HCMV Envelope Glycoprotein Diversity Demystified

Human cytomegalovirus (HCMV) is the leading viral cause of congenital birth defects and is responsible for morbidity and mortality in immunosuppressed individuals. Considerable efforts have been deployed over the last decade to develop a vaccine capable of preventing HCMV infection. However, in recent clinical trials, vaccines showed at best modest efficacy in preventing infection. These findings might be explained by the high level of sequence polymorphism at the genomic level. To investigate if genomic variation also leads to antigenic variation, we performed a bioinformatic sequence analysis and evaluated the percentage of conservation at the amino acid level of all the proteins present in the virion envelope. Using more than two hundred sequences per envelope glycoprotein and analyzing their degree of conservation, we observe that antigenic variation is in large part limited to three proteins. In addition, we demonstrate that the two leading vaccine candidates, the pentamer and gB complexes, are well conserved at the amino acid level. These results suggest that despite genomic polymorphism, antigenic variability is not involved in the modest efficacy observed in the recent clinical trials for a HCMV vaccine. We therefore propose that next-generation vaccines should focus on stabilizing and refining the gB domains needed to induce a protective humoral response

HCMV envelope glycoprotein diversity demystified

Human cytomegalovirus (HCMV) is the leading viral cause of congenital birth defects and is responsible for morbidity and mortality in immunosuppressed individuals. Considerable efforts have been deployed over the last decade to develop a vaccine capable of preventing HCMV infection. However, in recent clinical trials, vaccines showed at best modest efficacy in preventing infection. These findings might be explained by the high level of sequence polymorphism at the genomic level. To investigate if genomic variation also leads to antigenic variation, we performed a bioinformatic sequence analysis and evaluated the percentage of conservation at the amino acid level of all the proteins present in the virion envelope. Using more than two hundred sequences per envelope glycoprotein and analyzing their degree of conservation, we observe that antigenic variation is in large part limited to three proteins. In addition, we demonstrate that the two leading vaccine candidates, the pentamer and gB complexes, are well conserved at the amino acid level. These results suggest that despite genomic polymorphism, antigenic variability is not involved in the modest efficacy observed in the recent clinical trials for a HCMV vaccine. We therefore propose that next-generation vaccines should focus on stabilizing and refining the gB domains needed to induce a protective humoral response

Noise in transcription negative feedback loops: simulation and experimental analysis

Negative feedback loops have been invoked as a way to control and decrease transcriptional noise. Here, we have built three circuits to test the effect of negative feedback loops on transcriptional noise of an autoregulated gene encoding a transcription factor (TF) and a downstream gene (DG), regulated by this TF. Experimental analysis shows that self-repression decreases noise compared to expression from a non-regulated promoter. Interestingly enough, we find that noise minimization by negative feedback loop is optimal within a range of repression strength. Repression values outside this range result in noise increase producing a U-shaped behaviour. This behaviour is the result of external noise probably arising from plasmid fluctuations as shown by simulation of the network. Regarding the target gene of a self-repressed TF (sTF), we find a strong decrease of noise when repression by the sTF is strong and a higher degree of noise anti-correlation between sTF and its target. Simulations of the circuits indicate that the main source of noise in these circuits could come from plasmid variation and therefore that negative feedback loops play an important role in suppressing both external and internal noise. An important observation is that DG expression without negative feedback exhibits bimodality at intermediate TF repression values. This bimodal behaviour seems to be the result of external noise as it can only be found in those simulations that include plasmid variation

ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production.

Chemotaxis is an essential physiological process, often harnessed by tumors for metastasis. CXCR4, its ligand CXCL12 and the atypical receptor ACKR3 are overexpressed in many human cancers. Interfering with this axis by ACKR3 deletion impairs lymphoma cell migration towards CXCL12. Here, we propose a model of how ACKR3 controls the migration of the diffused large B-cell lymphoma VAL cells in vitro and in vivo in response to CXCL12. VAL cells expressing full-length ACKR3, but not a truncated version missing the C-terminus, can support the migration of VAL cells lacking ACKR3 (VAL-ko) when allowed to migrate together. This migration of VAL-ko cells is pertussis toxin-sensitive suggesting the involvement of a Gi-protein coupled receptor. RNAseq analysis indicate the expression of chemotaxis-mediating LTB4 receptors in VAL cells. We found that LTB4 acts synergistically with CXCL12 in stimulating the migration of VAL cells. Pharmacologic or genetic inhibition of BLT1R markedly reduces chemotaxis towards CXCL12 suggesting that LTB4 enhances in a contact-independent manner the migration of lymphoma cells. The results unveil a novel mechanism of cell-to-cell-induced migration of lymphoma

STRING: known and predicted protein–protein associations, integrated and transferred across organisms

