Separating Actin-Dependent Chemokine Receptor Nanoclustering from Dimerization Indicates a Role for Clustering in CXCR4 Signaling and Function.

A current challenge in cell motility studies is to understand the molecular and physical mechanisms that govern chemokine receptor nanoscale organization at the cell membrane, and their influence on cell response. Using single-particle tracking and super-resolution microscopy, we found that the chemokine receptor CXCR4 forms basal nanoclusters in resting T cells, whose extent, dynamics, and signaling strength are modulated by the orchestrated action of the actin cytoskeleton, the co-receptor CD4, and its ligand CXCL12. We identified three CXCR4 structural residues that are crucial for nanoclustering and generated an oligomerization-defective mutant that dimerized but did not form nanoclusters in response to CXCL12, which severely impaired signaling. Overall, our data provide new insights to the field of chemokine biology by showing that receptor dimerization in the absence of nanoclustering is unable to fully support CXCL12-mediated responses, including signaling and cell function in vivo.

Cryo-EM structure of Shiga toxin 2 in complex with the native ribosomal P-stalk reveals residues involved in the binding interaction.

Shiga toxin 2a (Stx2a) is the virulence factor of enterohemorrhagic Escherichia coli. The catalytic A1 subunit of Stx2a (Stx2A1) interacts with the ribosomal P-stalk for loading onto the ribosome and depurination of the sarcin-ricin loop, which halts protein synthesis. Because of the intrinsic flexibility of the P-stalk, a structure of the Stx2a-P-stalk complex is currently unknown. We demonstrated that the native P-stalk pentamer binds to Stx2a with nanomolar affinity, and we employed cryo-EM to determine a structure of the 72 kDa Stx2a complexed with the P-stalk. The structure identifies Stx2A1 residues involved in binding and reveals that Stx2a is anchored to the P-stalk via only the last six amino acids from the C-terminal domain of a single P-protein. For the first time, the cryo-EM structure shows the loop connecting Stx2A1 and Stx2A2, which is critical for activation of the toxin. Our principal component analysis of the cryo-EM data reveals the intrinsic dynamics of the Stx2a-P-stalk interaction, including conformational changes in the P-stalk binding site occurring upon complex formation. Our computational analysis unveils the propensity for structural rearrangements within the C-terminal domain, with its C-terminal six amino acids transitioning from a random coil to an α-helix upon binding to Stx2a. In conclusion, our cryo-EM structure sheds new light into the dynamics of the Stx2a-P-stalk interaction and indicates that the binding interface between Stx2a and the P-stalk is the potential target for drug discovery.

New insights into the role of endosomal proteins for African swine fever virus infection.

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection.

Emerging Themes in CryoEM─Single Particle Analysis Image Processing.

Cryo-electron microscopy (CryoEM) has become a vital technique in structural biology. It is an interdisciplinary field that takes advantage of advances in biochemistry, physics, and image processing, among other disciplines. Innovations in these three basic pillars have contributed to the boosting of CryoEM in the past decade. This work reviews the main contributions in image processing to the current reconstruction workflow of single particle analysis (SPA) by CryoEM. Our review emphasizes the time evolution of the algorithms across the different steps of the workflow differentiating between two groups of approaches: analytical methods and deep learning algorithms. We present an analysis of the current state of the art. Finally, we discuss the emerging problems and challenges still to be addressed in the evolution of CryoEM image processing methods in SPA.

Structural analysis of receptors and actin polarity in platelet protrusions.

During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular-extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the αIIbß3 integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.

Discrimination of Etiologically Different Cholestasis by Modeling Proteomics Datasets.

Cholestasis is characterized by disrupted bile flow from the liver to the small intestine. Although etiologically different cholestasis displays similar symptoms, diverse factors can contribute to the progression of the disease and determine the appropriate therapeutic option. Therefore, stratifying cholestatic patients is essential for the development of tailor-made treatment strategies. Here, we have analyzed the liver proteome from cholestatic patients of different etiology. In total, 7161 proteins were identified and quantified, of which 263 were differentially expressed between control and cholestasis groups. These differential proteins point to deregulated cellular processes that explain part of the molecular framework of cholestasis progression. However, the clustering of different cholestasis types was limited. Therefore, a machine learning pipeline was designed to identify a panel of 20 differential proteins that segregate different cholestasis groups with high accuracy and sensitivity. In summary, proteomics combined with machine learning algorithms provides valuable insights into the molecular mechanisms of cholestasis progression and a panel of proteins to discriminate across different types of cholestasis. This strategy may prove useful in developing precision medicine approaches for patient care.

