Comparison of the Hi-C, GAM and SPRITE methods using polymer models of chromatin.

Hi-C, split-pool recognition of interactions by tag extension (SPRITE) and genome architecture mapping (GAM) are powerful technologies utilized to probe chromatin interactions genome wide, but how faithfully they capture three-dimensional (3D) contacts and how they perform relative to each other is unclear, as no benchmark exists. Here, we compare these methods in silico in a simplified, yet controlled, framework against known 3D structures of polymer models of murine and human loci, which can recapitulate Hi-C, GAM and SPRITE experiments and multiplexed fluorescence in situ hybridization (FISH) single-molecule conformations. We find that in silico Hi-C, GAM and SPRITE bulk data are faithful to the reference 3D structures whereas single-cell data reflect strong variability among single molecules. The minimal number of cells required in replicate experiments to return statistically similar contacts is different across the technologies, being lowest in SPRITE and highest in GAM under the same conditions. Noise-to-signal levels follow an inverse power law with detection efficiency and grow with genomic distance differently among the three methods, being lowest in GAM for genomic separations >1 Mb.

Polymer models are a versatile tool to study chromatin 3D organization.

The development of new experimental technologies is opening the way to a deeper investigation of the three-dimensional organization of chromosomes inside the cell nucleus. Genome architecture is linked to vital functional purposes, yet a full comprehension of the mechanisms behind DNA folding is still far from being accomplished. Theoretical approaches based on polymer physics have been employed to understand the complexity of chromatin architecture data and to unveil the basic mechanisms shaping its structure. Here, we review some recent advances in the field to discuss how Polymer Physics, combined with numerical Molecular Dynamics simulation and Machine Learning based inference, can capture important aspects of genome organization, including the description of tissue-specific structural rearrangements, the detection of novel, regulatory-linked architectural elements and the structural variability of chromatin at the single-cell level.

Inference of chromosome 3D structures from GAM data by a physics computational approach.

The combination of modelling and experimental advances can provide deep insights for understanding chromatin 3D organization and ultimately its underlying mechanisms. In particular, models of polymer physics can help comprehend the complexity of genomic contact maps, as those emerging from technologies such as Hi-C, GAM or SPRITE. Here we discuss a method to reconstruct 3D structures from Genome Architecture Mapping (GAM) data, based on PRISMR, a computational approach introduced to find the minimal polymer model best describing Hi-C input data from only polymer physics. After recapitulating the PRISMR procedure, we describe how we extended it for treating GAM data. We successfully test the method on a 6 Mb region around the Sox9 gene and, at a lower resolution, on the whole chromosome 7 in mouse embryonic stem cells. The PRISMR derived 3D structures from GAM co-segregation data are finally validated against independent Hi-C contact maps. The method results to be versatile and robust, hinting that it can be similarly applied to different experimental data, such as SPRITE or microscopy distance data.

Vaccination with (1-11)E2 in alum efficiently induces an antibody response to ß-amyloid without affecting brain ß-amyloid load and microglia activation in 3xTg mice.

Immunization against ß-amyloid (Aß) is pursued as a possible strategy for the prevention of Alzheimer's disease (AD). In clinical trials, Aß 1-42 proved poorly immunogenic and caused severe adverse effects; therefore, safer and more immunogenic candidate vaccines are needed. Multimeric protein (1-11)E2 is able to induce an antibody response to Aß, immunological memory, and IL-4 production, with no concomitant anti-Aß T cell response. Antisera recognize Aß oligomers, protofibrils, and fibrils. In this study, we evaluated the effect of prophylactic immunization with three doses of (1-11)E2 in alum in the 3xTg mouse model of AD. Immunization with (1-11)E2 efficiently induced anti-Aß antibodies, but afforded no protection against Aß accumulation and neuroinflammation. The identification of the features of the anti-Aß immune response that correlate with the ability to prevent Aß accumulation remains an open problem that deserves further investigation.