A full description of a protein's function requires knowledge of all partner proteins with which it specifically associates. From a functional perspective, ‘association’ can mean direct physical binding, but can also mean indirect interaction such as participation in the same metabolic pathway or cellular process. Currently, information about protein association is scattered over a wide variety of resources and model organisms. STRING aims to simplify access to this information by providing a comprehensive, yet quality-controlled collection of protein–protein associations for a large number of organisms. The associations are derived from high-throughput experimental data, from the mining of databases and literature, and from predictions based on genomic context analysis. STRING integrates and ranks these associations by benchmarking them against a common reference set, and presents evidence in a consistent and intuitive web interface. Importantly, the associations are extended beyond the organism in which they were originally described, by automatic transfer to orthologous protein pairs in other organisms, where applicable. STRING currently holds 730 000 proteins in 180 fully sequenced organisms, and is available at http://string.embl.de/

Patients with ankylosing spondylitis present a distinct CD8 T cell subset with osteogenic and cytotoxic potential.

OBJECTIVES Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease affecting mainly the axial skeleton. Peripheral involvement (arthritis, enthesitis and dactylitis) and extra-musculoskeletal manifestations, including uveitis, psoriasis and bowel inflammation, occur in a relevant proportion of patients. AS is responsible for chronic and severe back pain caused by local inflammation that can lead to osteoproliferation and ultimately spinal fusion. The association of AS with the human leucocyte antigen-B27 gene, together with elevated levels of chemokines, CCL17 and CCL22, in the sera of patients with AS, led us to study the role of CCR4+ T cells in the disease pathogenesis. METHODS CD8+CCR4+ T cells isolated from the blood of patients with AS (n=76) or healthy donors were analysed by multiparameter flow cytometry, and gene expression was evaluated by RNA sequencing. Patients with AS were stratified according to the therapeutic regimen and current disease score. RESULTS CD8+CCR4+ T cells display a distinct effector phenotype and upregulate the inflammatory chemokine receptors CCR1, CCR5, CX3CR1 and L-selectin CD62L, indicating an altered migration ability. CD8+CCR4+ T cells expressing CX3CR1 present an enhanced cytotoxic profile, expressing both perforin and granzyme B. RNA-sequencing pathway analysis revealed that CD8+CCR4+ T cells from patients with active disease significantly upregulate genes promoting osteogenesis, a core process in AS pathogenesis. CONCLUSIONS Our results shed light on a new molecular mechanism by which T cells may selectively migrate to inflammatory loci, promote new bone formation and contribute to the pathological ossification process observed in AS

Patients with ankylosing spondylitis present a distinct CD8 T cell subset with osteogenic and cytotoxic potential

OBJECTIVES Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease affecting mainly the axial skeleton. Peripheral involvement (arthritis, enthesitis and dactylitis) and extra-musculoskeletal manifestations, including uveitis, psoriasis and bowel inflammation, occur in a relevant proportion of patients. AS is responsible for chronic and severe back pain caused by local inflammation that can lead to osteoproliferation and ultimately spinal fusion. The association of AS with the human leucocyte antigen-B27 gene, together with elevated levels of chemokines, CCL17 and CCL22, in the sera of patients with AS, led us to study the role of CCR4$^{+}$ T cells in the disease pathogenesis. METHODS CD8$^{+}$CCR4$^{+}$ T cells isolated from the blood of patients with AS (n=76) or healthy donors were analysed by multiparameter flow cytometry, and gene expression was evaluated by RNA sequencing. Patients with AS were stratified according to the therapeutic regimen and current disease score. RESULTS CD8$^{+}$CCR4$^{+}$ T cells display a distinct effector phenotype and upregulate the inflammatory chemokine receptors CCR1, CCR5, CX3CR1 and L-selectin CD62L, indicating an altered migration ability. CD8$^{+}$CCR4$^{+}$ T cells expressing CX3CR1 present an enhanced cytotoxic profile, expressing both perforin and granzyme B. RNA-sequencing pathway analysis revealed that CD8$^{+}$CCR4$^{+}$ T cells from patients with active disease significantly upregulate genes promoting osteogenesis, a core process in AS pathogenesis. CONCLUSIONS Our results shed light on a new molecular mechanism by which T cells may selectively migrate to inflammatory loci, promote new bone formation and contribute to the pathological ossification process observed in AS

Prophylactic and postexposure efficacy of a potent human monoclonal antibody against MERS coronavirus

Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes severe respiratory disease with a high mortality rate. There is no licensed vaccine or antiviral for MERS. Here we isolated for the first time, to our knowledge, a potent MERS-CoV–neutralizing antibody from memory B cells of an infected individual. This antibody binds to a novel site on the viral Spike protein, neutralizes by interfering with the binding to the cellular receptor CD26, and is highly effective both in prophylaxis and in therapy in a relevant mouse model. This antibody can be developed for prophylaxis, for postexposure prophylaxis, or for the treatment of severe MERS-CoV infections

Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody.

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer's formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion

Development of broad‐spectrum human monoclonal antibodies for rabies post‐exposure prophylaxis

Currently available rabies post‐exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad‐spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non‐RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20–RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post‐vaccination antibody response