LTR-like and one LINE-like element) in the genomes of three Paracoccidioides isolates. Retrotransposons were present in all of the genomes analyzed. P. brasiliensis and P. lutzii species harbored the same retrotransposon lineages but differed in their copy numbers. In the Pb01, Pb03 and Pb18 genomes, the number of LTR retrotransposons was higher than the number of LINE-like elements, and the LINE-like element RtPc5 was transcribed in Paracoccidioides lutzii (Pb01) but could not be detected in P. brasiliensis (Pb03 and Pb18) by semi-quantitative RT-PCR. CONCLUSION: Five new potentially active retrotransposons have been identified in the genomic assemblies of the Paracoccidioides species complex using a combined computational and experimental approach. The distribution across the two known species, P. brasiliensis and P. lutzii, and phylogenetics analysis indicate that these elements could have been acquired before speciation occurred. The presence of active retrotransposons in the genome may have implications regarding the evolution and genetic diversification of the Paracoccidioides genus.

Optimization of heterologous protein production in Chinese hamster ovary cells under overexpression of spliced form of human X-box binding protein.

BACKGROUND: The optimization of protein production is a complex and challenging problem in biotechnology. Different techniques for transcription, translation engineering and the optimization of cell culture conditions have been used to improve protein secretion, but there remain many open problems involving post-translational modifications of the secreted protein and cell line stability. RESULTS: In this work, we focus on the regulation of secreted protein specific productivity (using a recombinant human immunoglobulin G (IgG)) by controlling the expression of the spliced form of human X-box binding protein (XBP-(s)) in Chinese hamster ovary cells (CHO-K1) under doxycycline (DOX) induction at different temperatures. We observed a four-fold increase in specific IgG productivity by CHO cells under elevated concentrations of DOX at 30°C compared to 37°C, without detectable differences in binding activity in vitro or changes in the structural integrity of IgG. In addition, we found a correlation between the overexpression of human XBP-1(s) (and, as a consequence, endoplasmic reticulum (ER) size expansion) and the specific IgG productivity under DOX induction. CONCLUSIONS: Our data suggest the T-REx system overexpressing human XBP-1(s) can be successfully used in CHO-K1 cells for human immunoglobulin production.

A mouse variable gene fragment binds to DNA independently of the BCR context: a possible role for immature B-cell repertoire establishment.

B-cell maturation occurs in several steps and requires constant stimulus for its continuing development. From the emergence of the pre-B-cell receptor, signal transduction stimulates and supports B-cell development. Current viewpoints indicate that both positive selection pressure for autoantigens and tonic signaling constitutively stimulate B-cell maturation. In this work, we tested for the presence of a putative DNA binding site in a variable gene segment in a germline configuration, independently of VDJ recombination. After a survey of the public antibody databases, we chose a single mouse heavy variable gene segment that is highly represented in anti-nucleic acid antibodies and tested it for ssDNA binding. A phage display approach was used to search for intrinsic binding to oligo deoxythymidine. The results revealed that binding to an antigen can be influenced by the use of a specific DNA binding V[Formula: see text] gene segment. Our data support the idea that some variable genes have intrinsic reactivity towards specific types of endogenous autoantigens, and this property may contribute to the establishment of the immature B-cell repertoire.

Comparative genomic analysis of human fungal pathogens causing paracoccidioidomycosis.

Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.

Production of recombinant human factor VIII in different cell lines and the effect of human XBP1 co-expression.

Recombinant factor VIII is one of the most complex mammalian proteins and a biotechnology venture required for the treatment of hemophilia A. The complexity of the protein, post-translational modifications and limitations of expression elements make the production of active recombinant FVIII a challenge. Here we report the production of biologically active Factor VIII in two different cell lines, CHO and HepG2, by transient transfection. Two expression vectors based on the CMV promoter were used: one harboring CMV Intron A (InA) and the other without it. To bypass difficulties in secretion, we also studied the influence of co-expression of the human splice isoform of the XBP1 gene. We report the production of recombinant FVIII possessing bioengineered FVIII heavy and light chains, linked by a minimal B domain. In our study, HepG2, a human hepatocyte cell line, expressed Factor VIII ten-fold more than a CHO cell line, and in HepG2 cells, the expression of XBP1 improved Factor VIII activity. For CHO cells, expression was improved by the presence of InA, but no further improvement was noted with XBP1 co-expression. These data suggest that the minimal B domain rFVIII preserves Factor VIII biological activity and that different expression elements can be used to improve its production.

Production of a recombinant Fab in Pichia pastoris from a Monocistronic expression vector.

Recombinant Fab is usually expressed using dicistronic vectors producing the heavy and light chains separately. We developed an improved vector for Fab fragment expression in Pichia pastoris, which allows a stoichiometric expression of both chains based on a monocistronic arrangement. The protein is produced as a unique polypeptide harbouring a KEX2 processing site between both chains. After KEX cleavage, a correctly folded mature Fab is formed. The produced recombinant protein is characterized as a heterodimeric functional Fab. The vector described is a new tool for the proper expression of antibody fragments or any heterodimeric polypeptides.

Thermodynamic basis for antibody binding to Z-DNA: comparison of a monoclonal antibody and its recombinant derivatives.

Antibody engineering represents a promising area in biotechnology. Recombinant antibodies can be easily manipulated generating new ligand and effector activities that can be used as prototype magic bullets. On the other hand, an extensive knowledge of recombinant antibody binding and stability features are essential for an efficient substitution. In this study, we compared the stability and protein binding properties of two recombinant antibody fragments with their parental monoclonal antibody. The recombinant fragments were a monomeric scFv and a dimeric one, harboring human IgG1 CH2-CH3 domains. We have used fluorescence titration quenching to determine the thermodynamics of the interaction between an anti-Z-DNA monoclonal antibody and its recombinant antibody fragments with Z-DNA. All the antibody fragments seemed to bind DNA similarly, in peculiar two-affinity states. Enthalpy-entropy compensation was observed for both affinity states, but a marked entropy difference was observed for the monomeric scFv antibody fragment, mainly for the high affinity binding. In addition, we compared the stability of the dimeric antibody fragment and found differences favoring the monoclonal antibody. These differences seem to derive from the heterologous expression system used.