HIV Vaccination: A Roadmap among Advancements and Concerns.

Since the identification of the Human Immunodeficiency Virus type 1 (HIV-1) as the etiologic agent of AIDS (Acquired Immunodeficiency Syndrome), many efforts have been made to stop the AIDS pandemic. A major success of medical research has been the development of the highly active antiretroviral therapy and its availability to an increasing number of people worldwide, with a considerable effect on survival. However, a safe and effective vaccine able to prevent and eradicate the HIV pandemic is still lacking. Clinical trials and preclinical proof-of-concept studies in nonhuman primate (NHP) models have provided insights into potential correlates of protection against the HIV-1 infection, which include broadly neutralizing antibodies (bnAbs), non-neutralizing antibodies targeting the variable loops 1 and 2 (V1V2) regions of the HIV-1 envelope (Env), polyfunctional antibody, and Env-specific T-cell responses. In this review, we provide a brief overview of different HIV-1 vaccine approaches and discuss the current understanding of the cellular and humoral correlates of HIV-1 immunity.

E2 multimeric scaffold for vaccine formulation: immune response by intranasal delivery and transcriptome profile of E2-pulsed dendritic cells.

BACKGROUND: The E2 multimeric scaffold represents a powerful delivery system able to elicit robust humoral and cellular immune responses upon systemic administrations. Here recombinant E2 scaffold displaying the third variable loop of HIV-1 Envelope gp120 glycoprotein was administered via mucosa, and the mucosal and systemic immune responses were analysed. To gain further insights into the molecular mechanisms that orchestrate the immune response upon E2 vaccination, we analysed the transcriptome profile of dendritic cells (DCs) exposed to the E2 scaffold with the aim to define a specific gene expression signature for E2-primed immune responses. RESULTS: The in vivo immunogenicity and the potential of E2 scaffold as a mucosal vaccine candidate were investigated in BALB/c mice vaccinated via the intranasal route. Fecal and systemic antigen-specific IgA antibodies, cytokine-producing CD4(+) and CD8(+) cells were induced assessing the immunogenicity of E2 particles via intranasal administration. The cytokine analysis identified a mixed T-helper cell response, while the systemic antibody response showed a prevalence of IgG1 isotype indicative of a polarized Th2-type immune response. RNA-Sequencing analysis revealed that E2 scaffold up-regulates in DCs transcriptional regulators of the Th2-polarizing cell response, defining a type 2 DC transcriptomic signature. CONCLUSIONS: The current study provides experimental evidence to the possible application of E2 scaffold as antigen delivery system for mucosal immunization and taking advantages of genome-wide approach dissects the type of response induced by E2 particles.

Conformation regulation of the X chromosome inactivation center: a model.

X-Chromosome Inactivation (XCI) is the process whereby one, randomly chosen X becomes transcriptionally silenced in female cells. XCI is governed by the Xic, a locus on the X encompassing an array of genes which interact with each other and with key molecular factors. The mechanism, though, establishing the fate of the X's, and the corresponding alternative modifications of the Xic architecture, is still mysterious. In this study, by use of computer simulations, we explore the scenario where chromatin conformations emerge from its interaction with diffusing molecular factors. Our aim is to understand the physical mechanisms whereby stable, non-random conformations are established on the Xic's, how complex architectural changes are reliably regulated, and how they lead to opposite structures on the two alleles. In particular, comparison against current experimental data indicates that a few key cis-regulatory regions orchestrate the organization of the Xic, and that two major molecular regulators are involved.

Filamentous bacteriophage fd as an antigen delivery system in vaccination.

Peptides displayed on the surface of filamentous bacteriophage fd are able to induce humoral as well as cell-mediated immune responses, which makes phage particles an attractive antigen delivery system to design new vaccines. The immune response induced by phage-displayed peptides can be enhanced by targeting phage particles to the professional antigen presenting cells, utilizing a single-chain antibody fragment that binds dendritic cell receptor DEC-205. Here, we review recent advances in the use of filamentous phage fd as a platform for peptide vaccines, with a special focus on the use of phage fd as an antigen delivery platform for peptide vaccines in Alzheimer's Disease and cancer.