Scipion-EM-ProDy: A Graphical Interface for the ProDy Python Package within the Scipion Workflow Engine Enabling Integration of Databases, Simulations and Cryo-Electron Microscopy Image Processing.

Macromolecular assemblies, such as protein complexes, undergo continuous structural dynamics, including global reconfigurations critical for their function. Two fast analytical methods are widely used to study these global dynamics, namely elastic network model normal mode analysis and principal component analysis of ensembles of structures. These approaches have found wide use in various computational studies, driving the development of complex pipelines in several software packages. One common theme has been conformational sampling through hybrid simulations incorporating all-atom molecular dynamics and global modes of motion. However, wide functionality is only available for experienced programmers with limited capabilities for other users. We have, therefore, integrated one popular and extensively developed software for such analyses, the ProDy Python application programming interface, into the Scipion workflow engine. This enables a wider range of users to access a complete range of macromolecular dynamics pipelines beyond the core functionalities available in its command-line applications and the normal mode wizard in VMD. The new protocols and pipelines can be further expanded and integrated into larger workflows, together with other software packages for cryo-electron microscopy image analysis and molecular simulations. We present the resulting plugin, Scipion-EM-ProDy, in detail, highlighting the rich functionality made available by its development.

COVID-19 vaccine candidates based on modified vaccinia virus Ankara expressing the SARS-CoV-2 spike induce robust T- and B-cell immune responses and full efficacy in mice.

Vaccines against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are urgently needed. We developed two COVID-19 vaccines based on modified vaccinia virus Ankara (MVA) vectors expressing the entire SARS-CoV-2 spike (S) protein (MVA-CoV2-S); their immunogenicity was evaluated in mice using DNA/MVA or MVA/MVA prime/boost immunizations. Both vaccines induced robust, broad and polyfunctional S-specific CD4+ (mainly Th1) and CD8+ T-cell responses, with a T effector memory phenotype. DNA/MVA immunizations elicited higher T-cell responses. All vaccine regimens triggered high titers of IgG antibodies specific for the S, as well as for the receptor-binding domain; the predominance of the IgG2c isotype was indicative of Th1 immunity. Notably, serum samples from vaccinated mice neutralized SARS-CoV-2 in cell cultures, and those from MVA/MVA immunizations showed a higher neutralizing capacity. Remarkably, one or two doses of MVA-CoV2-S protect humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2. In addition, two doses of MVA-CoV2-S confer full inhibition of virus replication in the lungs. These results demonstrate the robust immunogenicity and full efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic.IMPORTANCE The continuous dissemination of the novel emerging SARS-CoV-2 virus, with more than 78 million infected cases worldwide and higher than 1,700,000 deaths as of December 23, 2020, highlights the urgent need for the development of novel vaccines against COVID-19. With this aim, we have developed novel vaccine candidates based on the poxvirus modified vaccinia virus Ankara (MVA) strain expressing the full-length SARS-CoV-2 spike (S) protein, and we have evaluated their immunogenicity in mice using DNA/MVA or MVA/MVA prime/boost immunization protocols. The results showed the induction of a potent S-specific T-cell response and high titers of neutralizing antibodies. Remarkably, humanized K18-hACE2 mice immunized with one or two doses of the MVA-based vaccine were 100% protected from SARS-CoV-2 lethality. Moreover, two doses of the vaccine prevented virus replication in lungs. Our findings prove the robust immunogenicity and efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic.

Enhancement of HIV-1 Env-Specific CD8 T Cell Responses Using Interferon-Stimulated Gene 15 as an Immune Adjuvant.