The Physics of DNA Folding: Polymer Models and Phase-Separation.

Within cell nuclei, several biophysical processes occur in order to allow the correct activities of the genome such as transcription and gene regulation. To quantitatively investigate such processes, polymer physics models have been developed to unveil the molecular mechanisms underlying genome functions. Among these, phase-separation plays a key role since it controls gene activity and shapes chromatin spatial structure. In this paper, we review some recent experimental and theoretical progress in the field and show that polymer physics in synergy with numerical simulations can be helpful for several purposes, including the study of molecular condensates, gene-enhancer dynamics, and the three-dimensional reconstruction of real genomic regions.

In-silico evaluation of adenoviral COVID-19 vaccination protocols: Assessment of immunological memory up to 6 months after the third dose.

Background: The immune response to adenoviral COVID-19 vaccines is affected by the interval between doses. The optimal interval is unknown. Aim: We aim to explore in-silico the effect of the interval between vaccine administrations on immunogenicity and to analyze the contribution of pre-existing levels of antibodies, plasma cells, and memory B and T lymphocytes. Methods: We used a stochastic agent-based immune simulation platform to simulate two-dose and three-dose vaccination protocols with an adenoviral vaccine. We identified the model's parameters fitting anti-Spike antibody levels from individuals immunized with the COVID-19 vaccine AstraZeneca (ChAdOx1-S, Vaxzevria). We used several statistical methods, such as principal component analysis and binary classification, to analyze the correlation between pre-existing levels of antibodies, plasma cells, and memory B and T cells to the magnitude of the antibody response following a booster dose. Results and conclusions: We find that the magnitude of the antibody response to a booster depends on the number of pre-existing memory B cells, which, in turn, is highly correlated to the number of T helper cells and plasma cells, and the antibody titers. Pre-existing memory T cytotoxic cells and antibodies directly influence antigen availability hence limiting the magnitude of the immune response. The optimal immunogenicity of the third dose is achieved over a large time window, spanning from 6 to 16 months after the second dose. Interestingly, after any vaccine dose, individuals can be classified into two groups, sustainers and decayers, that differ in the kinetics of decline of their antibody titers due to differences in long-lived plasma cells. This suggests that the decayers may benefit from a tailored boosting schedule with a shorter interval to avoid the temporary loss of serological immunity.

Physical mechanisms of chromatin spatial organization.

In higher eukaryotes, chromosomes have a complex three-dimensional (3D) conformation in the cell nucleus serving vital functional purposes, yet their folding principles remain poorly understood at the single-molecule level. Here, we summarize recent approaches from polymer physics to comprehend the physical mechanisms underlying chromatin architecture. In particular, we focus on two models that have been supported by recent, growing experimental evidence, the Loop Extrusion model and the Strings&Binders phase separation model. We discuss their key ingredients, how they compare to experimental data and some insight they provide on chromatin architecture and gene regulation. Progress in that research field are opening the possibility to predict how genomic mutations alter the network of contacts between genes and their regulators and how that is linked to genetic diseases, such as congenital disorders and cancer.

A multimeric immunogen for the induction of immune memory to beta-amyloid.

The development of active immunotherapy for Alzheimer's disease (AD) requires the identification of immunogens that can ensure a high titer antibody response toward beta-amyloid, whereas minimizing the risks of a cell-mediated adverse reaction. We describe here two novel anti-beta-amyloid vaccines that consist of 'virus like particles' formed by a domain of the bacterial protein E2 that is able to self-assemble into a 60-mer peptide. Peptides 1-11 and 2-6 of beta-amyloid were displayed as N terminal fusions on the surface of the E2 particles. E2-based vaccines induced a fast-rising, robust and persistent antibody response to beta-amyloid in all vaccinated mice. The immune memory induced by a single administration of vaccine (1-11) E2 can be rapidly mobilized by a single booster injection, leading to a very high serum concentration of anti-beta-amyloid antibodies (above 1 mg ml(-1)). E2 vaccination polarizes the immune response toward the production of the anti-inflammatory cytokine interleukin-4 and does not induce a T cell response to beta-amyloid. Thus, E2-based vaccines are promising candidates for the development of immunotherapy protocols for AD.

Increased Dickkopf-1 expression in transgenic mouse models of neurodegenerative disease.

To investigate the role of the Wnt inhibitor Dickkopf-1 (DKK-1) in the pathophysiology of neurodegenerative diseases, we analysed DKK-1 expression and localization in transgenic mouse models expressing familial Alzheimer's disease mutations and a frontotemporal dementia mutation. A significant increase of DKK-1 expression was found in the diseased brain areas of all transgenic lines, where it co-localized with hyperphosphorylated tau-bearing neurons. In TgCRND8 mice, DKK-1 immunoreactivity was detected in neurons surrounding amyloid deposits and within the choline acetyltransferase-positive neurons of the basal forebrain. Active glycogen synthase kinase-3 (GSK-3) was found to co-localize with DKK-1 and phospho-tau staining. Downstream to GSK-3, a significant reduction in beta-catenin translocation to the nucleus, indicative of impaired Wnt signaling functions, was found as well. Cumulatively, our findings indicate that DKK-1 expression is associated with events that lead to neuronal death in neurodegenerative diseases and support a role for DKK-1 as a key mediator of neurodegeneration with therapeutic potential.

Vaccination against ß-Amyloid as a Strategy for the Prevention of Alzheimer's Disease.

Vaccination relies on the phenomenon of immunity, a long-term change in the immunological response to subsequent encounters with the same pathogen that occurs after the recovery from some infectious diseases. However, vaccination is a strategy that can, in principle, be applied also to non-infectious diseases, such as cancer or neurodegenerative diseases, if an adaptive immune response can prevent the onset of the disease or modify its course. Immunization against ß-amyloid has been explored as a vaccination strategy for Alzheimer's disease for over 20 years. No vaccine has been licensed so far, and immunotherapy has come under considerable criticism following the negative results of several phase III clinical trials. In this narrative review, we illustrate the working hypothesis behind immunization against ß-amyloid as a vaccination strategy for Alzheimer's disease, and the outcome of the active immunization strategies that have been tested in humans. On the basis of the lessons learned from preclinical and clinical research, we discuss roadblocks and current perspectives in this challenging enterprise in translational immunology.

Analysis of the Consolidation Phase of Immunological Memory within the IgG Response to a B Cell Epitope Displayed on a Filamentous Bacteriophage.

Immunological memory can be defined as the ability to mount a response of greater magnitude and with faster kinetics upon re-encounter of the same antigen. We have previously reported that a booster dose of a protein antigen given 15 days after the first dose interferes with the development of memory, i.e., with the ability to mount an epitope-specific IgG response of greater magnitude upon re-encounter of the same antigen. We named the time-window during which memory is vulnerable to disruption a "consolidation phase in immunological memory", by analogy with the memory consolidation processes that occur in the nervous system to stabilize memory traces. In this study, we set out to establish if a similar memory consolidation phase occurs in the IgG response to a B cell epitope displayed on a filamentous bacteriophage. To this end, we have analyzed the time-course of anti-ß-amyloid IgG titers in mice immunized with prototype Alzheimer's Disease vaccine fdAD(2-6), which consists of a fd phage that displays the B epitope AEFRH of ß -amyloid at the N-terminus of the Major Capsid Protein. A booster dose of phage fdAD(2-6) given 15 days after priming significantly reduced the ratio between the magnitude of the secondary and primary IgG response to ß-amyloid. This analysis confirms, in a phage vaccine, a consolidation phase in immunological memory, occurring two weeks after priming.