Induction of the endogenous innate immune system by interferon (IFN) triggers the expression of many proteins that serve like alarm bells in the body, activating an immune response. After a viral infection, one of the genes activated by IFN induction is the IFN-stimulated gene 15 (ISG15), which encodes a ubiquitin-like protein that undergoes a reversible posttranslational modification (ISGylation). ISG15 protein can also act unconjugated, intracellularly and secreted, acting as a cytokine. Although ISG15 has an essential role in host defense responses to microbial infection, its role as an immunomodulator in the vaccine field remains to be defined. In this investigation, we showed that ISG15 exerts an immunomodulatory role in human immunodeficiency virus (HIV) vaccines. In mice, after priming with a DNA-ISG15 vector mixed with a DNA expressing HIV-1 gp120 (DNA-gp120), followed by a booster with a modified vaccinia virus Ankara (MVA) vector expressing HIV-1 antigens, both wild-type ISG15-conjugated (ISG15-wt) and mutant unconjugated (ISG15-mut) proteins act as immune adjuvants by increasing the magnitude and quality of HIV-1-specific CD8 T cells, with ISG15-wt providing better immunostimulatory activity than ISG15-mut. The HIV-1 Env-specific CD8 T cell responses showed a predominant T effector memory (TEM) phenotype in all groups. Moreover, the amount of DNA-gp120 used to immunize mice could be reduced 5-fold after mixing with DNA-ISG15 without affecting the potency and the quality of the HIV-1 Env-specific immune responses. Our study clearly highlights the potential use of the IFN-induced ISG15 protein as immune adjuvant to enhance immune responses to HIV antigens, suggesting that this molecule might be exploitable for prophylactic and therapeutic vaccine approaches against pathogens.IMPORTANCE Our study described the potential role of ISG15 as an immunomodulatory molecule in the optimization of HIV/AIDS vaccine candidates. Using a DNA prime-MVA boost immunization protocol, our results indicated an increase in the potency and the quality of the HIV-1 Env-specific CD8 T cell response. These results highlight the adjuvant potency of ISG15 to elicit improved viral antigen presentation to the immune system, resulting in an enhanced HIV-1 vaccine immune response. The DNA-ISG15 vector could find applicability in the vaccine field in combination with other nucleic acid-based vector vaccines.

Automatic local resolution-based sharpening of cryo-EM maps.

MOTIVATION: Recent technological advances and computational developments have allowed the reconstruction of Cryo-Electron Microscopy (cryo-EM) maps at near-atomic resolution. On a typical workflow and once the cryo-EM map has been calculated, a sharpening process is usually performed to enhance map visualization, a step that has proven very important in the key task of structural modeling. However, sharpening approaches, in general, neglects the local quality of the map, which is clearly suboptimal. RESULTS: Here, a new method for local sharpening of cryo-EM density maps is proposed. The algorithm, named LocalDeblur, is based on a local resolution-guided Wiener restoration approach of the original map. The method is fully automatic and, from the user point of view, virtually parameter-free, without requiring either a starting model or introducing any additional structure factor correction or boosting. Results clearly show a significant impact on map interpretability, greatly helping modeling. In particular, this local sharpening approach is especially suitable for maps that present a broad resolution range, as is often the case for membrane proteins or macromolecules with high flexibility, all of them otherwise very suitable and interesting specimens for cryo-EM. To our knowledge, and leaving out the use of local filters, it represents the first application of local resolution in cryo-EM sharpening. AVAILABILITY AND IMPLEMENTATION: The source code (LocalDeblur) can be found at https://github.com/I2PC/xmipp and can be run using Scipion (http://scipion.cnb.csic.es) (release numbers greater than or equal 1.2.1). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Potent Anti-hepatitis C Virus (HCV) T Cell Immune Responses Induced in Mice Vaccinated with DNA-Launched RNA Replicons and Modified Vaccinia Virus Ankara-HCV.