A Dynamic Folded Hairpin Conformation Is Associated with α-Globin Activation in Erythroid Cells.

We investigate the three-dimensional (3D) conformations of the α-globin locus at the single-allele level in murine embryonic stem cells (ESCs) and erythroid cells, combining polymer physics models and high-resolution Capture-C data. Model predictions are validated against independent fluorescence in situ hybridization (FISH) data measuring pairwise distances, and Tri-C data identifying three-way contacts. The architecture is rearranged during the transition from ESCs to erythroid cells, associated with the activation of the globin genes. We find that in ESCs, the spatial organization conforms to a highly intermingled 3D structure involving non-specific contacts, whereas in erythroid cells the α-globin genes and their enhancers form a self-contained domain, arranged in a folded hairpin conformation, separated from intermingling flanking regions by a thermodynamic mechanism of micro-phase separation. The flanking regions are rich in convergent CTCF sites, which only marginally participate in the erythroid-specific gene-enhancer contacts, suggesting that beyond the interaction of CTCF sites, multiple molecular mechanisms cooperate to form an interacting domain.

Thermodynamic pathways to genome spatial organization in the cell nucleus.

The architecture of the eukaryotic genome is characterized by a high degree of spatial organization. Chromosomes occupy preferred territories correlated to their state of activity and, yet, displace their genes to interact with remote sites in complex patterns requiring the orchestration of a huge number of DNA loci and molecular regulators. Far from random, this organization serves crucial functional purposes, but its governing principles remain elusive. By computer simulations of a statistical mechanics model, we show how architectural patterns spontaneously arise from the physical interaction between soluble binding molecules and chromosomes via collective thermodynamics mechanisms. Chromosomes colocalize, loops and territories form, and find their relative positions as stable thermodynamic states. These are selected by thermodynamic switches, which are regulated by concentrations/affinity of soluble mediators and by number/location of their attachment sites along chromosomes. Our thermodynamic switch model of nuclear architecture, thus, explains on quantitative grounds how well-known cell strategies of upregulation of DNA binding proteins or modification of chromatin structure can dynamically shape the organization of the nucleus.

A thermodynamic switch for chromosome colocalization.

A general model for the early recognition and colocalization of homologous DNA sequences is proposed. We show, on thermodynamic grounds, how the distance between two homologous DNA sequences is spontaneously regulated by the concentration and affinity of diffusible mediators binding them, which act as a switch between two phases corresponding to independence or colocalization of pairing regions.

Immunogenicity and therapeutic efficacy of phage-displayed beta-amyloid epitopes.

In vitro and in vivo studies indicate that Alzheimer's Disease (AD) could be prevented or treated by active immunization against self-peptide beta-amyloid. In this study, we compared the immunogenicity of different regions of beta-amyloid, displayed on filamentous phages. We established that a filamentous phage displaying epitope 2-6 (AEFRH) of beta-amyloid at the N-terminus of Major Capside Protein (phage fdAD(2-6)) is more immunogenic than a phage displaying epitope 1-7 (DAEFRHD) that differs only in flanking residues. Monthly injections of fdAD(2-6) trigger a robust anti-beta-amyloid antibody response, and afford a significant reduction of plaque pathology in a mouse model of AD, whereas the same treatment, performed with phage fdAD(1-7), induces a lower anti-beta-amyloid titer and does not protect from amyloid deposition. "Memory" anti-amyloid antibodies induced by a single prime-boost cycle with vaccine fdAD(2-6), that have a lower titer compared to antibodies induced by monthly restimulations, do not prevent plaque pathology. Our data show that optimization of epitope display is essential in vaccine design, and suggest that the titer of the anti-amyloid response is the crucial parameter to obtain therapeutic efficacy in vivo.