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV) affecting 71 million people worldwide with no licensed vaccines that prevent infection. Here, we have generated four novel alphavirus-based DNA-launched self-amplifying RNA replicon (DREP) vaccines expressing either structural core-E1-E2 or nonstructural p7-NS2-NS3 HCV proteins of genotype 1a placed under the control of an alphavirus promoter, with or without an alphaviral translational enhancer (grouped as DREP-HCV or DREP-e-HCV, respectively). DREP vectors are known to induce cross-priming and further stimulation of immune responses through apoptosis, and here we demonstrate that they efficiently trigger apoptosis-related proteins in transfected cells. Immunization of mice with the DREP vaccines as the priming immunization followed by a heterologous boost with a recombinant modified vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV (MVA-HCV) induced potent and long-lasting HCV-specific CD4+ and CD8+ T cell immune responses that were significantly stronger than those of a homologous MVA-HCV prime/boost immunization, with the DREP-e-HCV/MVA-HCV combination the most immunogenic regimen. HCV-specific CD4+ and CD8+ T cell responses were highly polyfunctional, had an effector memory phenotype, and were mainly directed against E1-E2 and NS2-NS3, respectively. Additionally, DREP/MVA-HCV immunization regimens induced higher antibody levels against HCV E2 protein than homologous MVA-HCV immunization. Collectively, these results provided an immunization protocol against HCV by inducing high levels of HCV-specific T cell responses as well as humoral responses. These findings reinforce the combined use of DREP-based vectors and MVA-HCV as promising prophylactic and therapeutic vaccines against HCV.IMPORTANCE HCV represents a global health problem as more than 71 million people are chronically infected worldwide. Direct-acting antiviral agents can cure HCV infection in most patients, but due to the high cost of these agents and the emergence of resistant mutants, they do not represent a feasible and affordable strategy to eradicate the virus. Therefore, a vaccine is an urgent goal that requires efforts to understand the correlates of protection for HCV clearance. Here, we describe for the first time the generation of novel vaccines against HCV based on alphavirus DNA replicons expressing HCV antigens. We demonstrate that potent T cell immune responses, as well as humoral immune responses, against HCV can be achieved in mice by using a combined heterologous prime/boost immunization protocol consisting of the administration of alphavirus replicon DNA vectors as the priming immunization followed by a boost with a recombinant modified vaccinia virus Ankara vector expressing HCV antigens.

Validation of electron microscopy initial models via small angle X-ray scattering curves.

MOTIVATION: Cryo electron microscopy (EM) is currently one of the main tools to reveal the structural information of biological macromolecules. The re-construction of three-dimensional (3D) maps is typically carried out following an iterative process that requires an initial estimation of the 3D map to be refined in subsequent steps. Therefore, its determination is key in the quality of the final results, and there are cases in which it is still an open issue in single particle analysis (SPA). Small angle X-ray scattering (SAXS) is a well-known technique applied to structural biology. It is useful from small nanostructures up to macromolecular ensembles for its ability to obtain low resolution information of the biological sample measuring its X-ray scattering curve. These curves, together with further analysis, are able to yield information on the sizes, shapes and structures of the analyzed particles. RESULTS: In this paper, we show how the low resolution structural information revealed by SAXS is very useful for the validation of EM initial 3D models in SPA, helping the following refinement process to obtain more accurate 3D structures. For this purpose, we approximate the initial map by pseudo-atoms and predict the SAXS curve expected for this pseudo-atomic structure. The match between the predicted and experimental SAXS curves is considered as a good sign of the correctness of the EM initial map. AVAILABILITY AND IMPLEMENTATION: The algorithm is freely available as part of the Scipion 1.2 software at http://scipion.i2pc.es/.

Fast multiscale reconstruction for Cryo-EM.

We present a multiscale reconstruction framework for single-particle analysis (SPA). The representation of three-dimensional (3D) objects with scaled basis functions permits the reconstruction of volumes at any desired scale in the real-space. This multiscale approach generates interesting opportunities in SPA for the stabilization of the initial volume problem or the 3D iterative refinement procedure. In particular, we show that reconstructions performed at coarse scale are more robust to angular errors and permit gains in computational speed. A key component of the proposed iterative scheme is its fast implementation. The costly step of reconstruction, which was previously hindering the use of advanced iterative methods in SPA, is formulated as a discrete convolution with a cost that does not depend on the number of projection directions. The inclusion of the contrast transfer function inside the imaging matrix is also done at no extra computational cost. By permitting full 3D regularization, the framework is by itself a robust alternative to direct methods for performing reconstruction in adverse imaging conditions (e.g., heavy noise, large angular misassignments, low number of projections). We present reconstructions obtained at different scales from a dataset of the 2015/2016 EMDataBank Map Challenge. The algorithm has been implemented in the Scipion package.

The first single particle analysis Map Challenge: A summary of the assessments.

The recent successes of cryo-electron microscopy fostered great expectation of solving many new and previously recalcitrant biomolecular structures. However, it also brings with it the danger of compromising the validity of the outcomes if not done properly. The Map Challenge is a first step in assessing the state of the art and to shape future developments in data processing. The organizers presented seven cases for single particle reconstruction, and 27 members of the community responded with 66 submissions. Seven groups analyzed these submissions, resulting in several assessment reports, summarized here. We devised a range of analyses to evaluate the submitted maps, including visual impressions, Fourier shell correlation, pairwise similarity and interpretation through modeling. Unfortunately, we did not find strong trends. We ascribe this to the complexity of the challenge, dealing with multiple cases, software packages and processing approaches. This puts the user in the spotlight, where his/her choices becomes the determinant of map quality. The future focus should therefore be on promulgating best practices and encapsulating these in the software. Such practices include adherence to validation principles, most notably the processing of independent sets, proper resolution-limited alignment, appropriate masking and map sharpening. We consider the Map Challenge to be a highly valuable exercise that should be repeated frequently or on an ongoing basis.

Distinct Roles of Vaccinia Virus NF-κB Inhibitor Proteins A52, B15, and K7 in the Immune Response.

Poxviruses use a complex strategy to escape immune control, by expressing immunomodulatory proteins that could limit their use as vaccine vectors. To test the role of poxvirus NF-κB pathway inhibitors A52, B15, and K7 in immunity, we deleted their genes in an NYVAC (New York vaccinia virus) strain that expresses HIV-1 clade C antigens. After infection of mice, ablation of the A52R, B15R, and K7R genes increased dendritic cell, natural killer cell, and neutrophil migration as well as chemokine/cytokine expression. Revertant viruses with these genes confirmed their role in inhibiting the innate immune system. To different extents, enhanced innate immune responses correlated with increased HIV Pol- and Gag-specific polyfunctional CD8 T cell and HIV Env-specific IgG responses induced by single-, double-, and triple-deletion mutants. These poxvirus proteins thus influence innate and adaptive cell-mediated and humoral immunity, and their ablation offers alternatives for design of vaccine vectors that regulate immune responses distinctly.IMPORTANCE Poxvirus vectors are used in clinical trials as candidate vaccines for several pathogens, yet how these vectors influence the immune system is unknown. We developed distinct poxvirus vectors that express heterologous antigens but lack different inhibitors of the central host-cell signaling pathway. Using mice, we studied the capacity of these viruses to induce innate and adaptive immune responses and showed that these vectors can distinctly regulate the magnitude and quality of these responses. These findings provide important insights into the mechanism of poxvirus-induced immune response and alternative strategies for vaccine vector design.

NFκB activation by modified vaccinia virus as a novel strategy to enhance neutrophil migration and HIV-specific T-cell responses.

Neutrophils are antigen-transporting cells that generate vaccinia virus (VACV)-specific T-cell responses, yet how VACV modulates neutrophil recruitment and its significance in the immune response are unknown. We generated an attenuated VACV strain that expresses HIV-1 clade C antigens but lacks three specific viral genes (A52R, K7R, and B15R). We found that these genes act together to inhibit the NFκB signaling pathway. Triple ablation in modified virus restored NFκB function in macrophages. After virus infection of mice, NFκB pathway activation led to expression of several cytokines/chemokines that increased the migration of neutrophil populations (Nα and Nß) to the infection site. Nß cells displayed features of antigen-presenting cells and activated virus-specific CD8 T cells. Enhanced neutrophil trafficking to the infection site correlated with an increased T-cell response to HIV vector-delivered antigens. These results identify a mechanism for poxvirus-induced immune response and alternatives for vaccine vector design.

Clathrin regulates lymphocyte migration by driving actin accumulation at the cellular leading edge.

Lymphocyte migration, which is essential for effective immune responses, belongs to the so-called amoeboid migration. The lymphocyte migration is up to 100 times faster than between mesenchymal and epithelial cell types. Migrating lymphocytes are highly polarized in three well-defined structural and functional zones: uropod, medial zone, and leading edge (LE). The actiomyosin-dependent driving force moves forward the uropod, whereas massive actin rearrangements protruding the cell membrane are observed at the LE. These actin rearrangements resemble those observed at the immunological synapse driven by clathrin, a protein normally involved in endocytic processes. Here, we used cell lines as well as primary lymphocytes to demonstrate that clathrin and clathrin adaptors colocalize with actin at the LE of migrating lymphocytes, but not in other cellular zones that accumulate both clathrin and actin. Moreover, clathrin and clathrin adaptors, including Hrs, the clathrin adaptor for multivesicular bodies, drive local actin accumulation at the LE. Clathrin recruitment at the LE resulted necessary for a complete cell polarization and further lymphocyte migration in both 2D and 3D migration models. Therefore, clathrin, including the clathrin population associated to internal vesicles, controls lymphocyte migration by regulating actin rearrangements occurring at the LE.

3DIANA: 3D Domain Interaction Analysis: A Toolbox for Quaternary Structure Modeling.

Electron microscopy (EM) is experiencing a revolution with the advent of a new generation of Direct Electron Detectors, enabling a broad range of large and flexible structures to be resolved well below 1 nm resolution. Although EM techniques are evolving to the point of directly obtaining structural data at near-atomic resolution, for many molecules the attainable resolution might not be enough to propose high-resolution structural models. However, accessing information on atomic coordinates is a necessary step toward a deeper understanding of the molecular mechanisms that allow proteins to perform specific tasks. For that reason, methods for the integration of EM three-dimensional maps with x-ray and NMR structural data are being developed, a modeling task that is normally referred to as fitting, resulting in the so called hybrid models. In this work, we present a novel application-3DIANA-specially targeted to those cases in which the EM map resolution is medium or low and additional experimental structural information is scarce or even lacking. In this way, 3DIANA statistically evaluates proposed/potential contacts between protein domains, presents a complete catalog of both structurally resolved and predicted interacting regions involving these domains and, finally, suggests structural templates to model the interaction between them. The evaluation of the proposed interactions is computed with DIMERO, a new method that scores physical binding sites based on the topology of protein interaction networks, which has recently shown the capability to increase by 200% the number of domain-domain interactions predicted in interactomes as compared to previous approaches. The new application displays the information at a sequence and structural level and is accessible through a web browser or as a Chimera plugin at http://3diana.cnb.csic.es.

Virological and immunological characterization of novel NYVAC-based HIV/AIDS vaccine candidates expressing clade C trimeric soluble gp140(ZM96) and Gag(ZM96)-Pol-Nef(CN54) as virus-like particles.

UNLABELLED: The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in the HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation, and vaccine regimen. In this study, we have generated two novel NYVAC vectors, expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef), and defined their virological and immunological characteristics in cultured cells and in mice. The insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages, and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited. Our findings showed important insights into virus-host cell interactions of NYVAC vectors expressing HIV antigens, with the activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors. IMPORTANCE: We have generated two novel NYVAC-based HIV vaccine candidates expressing HIV-1 clade C trimeric soluble gp140 (ZM96) and Gag(ZM96)-Pol-Nef(CN54) as VLPs. These vectors are stable and express high levels of both HIV-1 antigens. Gag-induced VLPs do not interfere with NYVAC morphogenesis, are highly attenuated in immunocompromised and newborn mice after intracranial inoculation, trigger specific innate immune responses in human cells, and activate T (Env-specific CD4 and Gag-specific CD8) and B cell immune responses to the HIV antigens, leading to high antibody titers against gp140. For these reasons, these vectors can be considered vaccine candidates against HIV/AIDS and currently are being tested in macaques and humans.